THE REGULATION OF G PROTEIN-COUPLED RECEPTOR (GPCR) SIGNAL TRANSDUCTION BY p90 RIBOSOMAL S6 KINASE 2 (RSK2)
by
DOUGLAS JAMES SHEFFLER
Submitted in partial fulfillment of the requirements
for the degree of Doctor of Philosophy
Dissertation Advisor: Bryan L. Roth, M.D., Ph.D.
Department of Biochemistry
CASE
January, 2006
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the dissertation of
Douglas James Sheffler ______candidate for the Ph.D. degree *.
(signed) Martin D. Snider, Ph.D. ______(chair of the committee)
Bryan L. Roth, M.D., Ph.D. ______
William C. Merrick, Ph.D. ______
Paul R. Ernsberger, Ph.D. ______
Gary E. Landreth, Ph.D. ______
______
th October 26 , 2005 (date) ______
* We also certify that written approval has been obtained for any
proprietary material contained therein.
TABLE OF CONTENTS
List of Tables…………………………………………………………………………. .viii
List of Figures………………………………………………………………………...... ix
List of Abbreviations………………………………………………………………...... xii
Abstract……………………………………………………………………………….xviii
CHAPTER 1: Background
1.1 G Protein-Coupled Receptors……………………………………….. 1
1.2 G Protein-Coupled Receptor Desensitization……………………….. 4
1.3 G Protein-Coupled Receptor Endocytosis…………………………… 9
1.4 Serotonin Receptors………………………………………….……… 12
1.5 5-HT2 Receptor Subtype…………………………………………….. 14
1.6 5-HT2 Receptor Signaling…………………………………………… 16
1.7 5-HT2A Receptors…………………..……………………………….. 20
1.8 Regulation of 5-HT2A Receptor Signaling…………………………... 25
1.9 5-HT2A Receptor Interacting Proteins……………………………….. 27
PSD-95 and PDZ Domain Containing Proteins……………………… 29
Arrestins……………………………………………………………… 31
Caveolins…………………………………………………………..… 34
Other 5-HT2A Receptor Interacting Proteins………………………… 36
1.10 The p90 Ribosomal S6 Kinases – Introduction……………………… 38
1.11 The Activation Mechanism of RSK…………………………………. 44
i 1.12 The Role of RSK in Transcriptional Regulation…………………….. 46
1.13 Other Cellular RSK Actions…………………………………………. 52
1.14 RSK Tissue Distribution…………………………………………….. 56
1.15 RSK2 Knock-Out Mice……………………………………………… 58
1.16 Coffin-Lowry Syndrome (CLS) – Introduction…………………….. 60
1.17 Identification of the Gene Mutated in CLS………………………….. 60
1.18 RSK2 Gene Mutations in CLS………………………………………. 61
1.19 Growth, Cognitive Development, and General Features of CLS……. 64
1.20 Cranio-Facial Features of CLS………………………………………. 64
1.21 Skeletal Defects of CLS……………………………………………... 65
1.22 Psychiatric Illness of CLS Patients………………………………….. 66
1.23 Cardiovascular Abnormalities of CLS………………………………. 66
1.24 Movement Disorders of CLS………………………………………… 67
CHAPTER 2: Materials and Methods
2.1 Materials…………………………………………………………….. 69
Chemicals……………………………………………………………. 69
Cell Culture Reagents……………………………………………….. 69
cDNA constructs…………………………………………………….. 70
Antibodies…………………………………………………………… 80
2.2 Methods……………….…………………………………………….. 81
Cell Culture and Transfection……………………………………….. 81
Serum Dialysis ………………………………………………………. 81
ii
Amphotrophic Retrovirus Production, Viral Infection, and Polyclonal Cell Line Production…..……………….………...... 82
Yeast 2-Hybrid Analysis…………………………………………….. 83
Immunocytochemistry, Immunohistochemistry, and Confocal Microscopy …………………………………………… 86
Rat Brain Synaptic Membrane Preparation …………………………. 88
Western Blotting and Immunoprecipitation ………………………… 88
Western Blot Analysis of p42/p44 ERK Phosphorylation…………... 91
Cell Surface Biotinylation …………...... 92
[32P]Orthophosphate Metabolic Labeling …………………………… 94
[γ−32P]-ATP In vitro Kinase Assay …………………………………. 95
Determination of Phosphoinositide (PI) Hydrolysis………………… 97
Intracellular Calcium Mobilization …………….…………………… 101
Determination of cAMP Production …………….………………….. 101
Saturation Binding Assays ………………………………………….. 103
Competition Binding Assays ……………………………………….. 104
Microarray and Pathway Analysis…………………………………... 104
RNA Isolation and Quantitative RT-PCR...... 107
Data Analysis………………………………………………………... 108
CHAPTER 3: The Interaction of p90 Ribosomal S6 Kinase 2 (RSK2) with the 5-HT2A Receptor
3.1 Introduction and Rationale…………………………………………… 109
iii 3.2 A Yeast Two-Hybrid Screen Identifies Potential 5-HT2A Receptor-Interacting Proteins …………………………..…………… 110
3.3 Additional Two-Hybrid Analyses Narrow the Region of Interaction Between the i3 Loop of the 5-HT2A Receptor and the RSK2 “Target” 33.5 ………………………………………… 122
3.4 Full-Length RSK2 and RSK2-GFP Interact with 5-HT2A Receptors In Vitro …………………………………………………… 125
3.5 Endogenous RSK2 Interacts with Endogenous 5-HT2A Receptors In Vivo…………………………………………………….. 131
3.6 5-HT2A Receptors Colocalize with RSK2 and RSK2-GFP in HEK-293 Cells…………………………………………………….. 134
3.7 5-HT2A Receptors Colocalize with RSK2 in the Rat Brain Prefrontal Cortex and Globus Pallidus……………………………….. 137
3.8 Discussion……………………………………………………………. 140
CHAPTER 4: The Regulation of 5-HT2A Receptor Signal Transduction by p90 Ribosomal S6 Kinase 2 (RSK2)
4.1 Introduction and Rationale……………………………………………. 143
4.2 Characterization of RSK2 -/- and RSK2 +/+ Fibroblasts ……………. 144
4.3 RSK2 Knock-out Augments 5-HT2A Receptor Signaling to Phosphoinositide Hydrolysis and to Calcium Mobilization………….. 148
4.4 Microarray Analysis Reveals no Global Alterations in Gene Expression Occur in the Absence of RSK2 ……………………. 152
4.5 RSK2 Knock-out does not Alter the Surface-Expression of 5-HT2A Receptors…………………………………………………... 158
4.6 RSK2 Does Not Alter the Signaling of Constitutively Active Gαq…… 161
4.7 RSK2 Modulates 5-HT2A Receptor-Mediated p42/44 ERK Phosphorylation……………………………………………………… . 164
4.8 RSK2 Can Directly Phosphorylate 5-HT2A Receptors………………... 168
iv
4.9 The Kinase Activity of RSK2 is Essential for the 5-HT2A Receptor Phosphoinositide Hydrolysis Phenotype ………..………… 171
4.10 The Kinase Activity of RSK2 is not Essential for an Interaction with the 5-HT2A Receptor……………………………………………. 175
4.11 Discussion……………………………………………………………. 178
CHAPTER 5: The Role of p90 Ribosomal S6 Kinase 2 (RSK2) in G Protein-Coupled Receptor (GPCR) Signal Transduction
5.1 Introduction and Rationale…………………………………………… 184
5.2 RSK2 Attenuates Gαq-Coupled GPCR Signaling to Phosphoinositide Hydrolysis …………...... 186
5.3 RSK2 Alters Gαq-Coupled GPCR Signaling as Measured by Calcium Mobilization …..……………………………………………. 191
5.4 RSK2 Knock-out Augments the Signaling of β1-Adrenergic Receptors …………………………………………….. 195
5.5 Discussion…………………………………………………………….. 198
CHAPTER 6: Microarray and Pathway Analysis of Genes Expressed in RSK2 -/- and in RSK2 +/+ Fibroblasts
6.1 Introduction and Rationale…………………………………………… 203
6.2 Gene Expression Profiles of Serotonin Receptors and GPCR Signaling Proteins …………………………….…………..….. 207
6.3 Gene Expression Profiles of Mitogen-Activated Protein Kinase Cascade Members ………………………………….………… 211
6.4 Gene Expression Profile of the p90 Ribosomal S6 Kinases (RSKs)….. 211
6.5 Gene expression profile of the G Protein-Coupled Receptors ……….. 213
v Adenosine A2B Receptor (Adora2B)………………………………… 216
Endothelin Receptor Type A (Ednra)………………………………… 217
Protease-Activated Receptor 1 (F2r)…………………………………. 218
Melanocortin-2 Receptor (Mc2r)…………………………………….. 221
Prostaglandin E Receptor 4, Subtype EP4 (Ptger4)………………….. 222
Calcitonin Gene-Related Peptide Type 1 Receptor (Calclr)…………. 223
CD97 Antigen (CD97)……………………………………………….. 225
Parathyroid Hormone Receptor 1 (Pthr1)……………………………. 226
6.6 The Role of RSK2 in Biogenic Amine Synthesis Pathways…………. 227
6.7 The Role of RSK2 in Cell Cycle Control Pathways…………………. 231
Cyclins and Cyclin-Dependent Kinase Inhibitors……………………. 234
CHK1…………………………………………………………………. 236
MCM Family Proteins………………………………………………… 237
MDM2………………………………………………………………… 238
6.8 The Role of RSK2 in Insulin Signaling Pathways……………………. 239
Serum- and Glucocorticoid-Regulated Kinase (SGK)……………….. 242
Phosphatidylinositol 3 Kinase Regulatory Subunit, Polypeptide 4 (PIK3R4)………………………………………………. 242
Grb14…………………………………………………………………. 243
SOS2………………………………………………………………….. 243
c-Cbl-Associated Protein (CAP)……………………………………… 244
EHD2…………………………………………………………………. 245
EGR1 and Trib3……………………………………………………… 245
6.9 Discussion……………………………………………………………. 246
vi CHAPTER 7: Implications and Future Directions
7.1 RSK2 Associates with the 5-HT2A Receptor and Attenuates Signaling…………………………………………………. 255
7.2 Future Directions………………………..…………………………..... 258
7.3 Implications of the Current Findings……………………………….... 260
7.4 The Alteration of GPCR Signaling by RSK2………………………… 262
7.5 Microarray Data Implications………………………………………… 263
7.6 Final Words………………………………………………………….. . 266
Bibliography…………………………………………………………………………. 267
vii LIST OF TABLES
1-1 The Proteins Associated with 5-HT2A Receptors……………………………. 28
3-1 The Results of a Yeast Two-Hybrid Screen Using the i3 Loop of the 5-HT2A Receptor as “Bait” and a Human Brain cDNA as a Pool of “Target” Proteins ……………………………..………………. .. 118
3-2 A yeast-two-hybrid screen reveals potential 5-HT2A receptor interacting proteins…………………………………………………………… 119
4-1 RSK2 knock-out augments 5-HT2A receptor signaling and the re-introduction of wild-type, but not ‘kinase-dead’ RSK2 reverts the phosphoinositide hydrolysis phenotype………………………………….. 174
4-2 Microarray analysis of mouse RSK2 +/+ and RSK2 -/- fibroblasts reveals that phosphatase expression patterns are similar…………………….. 180
5-1 Microarray analysis of RSK2 +/+ and RSK2 -/- fibroblasts reveals the expression of selected GPCR’s………………………………………………. 185
5-2 RSK2 knock-out augments Gαq-coupled GPCR signaling to phosphoinositide hydrolysis………………………………………………….. 190
5-3 RSK2 knock-out alters Gαq-coupled GPCR signaling to calcium mobilization…………………………………………………………. 194
5-4 A summary of Gαq-coupled GPCR functional studies using endogenous GPCR ligands…………………………………………………… 200
6-1 Microarray analyses reveal dysregulation of GPCR expression …………….. 215
viii LIST OF FIGURES
1-1 A schematic representation of a G protein-coupled receptor demonstrates conserved structural features. ………………………………… 3
1-2 Model of the desensitization, endocytosis, and trafficking of a prototypical G protein-coupled receptor …………………………………… 8
1-3 The signaling of Gαq-coupled G protein-coupled receptors………………….. 19
1-4 A schematic representation of p90 ribsomal S6 kinase 2 (RSK2) shows conserved features of RSKs…………………………………………… 41
2-1 Construction of a human FLAG-tagged 5-HT2A receptor with a cleavable N-terminal membrane insertion signal peptide ……………………. 72
2-2 Construction of mouse RSK2 in pcDNA3……………………………………. 75
2-3 Construction of RSK2-EcoRI-REM in pcDNA3……………………………... 77
2-4 Construction of pBABE-RSK2-EcoRI-REM ………………………………… 79
2-5 Stimulation of phosphoinositide (PI) hydrolysis following Gαq-coupled GPCR activation ……………………………………………….. 100
3-1 A schematic showing the molecular basis of a yeast-2-hybrid screen ……...... 113
3-2 A schematic of a general yeast two-hybrid protocol …………………………. 116
3-3 RSK2 is identified at a potential 5-HT2A receptor interacting protein ……….. 121
3-4 Yeast-two hybrid analyses narrow the region of interaction between the 5-HT2A receptor and RSK2……………………………………… 124
3-5 The expression of RSK2 and RSK2-GFP in HEK-293 cells …………………. 127
3-6 RSK2 and RSK2-GFP interact with human 5-HT2A receptors in vitro ………. 130
3-7 RSK2 interacts with 5-HT2A receptors in vivo ……………………………….. 133
3-8 RSK2 and RSK2-GFP colocalize with native human 5-HT2A receptors independently of agonist exposure ………………………………… 136
3-9 RSK2 co-localizes with 5-HT2A receptors in rat brain prefrontal cortex and globus pallidus ………………………………………… 139
ix
4-1 RSK2 knock-out augments 5-HT2A receptor signaling without altering 5-HT2A receptor expression ………………………..……… 147
4-2 RSK2 knock-out augments the signaling of 5-HT2A receptors as measured by phosphoinositide hydrolysis or by calcium mobilization ………………… 151
4-3 RSK2 +/+ and -/- fibroblasts do not show differences in GPCR signaling pathway gene expression……………………………………………………... 155
4-4 RSK2 +/+ and -/- fibroblasts do not show differences in MAPK cascade gene expression ……………………………………………………… 157
4-5 RSK2 knock-out does not alter FLAG-5-HT2A receptor cell surface expression but augments FLAG-5-HT2A receptor signaling ………………..... 160
4-6 RSK2 knock-out does not alter the signaling of a constitutively active Gαq ……………………………………………………… 163
4-7 RSK2 knock-out potentiates basal and agonist-stimulated p42/44 ERK phosphorylation ………………………………………………… 167
4-8 RSK2 phosphorylates the 5-HT2A receptor…………………………………… 170
4-9 RSK2 kinase activity is essential for exerting a “tonic brake” on 5-HT2A receptor signaling ………………………………………………… 173
4-10 N-terminal and C-terminal kinase dead RSK2 mutants interact with human 5-HT2A receptors in vitro……………………………………………… 177
5-1 RSK2 exerts a “tonic brake” on Gαq-coupled GPCR signaling ……………… 189
5-2 RSK2 knock-out alters the signaling of PAR-1-thrombinergic, P2Y-purinergic, and Bradykinin-B receptors as measured by calcium mobilization ……………………………………………………… 193
5-3 RSK2 knock-out augments β1-adrenergic signaling…………………………. 197
6-1 RSK2 +/+ and -/- fibroblasts have few differences in global gene expression …………………….………. ………………………… 206
6-2 RSK2 +/+ and -/- fibroblasts express few monoamine GPCRs …………....… 210
6-3 RSK2 +/+ and -/- fibroblasts display a few differences in biogenic amine synthesis enzyme expression ………………………………………..... 230
x 6-4 RSK2 +/+ and -/- fibroblasts display a number of differences in cell cycle control gene expression profiles …………………………………. . 233
6-5 RSK2 +/+ and -/- fibroblasts display a number of differences in insulin signaling pathway gene expression profiles………………………….. 241
xi LIST OF ABBREVIATIONS
5-HT 5-hydroxytryptamine
5-HT2A 5-hydroxytryptamine 2A (serotonin-2A) receptor
5-HTP 5-hydroxytryptophan
AA amino acid
AADC aromatic L-amino acid decarboxylase
AC adenylate cyclase
ACTH adrenocorticotropic hormone
AD transcriptional activation domain
AOP-2 antioxidant protein 2
AP-2 adaptor protein 2
APC anaphase-promoting complex
ApoD apolipoprotein D
ARF ADP-ribosylation factor
ARIP activin-receptor interacting protein
ATP adenosine 5'-triphosphate
ß2AR ß2-adrenergic receptor
BAD Bcl2-antagonist of cell death
Bcl-xL B-cell leukemia/lymphoma-xL
BDNF brain-derived neurotrophic factor
Bmp4 bone-morphogenic protein 4
xii BODIPY-FL 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3- pentanoic acid, succinimidyl ester
Bub1 budding uninhibited by benzimidazoles 1 homolog
CaM calmodulin
cAMP adenosine 3',5'-cyclic monophosphate
CAP c-Cbl-associated protein
Cav caveolin
CBP CREB-binding protein
Cdk cyclin-dependent kinase
CFP cyan fluorescent protein
CIPP channel-interacting PDZ domain protein
CKIs cyclin dependent kinase inhibitors
CLS Coffin-Lowry Syndrome
CNS central nervous system
CRE cAMP response element
CREB cAMP-responsive-element binding protein
CRLR Calcitonin Gene-Related Peptide Type 1 Receptor
CSF cytostatic factor
CTK C-terminal kinase domain
D2 D2 dopamine receptor
DAG diacylglycerol
DMEM Dulbecco's modified essential medium
DNA-BD DNA-binding domain
DOI (±)-2,5-dimethoxy-4-iodoamphetamine
xiii EGF-TM7 epidermal growth factor-seven transmembrane receptors
EGR1 immediate early growth response gene 1
Emi1 early mitotic inhibitor 1
eNOS endothelial nitric oxide synthase
EP4 prostaglandin E receptor 4, subtype EP4
EPS extrapyramidal side effects
ERα estrogen receptor
ERK extracellular-regulated kinase
ETA endothelin receptor type A
FBS fetal bovine serum
FLNa filamin A
GDP guanosine 5'-diphosphate
GEF guanine-nucleotide exchange factor
GFP green fluorescent protein
GPCR G protein-coupled receptor
GRASP general receptor for phosphoinositides-associated scaffold protein
Grb growth factor receptor-bound protein
GRK G protein-coupled receptor kinase
GSK3 glycogen synthase kinase-3
GTP guanosine 5'-triphosphate
HBD hormone-binding domain
HEK human embryonic kidney
xiv HRP horse radish peroxidase
HSE heat shock element
HSF1 heat shock factor 1
i1 first intracellular loop
i2 second intracellular loop
i3 third intracellular loop
IBMX 3-isobutyl-1-methylxanthine
IEGs immediate early genes
IP3 inositol-1,4,5-trisphosphate
JNK c-Jun N-terminal kinase
-L-W media lacking leucine and tryptophan
LSD lysergic acid diethylamide
MAP2 microtubule-associated protein 2
MAPK mitogen-activated protein kinase
MC2R melanocortin-2 receptor
MCM minichromosome maintainance proteins
MCS multiple cloning site
MDM2 murine double minute 2
MEKK mitogen-activated protein kinase kinase kinase
MPF maturation-promoting factor
MPP-3 MAGUK p55 subfamily member-3
MW molecular weight
NHE1 Na+/H+ exchanger 1
xv NMDA N-methyl-D-aspartate
NSAIDs non-steroidal anti-inflammatory drugs
NSF N-ethylmaleimide-sensitive factor
NTK N-terminal kinase domain
PAGE polyacrylamide gel electrophoresis
PAR protease-activated receptor
PBS phosphate-buffered saline
PDK1 3-phosphoinositide-dependent protein kinase-1
PDZ PSD-95/Discs-large/ZO-1 domain
PI phosphoinositide
PI3K phosphatidylinositol 3 kinase
PIK3R4 phosphatidylinositol 3 kinase kegulatory subunit, polypeptide 4
PIP2 phosphotidylinositol-4,5-bisphosphate
PKA protein kinase A
PKC protein kinase C
PLA2 phospholipase A2
PLC phospholipase C
PP1 protein phosphatase 1
Pthr1 parathyroid hormone receptor 1
QDO quadruple-drop-out media
RAMPs receptor activity modifying proteins
RFU relative fluorescence units
xvi RSK p90 ribosomal S6 kinase
SAP97 synapse-associated protein 97
SGK serum- and glucocorticoid-regulated kinase
SIDEs stimulus-induced drop episodes
SOS son-of-sevenless
Spp1 secreted phosphoprotein-1
SRF serum response factor
SSCP single-strand polymorphism analysis
STAT signal transducer and activator of transcription
TBS tris-buffered saline
TBST tris-buffered saline with Tween-20
TDO triple-drop-out media
TM transmembrane domain
TRAP thrombin receptor activating peptide
UAS upstream activating sequence
xvii The Regulation of G Protein-Coupled Receptor (GPCR) Signal Transduction by p90 Ribosomal S6 Kinase 2 (RSK2)
Abstract
by
DOUGLAS JAMES SHEFFLER
5-hydroxytryptamine 2A (5-HT2A) serotonin receptors are G-protein coupled receptors
(GPCRs) that play prominent roles in both the central nervous system and in the
periphery and are the site of action of many hallucinogens, antipsychotic drugs, and
antidepressants. We have discovered that p90 Ribosomal S6 Kinase 2 (RSK2) interacts
with 5-HT2A serotonin receptors in vitro and in vivo and modulates the signaling of 5-
HT2A and all tested G protein-coupled receptors (GPCRs). We initially evaluated 5-HT2A
receptor signaling in fibroblasts obtained from RSK2 wild type (+/+) and knock-out (-/-)
mice and found that 5-HT2A receptor-mediated phosphoinositide hydrolysis, calcium signaling, and both basal and 5-HT-stimulated p42/44 ERK phosphorylation were
augmented in RSK2 -/- fibroblasts. Importantly, the increased basal p42/44 ERK
phosphorylation was attenuated by a 5-HT2A receptor-selective antagonist, MDL100,907.
Endogenous signaling to phosphoinositide hydrolysis by other GPCRs examined,
including, P2Y-purinergic, PAR-1-thrombinergic and bradykinin-B receptors, was also
potentiated in RSK2 -/- fibroblasts. We also found that β1-adrenergic receptor signaling
to adenlylate cyclase was augmented in RSK2 -/- fibroblasts. Re-introduction of wild type
but not kinase-dead forms of RSK2 into RSK2 -/- fibroblasts reversed the
phosphoinositide hydrolysis and calcium signaling phenotypes. Finally, the 5-HT2A
xviii receptor was determined to be phosphorylated by active RSK2. Intriguingly, inactivating mutations in RSK2 lead to Coffin-Lowry Syndrome, which is characterized by mental retardation, learning and coordination deficits, skeletal and craniofacial deformities, cardiovascular abnormalities, and psychosis. Together, these data indicate that RSK2 constitutively attenuates GPCR signaling and therefore alterations in GPCR signaling may account for many of the clinical manifestations of Coffin-Lowry Syndrome.
.
xix CHAPTER 1: Background
1.1 G Protein-Coupled Receptors
G protein-coupled receptors (GPCRs) are a large, functionally diverse superfamily of receptors that transduce extracellular stimuli into an intracellular biochemical response. GPCRs comprise the largest family of cell surface molecules involved in signal transduction, comprising 1-2% of the human genome (Kroeze et al.,
2003b). GPCRs receive signals in the form of a large variety of ligands including photons, peptides, proteins, lipids, eicosanoids, purines, nucleotides, excitatory amino acids, ions, and small molecules. GPCRs have a number of characteristic structural features including an extracellular N-terminus, 7 transmembrane domains linked by three intracellular and three extracellular loops, and an intracellular C-terminus (Figure 1-1).
Due to the presence of 7 transmembrane domains, GPCRs are also referred to as 7TM or heptahelical receptors.
1
Figure 1-1 - A schematic representation of a G protein-coupled receptor demonstrates conserved structural features. G protein-coupled receptors (GPCRs) are characterized by having an extracellular N-terminus, 7 transmembrane (TM) domains linked by three intracellular and three extracellular loops, and an intracellular C-terminus.
GPCRs are coupled to heterotrimeric G proteins that consist of alpha (α), beta (β), and gamma (γ) subunits.
2 Figure 1-1
3 As their name implies, GPCRs initiate signaling via G-proteins. The G-proteins
coupled to GPCRs are heterotrimeric and consist of alpha (α), beta (β), and gamma (γ)
subunits (Figure 1-1). The GPCR, in essence, is a guanine-nucleotide exchange factor
(GEF) for the Gα subunit. GPCR signaling is initiated by the binding of an agonist to the
GPCR, causing a conformational change of the GPCR, which results in the activation of
GEF activity toward the Gα subunit. This GEF activity causes an increase in the dissociation of GDP from Gα, allowing the rapid exchange of GDP for GTP, which is present in high intracellular concentrations. The activated, GTP-bound Gα subunit then
promotes dissociation of the heterotrimeric complex (Downes and Gautam, 1999). The
GTP-bound Gα subunits and Gβγ dimers then go on to activate a number of second
messenger generating pathways including the activation of phospholipase C and the
activation/inhibition of adenylate cyclase in addition to a variety of other pathways
(Gilman, 1987; Kroeze and Roth, 1998b). The signaling is terminated by the intrinsic
GTPase activity of the Gα subunit, which cleaves GTP to form GDP, inactivating Gα and resulting in the re-association of Gα with Gβγ in a heterotrimeric, inactive complex.
1.2 G Protein-Coupled Receptor Desensitization
The agonist-induced activation of GPCRs leads to a number of processes that can attenuate GPCR signaling. The long-term activation of GPCRs via agonist administration has been shown to desensitize receptors and down-regulate receptor protein levels in a variety of receptor systems (Chuang et al., 1996; Freedman and Lefkowitz, 1996). The process of desensitization is defined as a reduced capability of agonist-induced second
4 messenger production. Two major patterns of rapid GPCR desensitization have been
characterized, homologous or agonist-specific, and heterologous or agonist-nonspecific.
Homologous desensitization is a process whereby stimulation of a particular GPCR leads to desensitization of that specific GPCR. Heterologous desensitization, on the other hand, is where GPCR stimulation leads to desensitization of other types of GPCRs (Chuang et al., 1996). Alternatively, down-regulation is defined as the reduction in total specific receptor binding sites (Bmax) without a change in apparent affinity (Kd), indicating a loss of total cell receptors (Koenig and Edwardson, 1997). Chronic antagonist exposure can also lead to supersensitivity, whereby there is an increase in second messenger production in response to agonist exposure (Roth et al., 1999a).
Desensitization is the result of the convergence of a number of different cellular processes including receptor-phosphorylation, the internalization of cell surface receptors, the down-regulation of receptors due to reduced receptor mRNA or protein synthesis, or increases in the lysosomal and plasma membrane degradation of pre- existing receptors (Gray and Roth, 2002). Receptor phosphorylation via the action of intracellular protein kinases is the most rapid mechanism of receptor desensitization, resulting in the uncoupling of GPCRs from their cognate heterotrimeric G proteins
(Figure 1-2). A number of protein kinases have been discovered to phosphorylate serine and threonine residues within the intracellular domains of GPCRs following agonist exposure, including the second messenger-dependent kinases such as cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), as well as specific G protein-coupled receptor kinases (GRKs) (Ferguson and Caron, 1998; Ferguson et al., 1998; Lefkowitz,
5 1993). The GRK family members have been demonstrated to selectively phosphorylate agonist-activated receptors and are therefore involved in mediating homologous desensitization. Alternatively, the second messenger-dependent protein kinases not only phosphorylate agonist-activated GPCRs, but also indiscriminately phosphorylate receptors that have not been exposed to agonist, hence these protein kinases are involved in the process of heterologous desensitization (Lefkowitz, 1993).
6
Figure 1-2 - Model of the desensitization, endocytosis, and trafficking of a prototypical G protein-coupled receptor. The current model for agonist-induced trafficking of GPCRs based primarily on work on the β2-adrenergic receptor and other
GPCRs. Following activation, receptors are feedback phosphorylated by kinases such as
G protein coupled receptors kinases (GRKs) or second messenger kinases such as protein kinase A (PKA) resulting in rapid desensitization due to G protein uncoupling. G protein uncoupling is further promoted by the binding of arrestins to the phosphorylated third intracellular loops and carboxy-terminal tails of agonist-activated GPCRs. In addition to their role in desensitization, arrestins act as scaffolding proteins promoting the targeting of desensitized receptors to clathrin coated pits for their subsequent internalization by the interaction of the carboxy-terminal portions of arrestin with both the clathrin heavy chain and the β2-adaptin subunit of AP-2. The GTPase dynamin induces neck formation of coated pits and their release into the cytoplasm as clathrin-coated vesicles. These coated vesicles fuse with early endosomes where the receptors may be dephosphorylated by specific phosphatases and recycled back to the plasma membrane fully resensitized or targeted to lysosomes for degradation. Adapted from (Gray and Roth, 2001).
7 Figure 1-2
8 1.3 G Protein-Coupled Receptor Endocytosis
Following agonist stimulation, many GPCRs are rapidly internalized from the plasma membrane into cytoplasmic compartments. The endocytosis of GPCRs to intracellular compartments is an important regulatory process for both the desensitization and down-regulation of GPCRs. There appears to be at least two distinct pathways for agonist-induced endocytosis. The first of the pathways for GPCR endocytosis is via clathrin-coated pits (Lefkowitz et al., 1998; Roth et al., 1981), while the second pathway occurs via caveolae (Anderson, 1998; Okamoto et al., 1998). The molecular mechanism for clathrin-mediated GPCR endocytosis has been extensively studied for the β2- adrenergic receptor (β2AR) (Lefkowitz et al., 1998), but the precise mechanism of internalization by caveolae remains incompletely understood.
As noted, the mechanism of β2AR endocytosis via clathrin-coated pits has been extensively studied and as such is used as a model system for the internalization of
GPCRs via clathrin-coated pits (Lefkowitz et al., 1998). In this model (Figure 1-2), following agonist binding, β2ARs are phosphorylated by the GRKs (Ferguson et al.,
1998; Lefkowitz, 1993). Receptor phosphorylation by GRKs then promotes the binding of cytoplasmic accessory proteins known as β-arrestins to the intracellular loops and carboxy-terminal tails of agonist-activated, phosphorylated GPCRs. The binding of arrestins to phosphorylated residues is accomplished by the disruption of a “polar core” within the arrestin molecule by the highly charged receptor-attached phosphate moiety, resulting in the transition of arrestin to its active high-affinity receptor binding state
9 (Gurevich and Benovic, 1995; Gurevich and Benovic, 1997; Vishnivetskiy et al., 1999).
Indeed, mutations that destabilize the “polar core” of arrestins result in enhanced binding
of arrestins with non-phosphorylated receptor (Gurevich and Benovic, 1995), and these
mutant “constitutively active” arrestins have proven useful for the understanding of the
role of arrestins in GPCR endocytosis. Furthermore, arrestin binding further desensitizes
β2ARs by sterically hindering further G protein coupling (Ferguson, 2001). Indeed, it has
been demonstrated that full desensitization of β2ARs requires both GRK mediated
receptor phosphorylation and arrestin binding (Lohse et al., 1992).
Following the association of arrestins with the phosphorylated receptor, GPCR-
arrestin complexes migrate to clathrin-coated pits for subsequent endocytosis via the
interaction of arrestin with both the heavy chain of clathrin (Goodman et al., 1996) and
with the β2-adaptin subunit of the adaptor protein AP-2, which also interacts with
clathrin (Laporte et al., 2000). Following the migration of GPCRs to clathrin-coated pits,
the GTPase dynamin is required for clathrin-coated vesicle formation. Dynamin is a
GTPase that is required for vesicle pinching from the plasma membrane and has been
shown to be essential for agonist-induced endocytosis (Zhang et al., 1996). In accord with
this, dominant negative dynamin mutants (K44A) have been demonstrated to block β2AR receptor internalization, and these mutants have proven invaluable for the characterization of GPCR endocytosis (Zhang et al., 1996). Following vesicle pinching and release of the vesicles into the cytoplasm as clathrin-coated vesicles, the coated vesicles fuse with early endosomes. After targeting to the endosomal compartment,
GPCRs can be rapidly de-phosphorylated and recycled back to the plasma membrane in a
10 process known as resensitization, or are targeted to lysosomes (or to the proteosome) for degradation (Figure 1-2).
In summary, for the process of clathrin-mediated endocytosis, following agonist- stimulation, many GPCRs become desensitized on the plasma membrane through the cooperation of both receptor phosphorylation and arrestin binding. Arrestin binding then targets the receptors to clathrin-coated pits where they are subsequently internalized.
Internalization leads to GPCR sorting to either the lysosomal compartment for degradation, or recycling back to the plasma membrane in the process of resensitization.
Although this model (Figure 1-2) represents over a decade of investigation, primarily on the β2AR, it is only a model by which other GPCRs can be compared. It is highly likely that receptor-specific differences in this model will become apparent as GPCR endocytosis is likely not universally regulated.
The second pathway of GPCR internalization is via caveolae. Caveolae are small
(50-100 nM), invaginated membrane microdomains that are enriched in cholesterol and glycosphingolipids, first discovered in 1953 through electron micrograph studies of endothelial cells (Palade, 1953). Caveolar microdomain integrity is dependent on cholesterol and requires the presence of the integral membrane protein, caveolin
(Hailstones et al., 1998; Rodal et al., 1999). As in clathrin-mediated endocytosis, dynamin has also been reported to play a function in the endocytosis of caveolae, but its role in caveolar endocytosis is still debated, as other proteins such as intersectin have been reported to be associated with dynamin during the fission and internalization of
11 caveolae in epithelial cells (Henley et al., 1998; Oh et al., 1998; Predescu and Palade,
1993). In addition to caveolae, other cholesterol-dependent, sphingolipid-based
microdomains, known as lipid rafts, exist in the plasma membrane, but little is known
about their role in internalization (Johannes and Lamaze, 2002). Normally, caveolae exist
as immobile, non-endocytic domains on the plasma membrane, but their endocytosis has
been induced in a number of cell types in various ways (Parton et al., 1994; Pelkmans et
al., 2001; Thomsen et al., 2002). As previously stated, little is understood involving
endocytosis via caveolae, however, this pathway of endocytosis is garnering greater
attention as a number of GPCRs (β-adrenergic receptors (Xiang et al., 2002), 5-HT2A serotonin receptors (Dreja et al., 2002), bradykinin receptors (de Weerd and Leeb-
Lundberg, 1997), and endothelin receptors (Chun et al., 1994)) have been reported to be localized in caveolae.
1.4 Serotonin Receptors
The biogenic indolamine neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) was first identified at the Cleveland Clinic Foundation in 1948 as one of the major vasoconstricting substances in defibrinated blood (Rapport et al., 1948). 5-HT is formed by the hydroxylation of tryptophan by tryptophan hydroxylase to form 5- hydroxytryptophan (5-HTP) and the subsequent decarboxylation of 5-HTP by the
Aromatic L-Amino Acid Decarboxylase (AADC). The greatest concentration of 5-HT
(90%) is found in the enterochromaffin cells of the gastrointestinal tract, with the remainder of the body’s 5-HT found in platelets and in the central nervous system (CNS).
12 Since its identification, serotonin has been demonstrated to be involved in variety of
processes in both the CNS and in the periphery. In the CNS, serotonin is involved in the
regulation of feeding behavior, aggression, mood, perception, pain, and anxiety (Bradley
et al., 1986; Roth, 1994). In periphery, serotonin regulates vascular and nonvascular
smooth muscle contraction, platelet aggregation, uterine smooth muscle growth, and
gastrointestinal function (Hoyer et al., 1994; Roth, 1994).
The diverse actions of the serotonin receptors are mediated by at least 15 distinct
serotonin receptors, which are divided into seven major classes: 5-HT1, 5-HT2, 5-HT3, 5-
HT4, 5-HT5, 5-HT6, and 5-HT7 (Hoyer et al., 1994; Roth et al., 2000). Most classes of
serotonin receptors are further divided into a number of subtypes, including the 5-HT2 class, which is subdivided into the 5-HT2A, 5-HT2B, and 5-HT2C receptor subtypes (Roth et al., 1998b). Most serotonin receptor genes do not contain introns, and therefore only a few splice variants have been discovered. Of these, the 5-HT2C receptor has been shown
to have an alternatively spliced, nonfunctional splice variant (Canton, 1996), the 5-HT4 receptor has been found to have at least 2 splice variants (Gerald et al., 1995) and the 5-
HT7 receptor has also been shown to have splice variants (Heidmann, 1997). In addition,
the 5-HT2C receptor also shows extensive RNA editing of the second intracellular loop, leading to a number of editing isoforms (Burns, 1997).
With the exception of the 5-HT3 receptor class, which are ligand-gated ion
channels (Maricq et al., 1991), the remainder of the serotonin receptors belong to the
GPCR superfamily. The serotonin receptors are coupled to three different classical
13 signaling pathways via their coupling to specific G-proteins. The 5-HT1 class of serotonin receptors are coupled to Gαi, an inhibitory type guanine-nucleotide binding protein. Gαi-coupled GPCRs, when activated, result in the inhibition of adenylate cyclase activity (AC), leading to a decrease in cyclic adenosine-monophosphate (cAMP) production. This is measured experimentally by determining the inhibition of forskolin- stimulated AC activity. The 5-HT2 class of serotonin receptors are coupled to Gαq, which results in phosphoinositide hydrolysis via the activation of phospholipase C. The Gαq signaling pathway will be further discussed in a later section of this Introduction. The 5-
HT4, 5-HT6, and 5-HT7 classes of serotonin receptors are coupled to Gαs, a stimulatory type guanine-nucleotide binding protein. Gαs-coupled GPCRs, when activated, result in
the activation of adenylate cyclase activity (AC), leading to an increase in intracellular
cAMP production. The G-protein coupling of the 5-HT5 class of GPCRs remains elusive.
Further understanding of 5-HT receptor subtype signaling and ligand binding will
provide valuable insight for the development of serotonin-based therapeutic drugs.
1.5 5-HT2 Receptor Subtype
It was evident that 5-HT receptor subtypes existed as early as 1954, when organ bath studies suggested two types of serotonin receptors which were designated M and D
(Gaddum and Hameed, 1954; Gaddum et al., 1955; Gaddum and Picarelli, 1957). The 5-
3 HT2 site, resembling the originally identified D site, was later defined by [ H]spiperone
binding studies, and is now designated as the 5-HT2A receptor. 5-HT2A receptors show a heterogeneous distribution of in the brain, with high expression in the cortex and lower
14 expression in the basal ganglia and in the hippocampus (Blue et al., 1988; Pazos et al.,
1985; Roth et al., 1987). Within the cortex, 5-HT2A receptors are found mainly in pyramidal cells (Willins et al., 1997b). In addition, 5-HT2A receptors are found in platelets (de Chaffoy de Courcelles et al., 1987), vascular smooth muscle (Cohen et al.,
1981; Roth et al., 1984; Roth et al., 1986), uterine smooth muscle (Wilcox et al., 1992), and in other tissues (Hoyer et al., 1994). Intriguingly, 5-HT2A receptors have low (near
micromolar) affinity for their endogenous ligand, 5-HT (Peroutka and Snyder, 1979).
This suggests that 5-HT2A receptors may only be activated when 5-HT is released during
neuronal exocytosis or during platelet degranulation where the local concentration of 5-
HT is high (Gray et al., 2003b).
Around the same time, [3H]mesulergine was demonstrated to bind to a distinct 5-
HT receptor in the choroid plexus. This 5-HT site was originally designated the 5-HT1C receptor and is now known as the 5-HT2C receptor (Pazos et al., 1984). 5-HT2C receptors are found in choroids plexus (Pazos et al., 1984), cortex, basal ganglia, hippocampus, and hypothalamus (Molineaux et al., 1989). A third 5-HT2 class member was identified in the
stomach fundus (Kursar et al., 1994; McKenna et al., 1990) and was later designated the
5-HT2B receptor. 5-HT2B receptors are found in the stomach fundus (Wainscott et al.,
1996), vascular smooth muscle (Ullmer et al., 1995), spinal cord (Helton et al., 1994),
and in the brain (Choi and Maroteaux, 1996).
15 1.6 5-HT2 Receptor Signaling
The 5-HT2 class of serotonin receptors couple to Gαq, resulting in the activation of
phospholipase C (PLC) (Berridge et al., 1982; Conn and Sanders-Bush, 1984; Conn et al.,
1986; Roth et al., 1984; Ullmer et al., 1995). PLC cleaves the membrane phospholipid
phosphotidylinositol-4,5-bisphosphate (PIP2), resulting in the production of the second messengers diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). DAG is known to activate protein kinase C (PKC), leading to the phosphorylation of a number of cellular
2+ targets. In addition, the production of IP3 leads to the mobilization of Ca release from
2+ the endoplasmic reticulum (ER) via the stimulation of IP3-gated Ca channels. 5-HT2
class receptors are also known to activate the mitogen-activated protein kinase (MAPK)
cascade, through a yet incompletely defined mechanism (Figure 1-3) (Roth et al.,
1998b).
5-HT2A and 5-HT2C receptors have been demonstrated to activate the expression of the immediate-early gene, c-fos, in the brain (Moorman and Leslie, 1996). 5-HT2A receptors also activate Na+/K+ exchange, the JAK/STAT pathway, and BDNF expression
(Kroeze and Roth, 1998b). In vascular smooth muscle, 5-HT2A receptors have been
shown to activate voltage-gated calcium channels via a PKC mediated mechanism (Roth,
1990). In addition, 5-HT2A receptors can also stimulate phospholipase A2 (PLA2), leading to the production of the second messenger, arachidonic acid (Berg et al., 1994).
PLA2 is an enzyme that hydrolyzes the sn-2 position of membrane phospholipids to release free fatty acids and lysophospholipids. Of the PLA2 enzymes, only cPLA2 is
16 receptor regulated and it preferentially hydrolyzes sn-2 positions containing arachidonic
acid (Gilman, 1987), which is believed to be the rate limiting step in the generation of the
proinflammatory lipid mediators, the eicosanoids. The activation of cPLA2 requires phosphorylation at Ser505 and Ca2+ binding, with the phosphorylation of Ser505 attributed to ERK1/2 of the MAPK cascade (Lin et al., 1993; Nemenoff et al., 1993). It has been found that 5-HT2A receptors couple to cPLA2 via both ERK1/2 and p38 MAPKs in 3T3 cells (Kurrasch-Orbaugh et al., 2003).
17
Figure 1-3 - The signaling of Gαq-coupled G protein-coupled receptors. The binding of agonists to GPCRs causes a conformational change in the GPCR, promoting the exchange of GDP for GTP on the α-subunit of the heterotrimeric G protein, activating the
α-subunit and promoting the dissociation of the heterotrimeric G protein into α- and βγ-
subunits (not shown). The α-subunit of Gαq then activates phospholipase C (PLC), which catalyzes the hydrolysis of phosphotidylinositol-4,5-bisphosphate (PIP2) into the second
messengers diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). DAG activates
protein kinase C (PKC), leading to the phosphorylation of a number of cellular targets. In
2+ addition, the production of IP3 leads and the mobilization of Ca release from the endoplasmic reticulum (ER) via the stimulation of IP3-gated Ca2+ channels. The increase
2+ in intracellular Ca serves to act as an additional second messenger. 5-HT2 class
receptors are also known to activate the mitogen-activated protein kinase (MAPK)
cascade, through a yet incompletely defined mechanism. Phosphates are sequentially
hydrolyzed from IP3 by a series of phosphatases. The final phosphate is removed by inositol P phosphatase to form inositol, which is reincorporated into membrane phospholipids.
18 Figure 1-3
19 1.7 5-HT2A Receptors
As previously described, the 5-HT2A receptor has been shown to be most highly
enriched in the deeper layers of the cerebral cortex (Pazos et al., 1985; Pazos et al., 1987;
Roth et al., 1987). 5-HT2A receptors are also expressed within the striatum and the globus
pallidus (GP) (Bubser et al., 2001). Within the cerebral cortex, 5-HT2A receptors are enriched in apical dendrites and found to a lesser extent in asymmetric synapses and dendritic spines of pyramidal neurons (Cornea-Hebert et al., 1999; Cornea-Hebert et al.,
2002; Miner et al., 2003; Willins et al., 1997b). These 5-HT2A receptors are relevant for pyramidal neuron activity as the direct application of serotonin to pyramidal neurons has been demonstrated to increase the frequency and amplitude of spontaneous excitatory postsynaptic potentials via activation of 5-HT2A receptors (Aghajanian and Marek, 1997).
Cortical pyramidal neurons play a pivotal role in information processing in the cortex (Pucak et al., 1996; Shepherd et al., 1985). Specifically, the apical dendrite is thought to function as the “information gate” for pyramidal neurons wherein information from the extensive dendritic tree integrates before descending onto the soma and the axon
(Whitford et al., 2002). Indeed, apical dendrites of pyramidal neurons have been described to represent a potential site for switching the firing of pyramidal neurons from a normal to a psychotic pattern (Jakab and Goldman-Rakic, 1998), as the dysregulation of pyramidal neuronal activity may underlie certain negative and positive symptoms of schizophrenia (Benes, 1995; Selemon et al., 1995). It has been suggested that an enhanced serotonergic input could contribute to the positive symptoms of schizophrenia
20 and the sensory overload that is common to psychotic states (Roth and Meltzer, 1995). As
previously described, 5-HT2A receptors are located at this information gate, where
activation of these receptors by hallucinogens could disrupt the integrative function of the
pyramidal neurons causing perceptual and cognitive alterations, possibly contributing to
schizophrenia (Willins et al., 1998). Alternatively, antagonism of 5-HT2A receptors at this
information gate may ameliorate cognitive deficits and diminish psychosis (Meltzer,
1999; Roth et al., 1999a).
As early as 1954 it was proposed that serotonin is involved in schizophrenia due
to the structural similarity between lysergic acid diethylamide (LSD) and 5-HT (Wooley
and Shaw, 1954). Further support for the role of 5-HT2 class receptors in the pathogenesis of schizophrenia came with the discovery of a serotonergic component to antipsychotic drug binding in 1978 (Leysen et al., 1978). Clozapine, an atypical antipsychotic drug that is effective in the treatment of both positive and negative symptoms of schizophrenia, is a
5-HT2A receptor antagonist (Fink et al., 1984), and has been demonstrated to down- regulate 5-HT2 receptors (Reynolds et al., 1983), further reinforcing the hypothesis that
5-HT2A receptor blockade may be beneficial in schizophrenia. In addition, clozapine has
been shown to suppress tardive dyskinesia, to lack an effect on serum prolactin levels,
and to rarely cause agranulocytosis (Kroeze and Roth, 1998b). Importantly, clozapine
lacks extrapyramidal side effects (EPS), parkinsonian-like side effects correlated with
high D2 dopamine receptor occupancy in the striatum (Bubser et al., 2001; Matz et al.,
1974). Interestingly, clozapine has been shown to cause 5-HT2A receptor internalization
21 in vitro and alters the subcellular distribution in vivo, providing a possible mechanism for
clozapine-induced down-regulation of 5-HT2A receptors (Willins et al., 1997a).
Since 5-HT2A receptors mediate some of the cognitive process involved in working memory (Williams et al., 2002), which becomes profoundly dysfunctional in schizophrenia, the discovery of clozapine as a 5-HT2A receptor antagonist led to the
subsequent development of a large number of antipsychotic drugs with pharmacology
similar to clozapine. This new class of antipsychotic drugs, which are relatively devoid of
the EPS encountered with other neuroleptics, are called atypical antipsychotic drugs. It
was soon discovered that atypical antipsychotic drugs, as a group, bound with higher
affinity to 5-HT2A receptors than to dopamine D2 receptors (Altar et al., 1986). Indeed,
atypical antipsychotic drugs can be classified based on a 5-HT2A/D2 affinity ratio of >1,
while the typical antipsychotic drugs all have 5-HT2A/D2 affinity ratios <1 (Meltzer et al.,
1989). Thus, all currently approved atypical antipsychotic drugs used in treatment of schizophrenia are relatively potent 5-HT2A receptor antagonists and weak dopamine D2 receptor antagonists (Meltzer et al., 1989; Roth et al., 1998a). In addition to high affinity for 5-HT2A receptors and low D2 dopamine receptor affinity, the atypical antipsychotics
also have affinity for 5-HT2B, 5-HT6, and 5-HT7 sites (Kroeze and Roth, 1998b). The clinically used atypical antipsychotic agents (e.g. risperidone, olanzipine, quetiapine, sertindole, ziprasidone) (Borison, 1997), have revolutionized the treatment of schizophrenia and related disorders.
22 In vivo studies have shown that 5-HT2A receptors mediate 5-HT-induced head
twitch behavior (Willins and Meltzer, 1997), wet dog shakes (Yap and Taylor, 1983), and
urinary bladder contraction (Saxena et al., 1985). Peripherally, 5-HT2A receptors
modulate plasma levels of renin and aldosterone, and mediate vascular smooth muscle
contractility via the mitogen-activated protein kinase (MAPK) pathway (Banes et al.,
1999; Frishman and Grewall, 2000; McKune and Watts, 2001; Van de Kar et al., 2001;
Watts, 1998; Watts et al., 2001). 5-HT2A-selective antagonists are potent vasodilators
and may be anti-hypertensive (Frishman and Grewall, 2000). 5-HT2A receptors also
regulate the neuroendocrine activity of hypothalamic and pituitary cells, promoting the release of a number of hormones, including corticotrophin-releasing factor, adrenocorticotropic hormone (ACTH), oxytocin, prolactin, and vasopressin (Hemrick-
Luecke and Evans, 2002; Knowles and Ramage, 1999; Van de Kar et al., 2001).
Additionally, 5-HT2A receptors may play a role in hyperglycemia and hyperglucagonemia
(Sugimoto and Yamada, 2000). In accord with these notions, atypical antipsychotic
drugs that block the 5-HT2A receptor do not cause hyperprolactinemia, but instead
produce other undesired side effects such as impaired glucose tolerance and lipid
abnormalities (Kapur and Remington, 2001; Kroeze et al., 2003a).
In summary, 5-HT2A receptors are the site of action of most (Aghajanian, 1994;
Glennon, 1990; Nichols, 2004; Roth et al., 2002), but not all (Roth et al., 2002; Sheffler and Roth, 2003) hallucinogens including lysergic acid diethylamide and N, N’- dimethyltryptamine, which function as 5-HT2A receptor agonists. 5-HT2A receptors are essential for mediating a large number of physiologic processes in the periphery and in
23 the CNS including platelet aggregation, smooth muscle contraction, and the modulation
of mood and perception (Roth et al., 1998a). Since the dysregulation of the 5-HT2A serotonergic system has been implicated in a large number of diseases including depression, anxiety, schizophrenia, serotonin receptors have been used as a molecular target for multiple drugs of diverse therapeutic classes that mediate their actions, at least in part, by interactions with 5-HT2A receptors. To this end, many 5-HT2A receptor antagonists are effective antidepressants and antipsychotic drugs (Meltzer et al., 2004;
Meltzer et al., 1989; Roth, 1994; Roth et al., 2004). Thus, activation of the 5-HT2A receptors produces hallucination, whereas blockade of the 5-HT2A receptors is therapeutic
(Kroeze and Roth, 1998a; Roth et al., 1999b). Given that 5-HT2A receptors play crucial roles in the modulation of perception, cognition, and emotion (Jakab and Goldman-Rakic,
1998; Kroeze and Roth, 1998b; Roth, 1994; Roth et al., 1999a), insights into the molecular and cellular mechanisms governing 5-HT2A function can potentially aid us in understanding the pathogenesis and pathophysiology of schizophrenia. Since the molecular cloning of the 5-HT2A receptor (Pritchett et al., 1988; Saltzman et al., 1991;
Stam et al., 1992), there has been steady progress in understanding the molecular biology
(Roth et al., 1998a), protein structure (Roth and Shapiro, 2001), intracellular trafficking of the 5-HT2A receptor (Gray and Roth, 2001) and importance of scaffolding proteins in
5-HT2A receptor regulation (Bhatnagar et al., 2004; Xia et al., 2003a; Xia et al., 2003b).
24 1.8 Regulation of 5-HT2A Receptor Signaling
5-HT2A receptors demonstrate a number of differences from the paradigms of
GPCR desensitization, internalization, and down-regulation that have been previously described. For example, 5-HT2A receptors are unique in that they exhibit desensitization and down-regulation following both agonist and antagonist treatment (Blackshear et al.,
1986; Peroutka and Snyder, 1980; Roth et al., 1999a). Studies in P11 cells have
demonstrated that 5-HT2A receptors undergo agonist-induced down-regulation. It was
shown in these studies that agonist stimulation results in a transient increase in 5-HT2A mRNA and in mRNA stability (Wohlpart and Molinoff, 1998). However, in NIH-3T3 cells 5-HT2A receptors are desensitized but not down-regulated by agonist exposure (Roth et al., 1995). Other studies have shown a role for PKC (Roth et al., 1986) and for the
calcium-calmodulin-dependent protein kinases (Kagaya et al., 1993; Rahman and
Neuman, 1993) in the desensitization of 5-HT2A receptors, although the role of agonist-
directed phosphorylation of 5-HT2A remains unresolved.
Studies in HEK-293 cells and in C6 glioma cells have investigated the role of
endocytosis in the desensitization of 5-HT2A receptors (Gray et al., 2001). In HEK-293
cells, blocking endocytosis with a variety of chemical inhibitors or with dominant
negative dynamin (K44A) had no effect on desensitization, although resensitization was
potentiated (Gray et al., 2001). However, these studies also demonstrated that in C6
glioma cells, blocking endocytosis potentiated desensitization and attenuated
resensitization. These studies also found that the over-expression of GRK2 or GRK5 had
25 no effect on the desensitization of 5-HT2A receptors in HEK-293 cells (Gray et al., 2001).
Together, these studies have indicated that 5-HT2A receptor desensitization,
internalization and down-regulation are independent processes and that cell-type specific
differences exist for 5-HT2A receptor regulation.
As previously discussed, receptor phosphorylation followed by binding of arrestin-like proteins is a possible key event in inducing receptor internalization.
However, both 5-HT2A receptor agonists and antagonists induce redistribution of
receptors in transfected cells and in pyramidal neurons of the rat medial prefrontal cortex
(Willins et al., 1997a). In addition, the 5-HT2A receptor antagonist clozapine has been shown to cause 5-HT2A receptor internalization in vitro and to alter the subcellular distribution of 5-HT2A receptors in vivo. From this, it has been suggested that a common feature of atypical antipsychotics may be to alter subcellular distribution of 5-HT2A
(Willins et al., 1998). Therefore, arrestin binding to phosphorylated receptors, as previously discussed, can not be a universal signal for receptor internalization as antagonist treatment does not result in any measurable receptor activity or the
phosphorylation of receptors (Roettger, 1997). The elucidation of the regulatory
mechanisms for 5HT2A receptor signaling and trafficking will provide important insight into the therapeutic mechanisms of drugs that target the 5-HT2A receptor and has
implications for the rational design of novel medications (Gray and Roth, 2002; Kroeze et
al., 2002).
26 1.9 5-HT2A Receptor Interacting Proteins
As previously stated, GPCRs transduce extracellular stimuli into intracellular
signals via the activation and dissociation of heterotrimeric G proteins into Gα and Gβγ subunits, which then activate or inhibit downstream effectors (Cabrera-Vera et al., 2003).
However, this is an extreme oversimplification of GPCR signaling. GPCR signaling is not a linear process as GPCRs can interact with a number of multi-domain scaffolding proteins (Hall and Lefkowitz, 2002) in addition to a variety of other accessory or chaperone proteins (Brady and Limbird, 2002). The interaction of GPCRs with these proteins allows the generation of signaling specificity through the alteration of ligand recognition, the compartmentalization of signaling complexes, and through the regulation of GPCR trafficking (Brady and Limbird, 2002). Further understanding of the regulation of 5-HT2A receptor, and other GPCR, signaling via interactions with scaffolding,
accessory, and other proteins will advance our understanding of GPCR signaling and will
present novel targets for drug development and for disease treatment. To date a number
of 5-HT2A receptor interacting proteins have been discovered (Table 1-1). The 5-HT2A receptor interacting proteins fall into several broad categories: (1) PDZ domain containing proteins, (2) arrestins, (3) caveolins, and (4) other proteins. In the following section, I will summarize these known 5-HT2A interacting proteins and their role in 5-
HT2A receptor regulation.
27 Table 1-1 -The Proteins Associated with 5-HT2A Receptors
PROTEIN NAME FUNCTIONS AND REFERENCE Post Synaptic Density Protein 95 PDZ Domain-Containing Scaffolding Protein, (PSD-95) Regulation of Signal Transduction, Regulation (Xia et al., 2003a) of Protein Targeting Arrestins Scaffolding Protein, Regulator of Signal (Gelber et al., 1999) Transduction, Regulator of Clathrin-Mediated Endocytosis Antioxidant Protein 2 (AOP-2) A Thiol-Specific Antioxidant, Protects Proteins (Becamel et al., 2004) and DNA from Oxidative Damage
Synapse Associated Protein 97 PDZ Domain-Containing Scaffolding Protein (SAP97) (Becamel et al., 2004) Activin Receptor Interacting Protein-1 PDZ Domain-Containing Scaffolding Protein, (ARIP-1) Function Unclear (Becamel et al., 2004) ADP Ribosylation Factor-1 (ARF-1) Small GTP-binding Protein Involved in (Robertson et al., 2003) Membrane Trafficking
Channel Interacting PDZ Domain PDZ Domain-Containing Scaffolding Protein Protein (CIPP) (Becamel et al., 2004) MAGUK p55 Subfamily Member-3 PDZ Domain-Containing Scaffolding/Adaptor (MPP-3) Protein (Becamel et al., 2004) Calmodulin (CaM) Regulation of Signal Transduction, Possible (Turner and Raymond, 2005) Regulation of Receptor Phosphorylation
Caveolin-1 (Cav-1) Formation of Membrane Microdomains Known (Bhatnagar et al., 2004) as Caveolae, Scaffolding Protein, Regulation of Cholesterol Homeostasis
28 PSD-95 and PDZ Domain Containing Proteins
The C-terminal domain of many proteins contain structural motifs necessary for
appropriate sorting (El Far and Betz, 2002) and intracellular trafficking (Gray and Roth,
2002). The last four amino acids of the 5-HT2A receptor contain a canonical type I PDZ-
binding motif (VSCV), (X-S/T-X-φ), a protein-protein interaction motif that could serve
as a targeting signal (Kornau et al., 1995). PDZ-binding domains associate with PSD-
95/Discs-large/ZO-1 domain (PDZ domain) containing proteins such as post synaptic density 95 (PSD-95), which is also known as synapse associated protein 90 (SAP-90)
(Hung and Sheng, 2002; Lim et al., 2002). Multi-domain proteins, such as PSD-95, act as protein scaffolds in addition to known roles in modulating the function of the ion channels and receptors with which they associate (Bezprozvanny and Maximov, 2001;
Bredt, 1998; Harris and Lim, 2001; Hung and Sheng, 2002; Kim, 1997). For example,
PSD-95 has been shown to associate with and inhibit the internalization of β1-adrenergic receptors, in addition to facilitating the interaction between β1-adrenergic receptors and the N-methyl-D-aspartate ionotropic glutamate receptor (NMDA receptor) (Hu et al.,
2000).
As PSD-95 is enriched in asymmetric synapses and dendritic spines of cortical pyramidal neurons (Aoki et al., 2001), the same regions that 5-HT2A receptors are
enriched in, Xia et al. hypothesized that PSD-95 may be involved in 5-HT2A receptor regulation (Xia et al., 2003b). It was determined that the PDZ-binding domain of 5-HT2A receptors is necessary for the dendritic targeting of 5-HT2A receptors in pyramidal
29 neurons in vitro. However, these studies also disclosed that the PDZ-binding domain
interactions are not involved in the axonal exclusion of 5-HT2A receptors (Xia et al.,
2003b). Further studies by Xia et al. revealed that PSD-95 directly interacts with the 5-
HT2A receptor in vitro (Xia et al., 2003a). The interaction of PSD-95 with the 5-HT2A receptor enhanced 5-HT2A receptor signaling, inhibited agonist-induced internalization, and promoted 5-HT2A receptor clustering on the plasma membrane (Xia et al., 2003a).
These data indicate that PSD-95 is involved in 5-HT2A receptor trafficking and in the
regulation of 5-HT2A receptor signal transduction.
Recently, the association of PDZ domain containing proteins with 5-HT2A and 5-
HT2C receptors was examined by affinity chromatography in combination with MALDI-
TOF mass spectrometry (Becamel et al., 2004) These studies disclosed a number of PDZ domain containing proteins found in complex with either 5-HT2A and 5-HT2C receptors and demonstrated that differences in 5-HT2A and 5-HT2C PDZ domain protein complexes exist in vitro. In this study, activin receptor-interacting protein 1 (ARIP-1), synapse- associated protein 97 (SAP97), PSD-95, MAGUK p55 subfamily member-3 (MPP-3), channel-interacting PDZ domain protein (CIPP), and antioxidant protein 2 (AOP-2) were found to be associated with 5-HT2A receptors in vitro (Becamel et al., 2004). A number of these PDZ domain-containing proteins including ARIP-1, SAP97, PSD-95, MPP-3, and
CIPP are known to act as molecular scaffolds for the assembly of protein signaling complexes. On the other hand, AOP-2 is a thiol-dependent antioxidant that functions to scavenge particular hydroperoxides in the cell and to mediate specific signals (Phelan,
30 1999). The functional consequence of 5-HT2A receptor interaction with these PDZ domain-containing proteins will require further investigation.
Arrestins
As previously described, in the classical model of GPCR regulation, agonist activation of GPCRs results in phosphorylation of GPCRs by GRKs, which promotes receptor association with arrestins and uncoupling from G-proteins (Krupnick and
Benovic, 1998; Lefkowitz et al., 1998). The interaction of arrestins with the phosphorylated receptor further desensitizes GPCRs in most cases, terminating receptor signaling by inhibiting G protein coupling. Αrrestins then act as adaptor molecules, linking GPCRs to clathrin coated pits (Kohout and Lefkowitz, 2003; Luttrell and
Lefkowitz, 2002), leading to internalization of the receptor and eventual degradation of the receptor via lysosomes or proteosomes or the recycling of the receptors to the plasma membrane (Ferguson, 2001). Since the interaction of arrestins with GPCRs is not defined by a motif unique to one or several receptors, it has been speculated that arrestins acts as protein scaffolds for most GPCRs. As such, arrestins act as scaffolds for a number of proteins involved in the endocytotic pathway such as clathrin (Goodman et al., 1996), adaptor protein 2 (AP-2) (Laporte et al., 1999), N-ethylmaleimide-sensitive factor (NSF)
(McDonald et al., 1999), the small G protein ADP ribosylation factor 6 (ARF6), and its guanine nucleotide exchange factor, ARNO (Claing et al., 2001). The interactions of arrestins with these proteins facilitate the targeting of agonist-induced GPCR into clathrin-coated pits.
31
Arrestins have also been shown to serve as a protein scaffold for proteins
involved in signal transduction. For example, arrestins can associate with the tyrosine
kinase Src (Luttrell et al., 1999; Miller et al., 2000). The recruitment of Src by arrestins has been shown to play key roles in GPCR signaling for such GPCRs as the β2- adrenergic receptor (Luttrell et al., 1999), neurokinin receptors (Defea et al., 2000),
protease-activated receptors (DeFea et al., 2000), and endothelin receptors (Imamura et
al., 2001). Other proteins known to associate with arrestins include members of two
distinct mitogen activated kinase (MAP) kinase cascades, c-Jun N-terminal kinase 3
(JNK3) and extracellular signal regulated kinases 1 and 2 (ERK1/2), where JNK3 activity
was shown to be potently regulated by β-arrestin 2 and activated angiotensin AT1 receptors (McDonald et al., 2000; Miller et al., 2001). Together, these findings implicate arrestins as key regulators of both GPCR endocytosis and signal transduction.
Due to the role of arrestins in GPCR trafficking and signaling, a number of studies have addressed the role of arrestins in 5-HT2A receptor signaling (Bhatnagar et al.,
2001; Gelber et al., 1999; Gray et al., 2003a). Initial studies demonstrated that the i3 loop of the 5-HT2A receptor can bind purified arrestins, demonstrating a probable role for arrestins in 5-HT2A receptor regulation. In addition, arrestin-3 and β-arrestin have been shown to colocalize with 5-HT2A receptors in cortical neurons (Gelber et al., 1999).
These studies lent support to a probable role of arrestins in the regulation of 5-HT2A receptor signaling. However, in HEK-293 cells, 5-HT2A receptor internalization has been shown to be arrestin independent but dynamin dependent. Furthermore, in these studies
32 arrestin-2 and arrestin-3 were sorted to compartments distinct from 5-HT2A receptors
(Bhatnagar et al., 2001).
It was hypothesized in these studies that the arrestin-independent regulation of 5-
HT2A receptors in HEK-293 cells is probably caused by an apparent lack of agonist- induced receptor phosphorylation (Gray and Roth, 2001). In accord with this, high levels of basal phosphorylation of 5-HT2A receptors are found in HEK-293 cells when 5-HT2A receptors are over-expressed, agonist exposure does not increase the level of 5-HT2A receptor phosphorylation, and PKC does not play a role in the desensitization of 5-HT2A
receptors in HEK-293 cells (Vouret-Craviari et al., 1995). In addition, studies by Gray et
al., where all Ser/Thr residues on the intracellular side of the 5-HT2A receptor were
mutated demonstrated that only the S188 (i2 loop) and S421 (C-terminal tail) mutations
to ala result in attenuation of desensitization. Furthermore, mutation of all of the PKC
sites in the 5-HT2A receptor did not affect 5-HT2A receptor desensitization by PKC. These studies hypothesized that S188 may be important for G-protein coupling (Gray et al.,
2003b).
However, studies with constitutively active arrestin (R169E) that can bind to non- phosphorylated receptors, as previously described, have demonstrated that 5-HT2A receptors interact with arrestins. In these studies, the constitutively active arrestin interaction with the 5-HT2A receptor was shown to drive substantial basal internalization
of the 5-HT2A receptor. In addition, this interaction locked 5-HT2A receptors into an
agonist high affinity state as arrestin binding prefers the high-affinity state of GPCRs.
33 Interestingly, an inverse agonist was able to reverse the spontaneously formed arrestin-
receptor interaction (Gray et al., 2003a). Together, it appears that arrestins are likely to
have cell type specific effects that depend on the cellular milieu. It is likely that arrestin-
and phosphorylation-independent mechanisms are involved in the cellular trafficking of
GPCRs and in particular of the 5-HT2A receptor (Gray and Roth, 2001).
Caveolins
As previously described, caveolae are small (50-100 nM), invaginated membrane
microdomains that are enriched in cholesterol and glycosphingolipids, first discovered in
1953 through electron micrograph studies of endothelial cells (Palade, 1953). Caveolar microdomain integrity is dependent on cholesterol and requires the presence of the integral membrane protein, caveolin, which is a cholesterol binding protein (Hailstones et al., 1998; Rodal et al., 1999). Three members of the caveolin gene family have been identified: caveolin-1 (Cav-1), caveolin-2 (Cav-2), and caveolin-3 (Cav-3) (Monier et al.,
1995). Cav-1 and Cav-2 are ubiquitously expressed whereas Cav-3 shows muscle specific expression (Parton, 1996). The expression of Cav-1 has been shown to be sufficient to drive the formation of caveolae in cells lacking caveolae, pointing to the requirement of
Cav-1 for caveolae formation (Fra et al., 1995).
Cav-1 has been shown to act as a protein scaffold for a number of proteins involved in signal transduction including endothelial nitric oxide synthase (eNOS) (Ju et al., 1997), G proteins, H-Ras, and c-Src (Li et al., 1996). Cav-1 appears to primarily act
34 as a negative regulator of the activity of these proteins, sequestering them into caveolae
and maintaining them in inactive conformations (Ostrom and Insel, 2004). In addition,
gene silencing and knock-out studies have shown an indispensable role of Cav-1 in
maintaining the Ras-p42/p44 MAPK cascade (Galbiati et al., 1998), regulating nitric
oxide synthesis, cholesterol metabolism, and cardiac function (Hnasko and Lisanti,
2003). As previously stated, little is understood involving endocytosis via caveolae, however, this pathway of endocytosis is garnering greater attention as a number of
GPCRs (β-adrenergic receptors (Xiang et al., 2002), 5-HT2A serotonin receptors (Dreja et
al., 2002), bradykinin receptors (de Weerd and Leeb-Lundberg, 1997), and endothelin
receptors (Chun et al., 1994)) have been reported to be localized in caveolae.
In previous studies we have demonstrated that 5-HT2A receptors interact with
Cav-1 in a number of cell systems including HEK-293 cells, C6 glioma cells, and in rat brain synaptic membrane preparations (Bhatnagar et al., 2004). Cav-1 was also shown to co-localize with 5-HT2A receptors in HEK-293 cells both prior to and following agonist exposure (Bhatnagar et al., 2004). Knock-down of Cav-1 expression in C6 glioma cells led to a number of consequences for 5-HT2A receptor signaling. In these studies, Cav-1
knock-down nearly abolished 5-HT2A receptor signaling to phospholipase C, as measured
2+ by Ca mobilization. In addition, there was a total dysregulation of 5-HT2A receptor
p42/p44 ERK phosphorylation in the absence of Cav-1 (Bhatnagar et al., 2004), as would be expected given the known role of Cav-1 in the regulation of the Ras-p42/p44 MAPK cascade (Galbiati et al., 1998). Together, these studies highlight the important role of
Cav-1 and caveolae in 5-HT2A receptor signaling.
35 Other 5-HT2A Receptor Interacting Proteins
The remainder of the identified 5-HT2A receptor interacting proteins do not fall
within a particular category and include calmodulin (CaM) (Turner and Raymond, 2005)
and the ADP ribosylation factor 1 (ARF-1) (Robertson et al., 2003). CaM is a small,
soluble, Ca2+ binding protein that functions as the major calcium sensor in most cells
(Saimi and Kung, 2002). Binding of Ca2+ to CaM induces a structural change in CaM, revealing hydrophobic domains capable of binding to other cellular targets (Babu et al.,
1985). Turner et al. recently demonstrated that CaM binds to the 5-HT2A receptor through two sites in an agonist-dependent manner. One of these sites is present in the i2 loop and the other is found at the boundary between TMVII and the C-terminal tail of the receptor
(Turner and Raymond, 2005). The binding of CaM to the 5-HT2A receptor was shown to have two important functional consequences. First, CaM binding to either the i2 loop or to the C-terminal tail resulted in a decrease in G protein coupling. Secondly, CaM was shown to negatively regulate the phosphorylation of 5-HT2A receptor peptides by active
PKC, demonstrating a possible role for CaM in modulating the phosphorylation of the 5-
HT2A receptor (Turner and Raymond, 2005). These studies suggest that CaM is a novel regulator of 5-HT2A receptor function.
The ADP ribosylation factors (ARFs) are a group of six ubiquitously expressed
small GTP-binding proteins that are essential components of the machinery that regulates
membrane trafficking along both endocytotic and biosynthetic pathways (Chavrier and
Goud, 1999; Donaldson and Klausner, 1994). ARF-1 is one of the best characterized of
36 the ARFs, is primarily localized to the Golgi complex, and is a regulator of vesicle coat
recruitment for both COP1- and clathrin-coated vesicles (Rothman, 1996; Schekman and
Orci, 1996). Although primarily localized in the Golgi network, ARF-1 has been shown
to be translocated to the plasma membrane following GPCR stimulation (Robertson et al.,
2003). Robertson et al. showed an interaction of ARF-1 with the 5-HT2A receptor that occurs within the C-terminal domain of the 5-HT2A receptor (Robertson et al., 2003).
These studies showed that the interaction of the 5-HT2A receptor with ARF-1 was necessary for the activation of phospholipase D by the 5-HT2A receptor (Robertson et al.,
2003).
As can be seen, 5-HT2A receptor signaling is complex and is regulated by a variety of proteins-protein interactions. As described, the majority of these proteins
interact with the intracellular loops and the C-terminal tail of the 5-HT2A receptor. As the i3 loop is known to be important for G-protein coupling (Hyde and Roth, 1997;
Oksenberg et al., 1995), we sought to identify other proteins that could bind to the i3 loop of the 5-HT2A receptor and to investigate the role of these proteins in 5-HT2A receptor signaling. To this end, we conducted a yeast two-hybrid screen using the i3 loop of the 5-
HT2A receptor as “bait” and a human brain cDNA library as a pool of “target” proteins.
We discovered a number of potential 5-HT2A receptor interacting proteins (see Chapter
3). Of these proteins, I had a particular interest in p90 ribosomal S6 kinase 2 (RSK2), which I selected for further study. As the remainder of my studies focus on the role of
RSK2 in 5-HT2A and other GPCR signaling, the remainder of this Introduction will focus on the role and regulation of RSKs in signal transduction.
37 1.10 The p90 Ribosomal S6 Kinases - Introduction
The p90 ribosomal S6 kinases (RSKs), also referred to as the MAPKAP Kinases and S6K, are a unique family of serine/threonine protein kinases that are activated by a variety of cellular stimuli, growth factors, insulin, phorbol-esters, heat shock, and ionizing radiation (Chen et al., 1991; Chen et al., 1992; Frodin and Gammeltoft, 1999;
Merienne et al., 2000; Rivera et al., 1993; Zhang et al., 2001). RSKs were originally isolated from Xenopus laevis oocytes as kinases that could phosphorylate the 40S subunit of the ribosomal subunit protein S6 (Erikson and Maller, 1988). The phosphorylation of the ribosomal protein S6 is believed to promote the translation of selected mRNAs important for cell growth, hence this isolation was a search for a growth-factor regulated kinase (Jefferies et al., 1997). In Xenopus laevis, two S6 protein kinases were identified in the original isolation, S6KI and S6KII. Xenopus S6KII was the first RSK purified and was designated S6 kinase II to indicate its order of elution on anion-exchange chromatography (Erikson and Maller, 1986). The RSK nomenclature was proposed by
Alcorta et al. in 1989 when the mouse homologues of Xenopus S6K, were identified: rskmo-1 (later designated RSK1) and rskmo-2 (later designated RSK2) (Alcorta et al., 1989).
The RSKs are unique serine/threonine protein kinases containing two separate
kinase domains connected by a linker domain (Figure 1-4). The N-terminal kinase
(NTK) domains of RSKs are responsible for the phosphorylation of exogenous substrates
and are most closely related to the kinases of the AGC family, such as protein kinase A
(PKA) and protein kinase C (PKC). Alternatively, the C-terminal kinase (CTK) domain
38 of RSKs, which are involved in the regulation of RSK activity, are most closely related to the calcium/calmodulin-dependent kinases and to phosphorylase b kinase (Alcorta et al.,
1989). Multiple phosphorylation events by upstream protein kinases are necessary for the complete activation of the N-terminal kinase (NTK) domain of RSKs (Figure 1-4). The activation mechanism of RSKs will be described in detail in a later section of this
Introduction.
39
Figure 1-4 - A schematic representation of p90 ribosomal S6 kinase 2 (RSK2) shows conserved features of RSKs. RSKs are characterized by two independent kinase domains. The N-terminal kinase (NTK) phosphorylates substrates, while the C-terminal kinase (CTK) is involved in the regulation of RSK activity. The activation scheme of
RSKs involves the phosphorylation of a number of residues by the extracellular-regulated kinases (ERKs), the phosphoinositide-dependent protein kinase-1 (PDK1), and the CTK of RSK. ERK1/2 phosphorylates Thr365, Ser369, and Thr577 (RSK2 numbering) following mitogen stimulation. Following this, the CTK of RSK becomes active and autophosphorylates RSK in the linker region on Ser386 (RSK2 numbering). This creates a docking site for PDK1, which then phosphorylates Ser227 (RSK2 numbering), resulting in the full activity of the NTK of RSKs.
40 Figure 1-4
41 The RSK family of protein kinases consists of four highly homologous isoforms
(RSK1-4). RSK1 and RSK2 are 80% homologous, whereas RSK3 is a novel RSK
isoform with a unique N-terminal domain containing a putative nuclear localization
signal (NLS), K-K-X10-L-R-R-K-S present from amino acids 6-20. RSK3 is more
closely related to RSK2 (84%) than to RSK1 (75%) (Moller et al., 1994). RSK4 has been
recently identified as the fourth member of the RSK family, which appears to not be
regulated by growth factors and demonstrates constitutive activity (Dummler et al.,
2005). The RSK family of protein kinases are also closely related to the mitogen- and stress-activated protein kinases (MSKs) and RSK-B, which are 40% identical to the
RSKs and induced by TNFα and cellular stress stimuli (Deak et al., 1998; Moller et al.,
1994; Pierrat et al., 1998). The RSKs are phosphorylated and activated by extracellular regulated kinases (ERKs) following mitogen stimulation as will be described shortly
(Chen et al., 1992). Alternatively, the MSKs and RSK-B are activated by p38 MAPK in addition to the ERKs (Deak et al., 1998; New et al., 1999; Pierrat et al., 1998).
In their studies, Erikson and Maller primarily characterized the Xenopus S6KI,
which is the homolog of RSK1 (Erikson and Maller, 1985). The identity of S6KII was
not determined for another 12 years when Bhatt and Ferrel identified S6KII as the
homolog of RSK2 and noted that S6KII is the predominant RSK in Xenopus oocytes
(Bhatt and Ferrell, 2000; Erikson and Maller, 1988). Interestingly, the RSK nomenclature
is a misnomer as the S6 ribosomal subunit is likely not a target of RSKs in vivo. Since
the original identification of the RSKs, another S6 kinase of 70-kDa, designated
p70S6K, has been discovered which has the highest sequence homology to the NTK of
42 RSKs (Banerjee et al., 1990). It has been determined that the Km for the phosphorylation
of the 40S ribosomal protein subunit by p70S6K is 0.5 µM, whereas the Km for RSK is
10 µM (Zhao et al., 1995). Hence, p70S6K is the S6 kinase physiologically and not the
RSKs (Ballou et al., 1991; Blenis, 1993).
Null mutations in RSK2 result in the X-linked mental retardation syndrome,
Coffin-Lowry Syndrome (CLS) (Trivier et al., 1996). CLS is characterized by severe mental retardation, pathognomonic craniofacial and skeletal deformities, growth retardation (Lowry et al., 1971), movement disorders (Stephenson et al., 2005),
cardiovascular disorders, and a schizophrenia-like psychosis in heterozygote females
(Hanauer and Young, 2002; Sivagamasundari et al., 1994). Recently, RSK4 was
identified as the newest member of the RSK family and found to be commonly deleted in
patients with complex X-linked mental retardation. However, little has been discovered
about the function of RSK4 since its identification in 1999 (Yntema et al., 1999). The
phenotype of CLS provides insight into the role of RSK2 in signal transduction and will
be described in depth later in this Chapter. As RSK2 null mutations result in CLS, there
has been an intense focus on understanding the method of RSK activation, the
identification of RSK2 targets, the role of RSK2 in signal transduction, and in the
expression pattern of RSK2.
43 1.11 The Activation Mechanism of RSK
Growth factor and cytokine receptors linked to intracellular tyrosine kinases activate three major signaling pathways in the regulation of cell proliferation and differentiation. These include phosphoinositol-3-kinase (PI3K) and protein kinase B
(PKB) activation leading to cell survival, p70S6K activation leading to the stimulation of growth-associated protein synthesis, and the Ras-ERK MAPK cascade involved in cell division and differentiation (Hubbard et al., 1998; Superti-Furga and Courtneidge, 1995).
The activation mechanism of the RSKs is complex, requiring the coordinate phosphorylation of RSKs by a number of upstream protein kinases. The primary pathway of RSK activation is through the ERK MAP kinase cascade, with RSKs lying directly downstream of ERK1/2 in this pathway.
ERK1/2, preferentially in the inactive form, associates with the C-terminal 60 amino acids of RSKs within the consensus motif, L-A-Q-R-R-X-X-X-X-(L/I). The formation of this ERK-RSK complex is essential for the efficient ERK activation of RSK
(Roux et al., 2003; Smith et al., 1999). Although RSK3 is activated by ERK in vivo, it is not activated by ERK in vitro, suggesting a missing, yet unidentified co-factor may be necessary in vitro (Zhao et al., 1996). For RSK2, ERK1/2 phosphorylates Ser369 in the linker and Thr577 in CTK domain activation loop following stimulation of the ERK
MAPK cascade (Figure 1-4). Following phosphorylation of Thr577, the CTK of RSK2 becomes active and autophosphorylates RSK2 on Ser386 in the linker region. The RSK2
CTK consensus phosphorylation motif is, as would be expected, a match to that of CaM
44 Kinase I, B-X-R-X-X-(S/T)-X-X-X-B, where B is a subset of hydrophobic residues where Phe>Leu>Val>>Ala (Chrestensen and Sturgill, 2002)
The mechanism of activation of RSK was controversial for a long period of time.
Even though RSK was one of the first substrates of ERK identified, it was found that the deactivation of RSK by the action of phosphatases, followed by incubation with active
ERK in vitro only result in the partial activation of RSK. This led to a search for additional kinases necessary for the full activation of RSK2 (Sturgill et al., 1988). The full activation of RSK2 was found to require 3-phosphoinositide-dependent protein kinase-1 (PDK1), a broadly expressed kinase that has a high level of constitutive activity and does not appear to be regulated (Jensen et al., 1999). PDK1 is found both in the cytoplasm and at the plasma membrane, and the membrane association of PDK1 via a PH domain may serve as a method of regulation of PDK1’s activity. The phosphorylation of
Ser386 by the CTK of RSK2, as described above, creates a binding site that recruits
PDK1 to RSK2, regulating the activity of PDK1 (Frodin et al., 2000). Following the recruitment of PDK1 to RSK2, PDK1 phosphorylates RSK2 at Ser227. The phosphorylation of Ser227 leads to substantial activation of RSK2 in vitro and in vivo in the absence of mitogen stimulation (Frodin and Gammeltoft, 1999). Therefore, the full activation of RSK2 requires phosphorylation by both ERK1/2 and PDK1 (Dalby et al.,
1998; Jensen et al., 1999; Richards et al., 1999).
The activated NTK domain of RSKs can phosphorylate exogenous substrates with a consensus phosphorylation motif (K/R)-X-X-(S/T) (Alessi et al., 1996; Leighton et al.,
45 1995). When cells are appropriately stimulated, RSK2 has been found to translocate from
cytoplasm to cell membrane, prior to nuclear translocation. Transient membrane
association may be necessary for RSK activation as a myristylated RSK has been shown
to have elevated basal activity on cell membranes and does not require ERK for
activation (Richards et al., 2001; Shimamura et al., 2000). There are likely other mechanisms of RSK2 activity modulation as the RSKs contain a conserved autoinhibitory alpha helix motif downstream of their CTK domain. In RSK2 this motif is
697H-L-V-K-G-A-M-A-A-T-Y-S-A-L-N-R712 and the Y707A mutation has been shown to disrupt this helix and to cause constitutive activity (Poteet-Smith et al., 1999). As can be seen, the regulation of RSK activity, and hence RSK signal transduction, is complex.
1.12 The Role of RSK in Transcriptional Regulation
The active NTK of RSK2 phosphorylates a broad range of substrates including a number of transcription factors, proteins involved in chromatin remodeling, and transcriptional co-activators. These substrates include c-fos (De Cesare et al., 1998), histone H3 (Sassone-Corsi et al., 1999), cAMP-responsive-element binding protein
(CREB) (De Cesare et al., 1998), IκB/NFκB (Blenis, 1993), the estrogen receptor α (Joel et al., 1998), STAT3 (Zhang et al., 2003), the serum response factor (SRF) (Hanlon et al.,
2001), heat shock factor 1 (HSF1) (Wang et al., 2000), and ATF4 (Yang et al., 2004).
Additionally, RSK2 binds to and regulates the activity of the transcriptional co-activators p300 and CREB-binding protein (CBP) (Nakajima et al., 1996). The following section
46 will elaborate on a number of these RSK2 substrates and the role of RSK2 in their
regulation.
The primary role of RSK2 appears to be one of transcriptional regulation via the
phosphorylation of a number of transcription factors. For example, the expression of heat shock proteins is regulated through the activity of a specific transcription factor HSF1.
HSF1 is phosphorylated by RSK2 in vitro and this phosphorylation is repressed by non- steroidal anti-inflammatory drugs (NSAIDs), which have been previously shown to repress RSK2 activity (Stevenson et al., 1999). It has been suggested that RSK2 represses HSF1-heat shock element (HSE) binding activity during heat shock (Wang et al., 2000), but this area of RSK2 signal transduction has not been well explored. A number of genes are under the transcriptional control of the serum response element enhancer and are transcriptionally regulated by SRF transcription factor binding. RSK2 has been shown to phosphorylate SRF on Ser103 and dominant negative RSK2 mutations have been shown to inhibit the interaction of SRF with the transcription factor C/EBPβ, leading to decreased transcription driven from SRF-C/EBPβ regulated promoters (Hanlon et al., 2001). Another important cellular target of RSK2 is the IκB protein, which normally represses NFκB-mediated transcription. RSK2 has been shown to phosphorylate IκB at Ser32, leading to IκB degradation and subsequent stimulation of
NFκB-driven transcription (Blenis, 1993).
One highly important transcriptional role of RSK2 is in the phosphorylation of
CREB (Xing et al., 1996). CREB mediates the transcriptional inducibility of c-fos by
47 cAMP through a cAMP response element (CRE) located at -60 in the promoter (Sassone-
Corsi et al., 1988). C-fos is a member of the immediate early genes (IEGs), whose expression is rapidly and transiently activated as part of a mitogenic response (Sheng and
Greenberg, 1990). CREB phosphorylation at Ser133 by RSK2 is a prerequisite for c-fos induction in CLS cells, as cells from CLS patients are deficient in EGF stimulation of c- fos. However, serum-induced CREB phosphorylation is unaffected in CLS cells, showing that only some pathways of CREB phosphorylation are affected in the absence of RSK2
(De Cesare et al., 1998).
Defects in CREB phosphorylation are potential causes of cognitive impairment since CREB phosphorylation is an essential step in learning and long term memory formation (Gass et al., 1998; Silva et al., 1998) Intriguingly, the ERK MAPK cascade is also required for long term memory formation and synaptic plasticity and RSKs are a downstream targets of ERK (Atkins et al., 1998; Blum et al., 1999). As will be described later in this Chapter, cognitive impairment is a prominent feature of CLS, and it appears that a number of pathways involved in cognition converge on RSK2. In addition, many other genes involved in learning and memory are under the control of CRE-mediated expression such as BDNF, C/EBP, and the extracellular protease tissue plasminogen factor.
Beyond the role of RSK2 in the induction of c-fos, RSK and ERK coordinately phosphorylate c-fos, thereby enhancing the growth-promoting effect of c-fos in fibroblast cell lines (Chen et al., 1993; Chen et al., 1996). RSK2 is known to phosphorylate c-fos on
48 Ser374 and Ser362, stabilizing c-fos, allowing ERK phosphorylation of c-fos on Thr325 and Thr 331, resulting in c-fos induced cell transformation. Importantly, c-fos plays a role in mediating osteoclast differentiation and transformation (Eferl and Wagner, 2003;
Jochum et al., 2001) and mice lacking c-fos show osteopenia due to a complete block of osteoclast differentiation (Johnson et al., 1992; Wang et al., 1992). In addition, c-fos- dependent osteosarcoma formation is dependent on RSK2 (David et al., 2005). Since
RSK2 is required for the expression and maintenance of c-fos expression, this can help explain a number of the skeletal defects of CLS, which will be elaborated on in a later section of this Chapter.
In addition to the potential contribution of c-fos to the phenotype seen in CLS patients, another recently identified substrate of RSK2, the transcription factor ATF4, likely plays a large role in the skeletal defects demonstrated in CLS patients. RSK2 has been found to phosphorylate ATF4 on Ser245 (Yang et al., 2004). ATF4 has been established to be required for the regulation of the onset of osteoblast differentiation.
Additionally, RSK2 and ATF4 have been found to regulate type I collagen synthesis through a posttranslational mechanism, a protein that makes up 90% of the bone ECM protein content (Yang et al., 2004). ATF4 knock-out mice display skeletal defects similar to those found in CLS patients, which will be elaborated on later in this Chapter.
RSKs are also known to interact with the transcriptional co-activators p300/CBP.
RSK1 and RSK2 have been demonstrated to be recruited to p300/CBP and to repress
CREB-mediated transcription. RSK has been shown to phosphorylate CBP at Ser1772
49 but the phosphorylation does not seem to alter p300/CBP activity (Nakajima et al., 1996).
CBP and RSK2 have been shown to associate in quiescent cells and to dissociate within a few minutes following mitogenic stimulation. In these studies, CBP was shown to prefer to interact with unphosphorylated RSK2, wherein both RSK2 kinase activity and CBP acetylase activity are inhibited. Following mitogen stimulation and RSK2 activation,
RSK2 dissociates from CBP (Merienne et al., 2001). RSK2 is likely transported to CREB following mitogen stimulation via its interaction with p300/CBP, where RSK2 phosphorylates CREB on Ser133, as previously described. The dissociation of RSK2 from the p300/CBP complex is logical as active, phosphorylated CREB associates with p300/CBP and subsequently activates the transcription of genes under the control of
CREs.
In addition to the role of RSK2 in the regulation of transcription factor and co- activator activity, RSK2 has been demonstrated to directly interact with nuclear receptors. RSK2 directly interacts with the estrogen receptor α (ERα) via the hormone- binding domain (HBD) of ERα. This interaction is stimulated following EGF treatment and results in the phosphorylation of Ser167 in the ERα by RSK2 and the subsequent activation of ERα-mediated transcription (Joel et al., 1998). Binding of RSK2 to the
HBD induces a conformational change in ERα that results in ERα activation. Structural studies have indicated that the docking of RSK2 with the HBD likely exposes Ser167 via this conformational change. The exposure of Ser167 via RSK2 binding to the HBD then allows RSK2 phosphorylation of Ser167, leading to increased ERα activity toward nuclear targets. Interestingly, studies have demonstrated that the binding of RSK2 to the
50 HBD of ERα can be blocked by 4-hydroxytamoxifen but not by estradiol (Clark et al.,
2001).
Finally, RSK2 may also play a role in the regulation of chromatin structure. RSK2 can phosphorylate histone H3 in vitro and in vivo on Ser10 (Sassone-Corsi et al., 1999).
- In addition, the arsenite (AsO2 ) induced phosphorylation of Ser10 in RSK2 -/- cells is blocked, further demonstrating the requirement for RSK2 in the phosphorylation of histone H3 (He et al., 2003). The phosphorylation of histone H3 is believed to play a role in chromatin remodeling and chromosome condensation (Thomson et al., 1999). In addition, p53 is also a substrate of RSK2, phosphorylated on Ser15, and has been shown to be an intermediate in the RSK2-H3 interaction, mediating RSK2 phosphorylation of histone H3 (Cho et al., 2005).
RSK2 was recently identified to interact with PEA-15 (Vaidyanathan and Ramos,
2003), a small death effector that regulates the ERK MAPK pathway by binding to the
ERKs and preventing their translocation to the nucleus (Formstecher et al., 2001). PEA-
15 has been shown to bind to RSK2 and to prevent RSK2 translocation to the nucleus, hence preventing RSK2 phosphorylation of nuclear substrates. In addition, the PEA-15 interaction with RSK2 decreases RSK2 kinase activity. Together, the decreased activation of RSK2 in combination with the decreased nuclear translocation of RSK2 inhibits RSK2 phosphorylation of CREB and histone H3. As RSK2 has been shown to be required for c-fos activation (Bruning et al., 2000), as previously described, PEA-15 may play a role in limiting RSK2’s activity toward targets such as c-fos, CREB and H3
51 (Vaidyanathan and Ramos, 2003). As can be seen, RSKs are highly involved in transcriptional regulation, although the role of RSKs in these areas has only begun to be explored given the complex manner of RSK signaling pathways regulation.
1.13 Other Cellular RSK Actions
A large number of other RSK cellular targets have been identified. These targets include son-of-sevenless (SOS) (Douville and Downward, 1997), the neural cell adhesion molecule L1 (Wong et al., 1996), Filamin A (FLNa) (Woo et al., 2004), lamin-C (Erikson and Maller, 1988), the Bcl2-antagonist of cell death (BAD) (Bonni et al., 1999), glycogen synthase kinase-3 (Eldar-Finkelman et al., 1995), troponin I, (Erikson and Maller, 1988), microtubule-associated protein 2 (MAP2) (Chung et al., 1991), and the Yersinia pestis cytoxin protein YopM (McDonald et al., 2003). Because RSK2 has a broad range of substrates and actions, it is likely to participate in many other cellular processes. In the following section, a number of these targets will be discussed in depth.
RSK can phosphorylate glycogen synthase kinase-3 (GSK3) inhibiting it’s activity toward phosphorylating glycogen synthase. The phosphorylation of glycogen synthase results in the inhibition of glycogen synthesis, therefore RSK phosphorylation of
GSK3 should result in an increase in glycogen synthesis (Sutherland et al., 1993). RSK2 has also been demonstrated to phosphorylate SOS in response to EGF stimulation, and this phosphorylation decreases the interaction of SOS with growth factor receptor-bound protein 2 (Grb2), resulting in decreased activation of the ERK MAPK cascade (Douville
52 and Downward, 1997). This phosphorylation of SOS is relevant to signaling differences
seen in RSK2 knock-out mice, as will be described later in this Chapter.
RSKs have also been implicated in the phosphorylation of proteins involved in
cytoskeletal signaling. Filamin A (FLNa), a membrane associated cytoskeletal protein,
has been demonstrated to be a substrate of RSK. RSK was shown to phosphorylate FLNa
on Ser2152. It has been previously established that the phosphorylation of FLNa on
Ser2152 is necessary for membrane ruffling, suggesting a role for RSK in the control of
actin cytoskeletal dynamics (Woo et al., 2004). In addition, RSK1 has been shown to
phosphorylate the neural cell adhesion molecule L1 at Ser1152 (Wong et al., 1996).
Importantly, the disruption of RSK1 binding to L1 in the vicinity of 1152 resulted in a
reduction of neurite outgrowth in PC12 cells (Wong et al., 1996), implying a role for
RSK1 in the PC12 cell differentiation.
RSKs also play a number of roles in cell cycle progression. Cell-cycle related
substrates of the RSKs include the kinase Myt1 (Palmer et al., 1998), the budding
uninhibited by benzimidazoles 1 homolog (Bub1) (Schwab et al., 2001), and the early
mitotic inhibitor 1 (Emi1) (Paronetto et al., 2004). The p34cdc2/cyclin B complex, otherwise known as maturation-promoting factor (MPF), is inactivated by phosphorylation on Thr14 or Tyr15 by the Wee1 family kinases. MAPK cascade activation triggers MPF activation by decreasing the activity of the Wee1 kinases, driving the G2-to-M phase transition during the cell cycle. In Xenopus oocytes, only Tyr15 is phosphorylated by the Xenopus homolog of the Wee1 kinase. Myt1 was identified as a
53 kinase in Xenopus that can phosphorylate both Thr14 and Tyr15. RSK2 has been found to
phosphorylate Myt1 in a C-terminal regulatory domain independent of MPF activity,
which down-regulates Myt1 activity toward p34cdc2/cyclin B complexes in vitro (Palmer
et al., 1998). Therefore, RSK2 activity plays a role in the activation of MPF activity via the inhibition of My1 activity.
Bub1 is a protein kinase in Xenopus that is a crucial component of the signaling cascade from unattached kinetochores that prevents activation of the anaphase promoting complex (APC). During progesterone-induced Xenopus oocyte maturation, the MAPK
cascade is activated. Following MAPK activation, oocytes go through two meiotic
divisions and arrest with an intact spindle in metaphase of meiosis II due to an activity
defined as cytostatic factor (CSF). This CSF arrest has been attributed to RSK through
the RSK-mediated phosphorylation of Bub1 (Schwab et al., 2001). CSF activity also
requires the activation of the Mos/MAPK/RSK2 pathway during oocyte maturation and
the maintenance of Cdc20 inhibitory regulators Emi1 and Mad1 once CSF is established.
Emi1 is an essential inhibitor of APC at metaphase and RSK2 has been shown to
phosphorylate the protein Emi1, stabilizing the interaction of Emi1 with the APC
activator Cdc20 (Paronetto et al., 2004). Together, Emi1 and Cdc20 cooperate to
establish CSF arrest during mouse oocyte maturation.
RSKs also play a role in the control of apoptotic pathways. Inactivation of the
pro-apoptotic factor Bad by phosphorylation at a number of serine residues inhibits
binding of BAD to B-cell leukemia/lymphoma-xL (Bcl-xL) and induces the binding of
54 14-3-3 proteins, which sequesters Bad away from Bcl-xL (Zha et al., 1996). RSK2 was
originally found to phosphorylate Bad at Ser112 and to suppress BAD-mediated cell
death (Tan et al., 1999), however a later study showed that the major site of BAD
phosphorylation by RSK2 is not Ser112 but Ser155 (Lizcano et al., 2000). Regardless,
the phosphorylation of Ser155 triggers the dissociation of Bad from Bcl-xL and the
interaction of Bad with 14-3-3 proteins and is induced by UVB irradiation (She et al.,
2002).
The 14-3-3 proteins are a family of 30-kDa acidic proteins that interact with a
wide variety of cellular proteins including protein kinases, receptors, enzymes, structural
and cytoskeletal proteins, and small G-proteins (Cavet et al., 2003). 14-3-3 proteins are
known to act as a scaffold for several proteins in the MAPK cascade members including
mitogen-activated protein kinase kinase kinase 1,2, and 3 (MEKK1,2,3) (Fanger et al.,
1998) and to regulate the activity of Raf (Tzivion et al., 1998). Specifically, 14-3-3β has
also been demonstrated to interact with RSK and to inhibit RSK activation. Both RSK
and 14-3-3β have been demonstrated to translocate to the plasma membrane, where 14-3-
3 dissociates from RSK. Interestingly, RSK has been shown to phosphorylate the Na+/H+ exchanger (NHE1) and 14-3-3β has been previously shown to bind to the site of RSK
phosphorylation on NHE1. Hence, active RSK at the plasma membrane may
phosphorylate NHE1, which then competes for 14-3-3 binding (Cavet et al., 2003). A
similar mechanism may occur with BAD, with a 14-3-3/RSK complex phosphorylating
Bad, which then competes with RSK for 14-3-3 protein binding.
55 1.14 RSK Tissue Distribution
The tissue distribution of the RSKs has been explored in depth in order to further understand the role of the RSKs in signal transduction and in development. There has been an additional focus on the expression pattern of RSK2 and RSK4 since null mutations in these two RSKs are involved in mental retardation syndromes. RSK2 mRNA is detected as two transcripts of 3.5 and 8.5 kb in all tissues and it has been determined that the alternative use of two different polyadenylation sites gives rise to these transcripts. RSK1, RSK3, and RSK4, on the other hand, give rise to single transcripts. There are no studies which have addressed whether each of these two RSK2 transcripts contribute equally to the translation of RSK2. Overall, the strongest expression of RSK2 was found in the skeletal muscle, heart, and pancreas (Zeniou et al.,
2002a). During development, RSK2 shows high levels of expression during somitogenesis, which implies that RSK2 plays a specific role in skeletal and muscle development (Kohn et al., 2003). On the other hand, RSK3 has been found to be predominantly expressed in lung and skeletal muscle and RSK1 is primarily expressed in the kidney, lung, and pancreas. (Zhao et al., 1995).
Studies have also focused on RSK expression in both the developing and adult mouse brain. In the mouse brain, RSK1 shows the highest expression in cerebellum, with
RSK3 strongly expressed in the medulla. RSK2 shows a high level of expression in the cerebellum, occipital pole and frontal lobe of the cortex. Within the cerebellum of the adult mouse brain, RSK2 was strongly expressed in the Purkinje cells and some cells of
56 the deep cerebellar nuclei. The highest RSK2 expression in the mouse brain is in the
CA3 region of the hippocampus, although CA1-CA2 regions also show staining.
Substantial RSK2 expression is detected through-out the neocortex with the strongest
detection in layers V and VI. The RSK2 expression in Purkinje cell layer of the
cerebellum, CA1-CA3 in the hippocampus, and in the cerebral cortex was also verified in
these studies by immunohistochemistry (Zeniou et al., 2002a).
The neurons in these brain regions are known to display high synaptic activity,
supporting a role of RSK2 in neural transmission. In addition, the prefrontal cortex plays
a crucial role in long-term memory (LTM) formation and in integrating different types of
information in working memory. Furthermore, LTM formation requires the cAMP
response element (CRE) binding protein (CREB) mediated transcription, CREB is an in
vivo target of RSK2 (Silva et al., 1998), and a direct relationship has been demonstrated
between RSK2-mediated CREB phosphorylation and intelligence level in CLS patients
(Harum et al., 2001). Because the transcription factor CREB is implicated in learning and memory (Josselyn et al., 2001), changes in the CREB pathway associated with RSK2 deficiency may lead, in part, to the symptoms of CLS. Together, the expression data for
RSK1-RSK3 demonstrate that RSK1 and RSK3 should be able to compensate for RSK2 knock-out in most tissues but not in those regions of the brain where RSK2 is detected, as
RSK2 appears to have a non-overlapping distribution with RSK1 and RSK3. These data support the hypothesis that RSK1 and RSK3 cannot fully compensate for the lack of
RSK2, help to explain the cognitive dysfunction in CLS patients, and further support a
57 role of RSK2 in cognition (Prabhakaran et al., 2000; Shimamura, 1995; Zeniou et al.,
2002a).
1.15 RSK2 Knock-Out Mice
In order to further understand the function of RSK2, several groups have created
RSK2 knock-out mice. RSK2 mutant mice weigh 10% less and are 14% shorter, consistent with RSK2’s role in growth. The mice also exhibit poor learning and coordination, consistent with what is found in CLS patients (Xing et al., 1996).
Intriguingly, RSK2 knock-out mice have a specific but partial loss of adipose tissue, demonstrating a role for RSK2 in body weight regulation. These mice are resistant to weight gain when fed a high fat diet, are insulin resistant, and have fatty livers.
Therefore, not only are the RSK2 knock-out mice a good animal model for CLS, they may also be a model for lipodystrophy. It was found that low-level leptin treatment normalizes insulin and glucose in these mice (El-Haschimi et al., 2003).
The RSK2 knock-out mice also have a few other interesting features. The original purpose of creating the RSK2 knock-out mice was to investigate the role of
RSK2 in insulin signaling and in insulin-stimulated glycogen synthesis. The rationale for this is that RSK had been shown to be an insulin-stimulated kinase that phosphorylates the protein phosphatase 1 (PP1) G-subunit, which is responsible for the regulation of muscle glycogen metabolism by insulin (Moller et al., 1994). It was expected that RSK2 knock-out mice would have defects in glycogen metabolism; however, the RSK2 knock-
58 out mice instead demonstrated that RSK2 is not required for insulin-stimulated glycogen synthesis. RSK2 was also found to not be required for insulin or exercise stimulated c-fos or c-jun expression in skeletal muscle (Dufresne et al., 2001).
Even though RSK2 was not found to be required for glycogen synthesis, there were interesting alterations in the ERK MAP cascade found in the RSK2 knock-out mice.
Insulin- and exercise-stimulated ERK1/2 phosphorylation was found to be both increased and prolonged in the skeletal muscle of RSK2 knock-out mice. In addition, there was an increase in the basal level of phosphorylated ERK1/2 in the skeletal muscle. Due to these studies, RSK2 was hypothesized to play a role in ERK feedback inhibition. The authors of these studies argued for the possibility of lower ERK phosphatase expression in RSK2 knock-out mice, thereby altering the activity of both basal and stimulated ERK (Dufresne et al., 2001). Another pathway by which feedback may occur is the phosphorylation of
SOS by RSK. SOS phosphorylation causes SOS dissociation from Grb2 leading to decreased activation of Ras and thus decreased ERK activation (Douville and Downward,
1997). The RSK2 knock-out mice provide a valuable tool for the study of RSK2s function. Indeed, most of the functional studies performed in this thesis were accomplished through the use of mouse fibroblasts cell lines derived from these mice
(Bruning et al., 2000).
59 1.16 Coffin-Lowry Syndrome (CLS) - Introduction
It has been well established that mutations in X-linked genes are the most
common genetic causes of mental retardation, owing to the observation that a higher
percentage of males than females are affected. X-linked mental retardation is divided into
two primary groups: syndromal forms in which the mental retardation is part of a complex syndrome with consistent and distinctive clinical findings, and non-specific mental retardation where mental retardation is the primary symptom (Chiurazzi and
Oostra, 2000). Coffin-Lowry Syndrome (CLS; MIM 303600) is a syndromic form of X- linked mental retardation that was first described independently by both Coffin et al. in
1966 (Coffin et al., 1966) and Lowry et al. in 1971 (Lowry et al., 1971). This disorder was renamed as CLS in 1975 when it was recognized that Coffin and Lowry were reporting the same disorder (Temtamy et al., 1975). The incidence of CLS is unknown but it is believed to be in the range of 1 in 50-100,000 males (Touraine et al., 2002).
1.17 Identification of the Gene Mutated in CLS
Initially, genetic linkage studies were undertaken to identify the gene or genes mutated in CLS. Linkage studies narrowed the gene or genes responsible for CLS to a 2 centimorgan region of Xp22.2 between markers DXS365 and DXS7161 (Biancalana et al., 1994). These linkage studies allowed for the identification of the mutated gene in
CLS, which was identified by Trivier et al. in 1996 were they found mutations in the gene
RPS6KA3 at Xp22.2 encoding the serine/theonine kinase RSK2 in CLS patients (Trivier
60 et al., 1996). As previously described, RSK2 is a 740 amino acid kinase whose gene
covers approximately 80 kb and is comprised of 22 exons (Jacquot et al., 1999). Thus far,
RSK1 (chromosome 3) and RSK3 (6q27), have not been associated with a disorder, but
RSK4 (Xq21) mutation may also lead to mental retardation (Yntema et al., 1999).
1.18 RSK2 Gene Mutations in CLS
To date, more than 110 mutations have been described in CLS patients
(http://www-ulpmed.u-strasbg.fr/chimbio/diag/coffin/listmut.html) and the most common
mutations are I38S, R114W, and R110X. Of the mutations described, approximately 40%
are missense mutations, 20% nonsense mutations, 20% splicing errors, 20% short
deletion or insertion (Touraine et al., 2002). A number of the mutations seen in CLS
patients are in residues that are absolutely required for the function of kinases. For
example, the RSK2 mutations G75V, V82F, and G431D are all located nearby or within
the glycine-rich ATP-binding sites in the NTK and CTK domains. Additionally, the
D154Y mutation affects a residue that in PKA binds the ribose moiety of ATP. All of these mutations are likely to impair ATP binding of RSK2. Finally, H127Q affects a residue conserved in all protein kinases that has a yet undefined role (Frodin and
Gammeltoft, 1999). Other mutations commonly found are those residues involved in the
activation of RSK2, including the A225V, S227A, and T231I mutations, all of which
affect Ser227 phosphorylation in activation loop of NTK. In all, about 60% of mutations
in RSK2 result in premature truncation of the protein and it is presumed that the truncated
RSK2 proteins are unstable as the majority of these proteins are undetectable by Western
61 blot. This is partially confirmed by the fact that the extent of truncation does not appear to determine the phenotype of CLS (Jacquot et al., 1999).
As can be seen from the high number of mutations that have been identified in the
RSK2 gene, there is a strong allelic heterogeneity in CLS. In addition to allelic heterogeneity, there is an excessively high rate of de novo mutation in the RSK2 gene
(Delaunoy et al., 2001). However, the mutations in the RSK2 gene are quite scattered throughout the gene without any significant clustering (Jacquot et al., 1998). Quite unfortunately from a genetic counseling standpoint, there is evidence for germline mosaicism in CLS. Germline mosaicism, with or without somatic mosaicism, has been identified in autosomal dominant entities such as osteogenesis imperfecta, neurofibromatosis, and has been reported for many X-linked disorders. Two possibilities for mosaicism exist: the mutation can occur in a germ cell that continues to divide, or the mutation occurs early in a somatic cell before the separation to germinal cells and is present in both somatic and germinal cells. There have been multiple instances of the mothers of CLS patients showing germline mosaicism and it is believed that CLS mosaicism is as high or higher than that found in other X-linked disorders (Horn et al.,
2001). Therefore, unfortunately, the absence of a mutation in the mother does not rule out recurrence in her children (Delaunoy et al., 2001).
Intriguingly, 66% of patients screened do not show a RSK2 gene mutation.
However, this may be due to the screening method that has been employed in the vast majority of studies: Single-Strand conformation polymorphism analysis (SSCP). SSCP
62 analyses will miss intronic mutations, and it has been estimated that 25% of RSK2 gene mutations are intronic. In addition, it is likely that many CLS patients are misdiagnosed.
There have been a few reported cases of patients diagnosed with CLS-like disorders such as an adult who showed a deletion of q25.1-q25.3 and has CLS like symptoms. It is interesting to note that GPR10, GPR26, and GRK5 map into or near the region
(McCandless et al., 2000). Because of the presence of a number of patients without RSK2 mutations identified, one must remain open to the possibility of locus heterogeneity of
CLS or the possibility that other genes upstream of RSK2 may be mutated in these patients (Zeniou et al., 2002b).
The purpose of this following portion of the Introduction is to provide background on the symptomology and pathology of CLS. Focus will be placed on growth and development, facial features, skeletal defects, psychiatric illness, cardiovascular disorders, and movement disorders. In addition, differences between male patients and female heterozygote carriers will be discussed where applicable. It should be noted that X inactivation studies of female carriers have suggested random X inactivation with no selection mechanism (Manouvrier-Hanu et al., 1999). In general, less severe symptoms are observed in female heterozygotes, as female carriers can display symptoms ranging from mild to full blown CLS.
63 1.19 Growth, Cognitive Development, and General Features of CLS
Pregnancy usually progresses normally and at birth most parameters are within normal ranges. Intrauterine growth is often noted to be slow but in the normal range.
Normally, at birth, any diagnosis of CLS is difficult as most of the classical signs of CLS become more prominent during early childhood. Often, a broad, high forehead at birth may give a false impression of macrocephaly, although frequent microcephaly is found.
Males are usually short in stature and this becomes more apparent in early childhood, whereas females are variably affected (Jacquot et al., 1998). CLS patients show a great variability in mental retardation where males are often characterized as having severe mental retardation with an IQ often below 40. Again, females are variably affected with respect to mental retardation. As the patients age into childhood, other features of CLS become more apparent. There is a fullness of forearms due to increased subcutaneous fat, laxity of the joints, and it is estimated that approximately 33% of CLS patients have seizures (Hanauer and Young, 2002). It is also estimated that 33% of patients have sensorineural hearing loss (Jacquot et al., 1998).
1.20 Cranio-Facial Features of CLS
There are a number of facial features found typically in CLS patients. These features usually develop more prominently during early childhood, with males showing the majority of features and females being variably affected. CLS patients show a prominent forehead, orbital hypertelorism, narrow downslanting palpebral fissures, and a
64 coarse face. The nasal bridge is most often flat with anteverted nares, and the jaw is prominent with full, everted lips. The ears of CLS patients are large and prominent
(Jacquot et al., 1998). Finally, CLS patients have small widely spaced teeth with premature loss of primary dentition (Manouvrier-Hanu et al., 1999).
1.21 Skeletal Defects of CLS
Patients with CLS show a number of progressive skeletal deformations. At birth, during early development, there is a delay in the closure of the anterior fontanelle, and delayed bone development becomes apparent during early childhood. CLS patients have characteristic large, broad hands with stubby tapering fingers (Jacquot et al., 1998;
Manouvrier-Hanu et al., 1999). These stubby characteristic fingers have been noted in all
CLS patients including female heterozygote carriers and are an extremely useful diagnostic feature of CLS (Hanauer and Young, 2002). In addition to the abnormalities with the fingers, there are also abnormalities of the trunk, with 80% of male patients having either pectus carinatum or excavatum (Jacquot et al., 1998). CLS patients usually have severe spinal kyphosis and/or scoliosis, which lead to abnormal posture and cause the patients to appear even shorter. The kyphosis and/or scoliosis is usually so severe as to require surgical correction (Jacquot et al., 1998).
65 1.22 Psychiatric Illness of CLS Patients
Male CLS patients are normally cheerful, easygoing, and friendly (Gilgenkrantz et al., 1988). Female heterozygote carriers have incidence of psychotic behavior, schizophrenia, and depressive psychosis (Manouvrier-Hanu et al., 1999). The typical onset of psychiatric illness in the female patients is around 20 years of age. This pattern is observed only rarely in males (Hanauer and Young, 2002). In one study, 6 of 22 female
CLS patients, 38 heterozygote mothers, and 8 sisters of “affected” CLS patients showed incidence of psychosis (Hunter, 2002). The high incidence of psychiatric illness among female heterozygotes, considering the rarity of CLS, indicates that this psychotic behavior is a true complication of CLS (Hanauer and Young, 2002).
1.23 Cardiovascular Abnormalities of CLS
There are three major types of cardiomyopathy. The first and most common type of cardiomyopathy is dilated cardiomyopathy. This is caused by the damage induced weakening of heart chamber walls with subsequent dilation of the heart chambers. The second type of cardiomyopathy is hypertrophic cardiomyopathy, which is caused by the thickening of the heart wall chambers due to disorganized growth of heart muscle cells within the ventricles. The third type of cardiomyopathy is restrictive cardiomyopathy, which is the least common form and is extremely rare in children (Facher et al., 2004).
Restrictive cardiomyopathy is caused by impaired ventricular filling and decreased or normal diastolic volumes (Richardson et al., 1996). There have been documented cases of
66 restrictive cardiomyopathy in CLS patients and due to the rarity of this condition, it is likely to be a true complication of CLS. Cardiomyopathy has also been reported that was characterized by left ventricular (LV) dilatation with decreased contractile function, and mitral valve insufficiency (Massin et al., 1999).
In addition to reports of cardiomyopathy, there have been a large number of cardiovascular abnormalities reported in CLS patients. Cardiovascular abnormalities have appeared to play a role in the mortality of at least six patients. Cardiac abnormalities have been described in 14% of male CLS patients and include mitral, aortic, and tricuspid valve abnormalities, and pulmonary and aortic root dilation (Hunter, 2002). In addition to these abnormalities, a number of cases have been described where abnormally short chordae tethering the mitral valve have been found (Hunter, 2002; Massin et al., 1999).
1.24 Movement Disorders of CLS
Stimulus-induced drop episodes (SIDEs) have been described for a great number of CLS patients which are induced by unexpected tactile or auditory stimuli. These
SIDEs are not an epileptic phenomenon, as epileptic activity has been shown to be absent
(Caraballo et al., 2000; Crow et al., 1998; Fryns and Smeets, 1998; Nakamura et al.,
1998). These “drop-attacks” are interesting in that electroencephalograms remain unchanged during the episodes while electromyograms of the lower limbs become silent for 60-80 msec. SIDEs appear to worsen in frequency and severity as the patients age.
SIDEs resemble cataplexy and hyperekplexia but are distinct from these movement
67 disorders (Hunter, 2002). For example, triggers for hyperekplexia are also normal triggers
for CLS, but the response better resembles cataplexy, though the typical duration is
shorter. (Nelson and Hahn, 2003). As the disorders of hyperekplexia and cataplexy are not that common, a description of these disorders follows with their relevance to CLS.
Hyperekplexia is defined as an exaggerated motor response to auditory, somethetic, or visual stimuli. This exaggerated motor response is resistant to habituation and may be characterized by either a brief pathologic startle reflex or a sustained tonic spasm. During the hyperekplectic response, the patients remain conscious (Brown et al.,
1991; Saenz-Lope et al., 1984). Hyperekplexia is believed to be mediated by glycine receptor sensitivity, leading to reduced glycinergic inhibition in the spinal cord and subsequent neuronal hyperexcitability. Hyperekplexia is believed to be mediated by pathways concentrated in pontomedullary reticular formation. (Yeomans and Frankland,
1995). On the other hand, cataplexy is defined as the sudden loss of bilateral muscle tone that is more pronounced in the antigravity muscles and is provoked by a strong emotion with a preservation of consciousness. For cataplexy, laughter appears to be the most common provoking response (Guilleminault and Gelb, 1995). Cataplexy is believed to be caused by hyperactivation of the muscarinic cholinergic system. Cataplexy is believed to be mediated by pathways concentrated in the mediodorsal pontine tegmentum.
(Guilleminault and Gelb, 1995). SIDEs continue to remain a mystery, although it has been proposed that hyperekplexia and cataplexy may involve parallel neuronal pathways and that the SIDEs of CLS may be the result of an improper balance between these two parallel pathways (Nelson and Hahn, 2003).
68 CHAPTER 2: Materials and Methods
2.1 MATERIALS
Chemicals
5-Hydroxytryptamine creatinine sulfate (5-HT), ATP, bradykinin, Quipazine, (±)-
2,5-Dimethoxy-4-iodoamphetamine (DOI), 5-Methoxy-N,N-dimethyltryptamine,
chlorpromazine, and ketanserin were acquired from Sigma (St. Louis, MO). Thrombin
receptor activating peptide (TRAP) was a gift from Dr. Paul Di Corleto (Lerner Research
Institute, Case Western Reserve University). [32P]orthophosphate was obtained from
Amersham Biosciences (Piscataway, NJ). [3H]inositol (21.0 Ci/mmol), [3H]ketanserin
(76.0 or 88.0 Ci/mmol) and were obtained from New England Nuclear (Boston, MA).
3a70B liquid scintillation cocktail was obtained from Research Products International
(Elk Grove Village, IL). Ecoscint A liquid scintillation cocktail was obtained from
National Diagnostics (Atlanta, GA).
Cell Culture Reagents
Human Embryonic Kidney-293 (HEK-293), Human Embryonic Kidney 293TS
(HEK-293TS), and C6 glioma cells were purchased from the American Type Culture
Collection (Manassas, VA). Fugene6™ transfection reagent was obtained from Roche
(Indianapolis, IN). Dulbecco’s modified essential medium (DMEM), OPTI-MEM,
69 inositol-free DMEM, F12 nutrient mixture, fetal bovine serum, dialyzed fetal bovine
serum, sodium pyruvate, penicillin, and streptomycin were purchased from
Invitrogen/Gibco BRL (Gaithersburg, MD).
cDNA Constructs
The construct coding for FLAG-tagged wild type (wt) human 5-HT2A receptor
(FLAG-h5-HT2A) was constructed in a manner similar to that previously described (Xia et al., 2003a). Briefly, the human 5-HT2A receptor was cloned from the pM05 plasmid containing the human 5-HT2A receptor coding sequence (provided by T.A. Branchek,
Synaptic Pharmaceutical), into the plasmid pUniversal-Signal, which added a cleavable
signal sequence (MKTIIALSYIFCLVFA) from influenza haemagglutinin and a FLAG
tag (DYKDDDDK) to the amino-terminus of the human 5-HT2A receptor (Figure 2-1).
Addition of this signal sequence to a FLAG-tagged β2AR enhanced membrane insertion,
expression, and function of the receptor (Guan et al., 1992). Additionally, the signal
peptide was shown to be cleaved effectively converting the β2AR from a type IIIb to a
type IIIa membrane protein (Guan et al., 1992). Clones containing inserts in the
appropriate orientation were verified by automated sequencing (Cleveland Genomics,
Cleveland, OH) of the entire insert.
70
Figure 2-1 - Construction of a human FLAG-tagged 5-HT2A receptor with a cleavable N-terminal membrane insertion signal peptide. A 5-HT2A receptor construct was constructed that added a cleavable signal sequence
(MKTIIALSYIFCLVFA) from influenza haemagglutinin and a FLAG tag
(DYKDDDDK) to the amino-terminus of the human 5-HT2A receptor. The FLAG-h5-
HT2A receptor construct was created by PCR amplifying the human 5-HT2A receptor from the plasmid pM05 (provided by T.A. Branchek, Synaptic Pharmaceutical), into the NotI restriction site of the plasmid pUniversal-Signal, a modified pIRES-NEO (Clontech, Palo
Alto, CA) vector with the EcoRV – EcoRI fragment of the MCS replaced as indicated.
The plasmid pUniversal-Signal was created by Wes Kroeze.
71 Figure 2-1
72 The vector pMT2-HA-RSK2 (containing influenza A virus haemagglutinin (HA)- tagged mouse RSK2) has been described (Zhao et al., 1996). The native mouse RSK2 cDNA was subcloned from pMT2-HA-RSK2 (removing the HA tag) into the vector pcDNA3 (Invitrogen) to create the vector RSK2, introducing a HindIII site upstream and a KpnI site downstream. The forward and reverse primers were 5’-AAA AAA GCT
TTA GCC ACC ATG CCG CTG GCG CAG CTG-3’ and 5’-ACC TCA ACA GCC CTG
TGA GGT ACC TTT T-3’, respectively, with restriction sites in bold type (Figure 2-2).
To construct a viral vector for the expression of RSK2, bases 1938-1940 of the vector RSK2 were first mutated from TTC to CAG using Stratagene’s Quick Change
Site-Directed Mutagenesis Kit to remove an EcoRI restriction site at position 1935 of the wild type mouse RSK2 sequence, while maintaining amino acid identity to create the vector pRSK2-EcoRI-REM (Figure2-3). The RSK2 insert of pRSK2-EcoRI-REM was then subcloned into the EcoRI and SalI sites of pBABE-puro (Morgenstern and Land,
1990) to create pBABE-RSK2-EcoRI-REM. The forward and reverse primers were 5’-
AAA AGA ATT CGC CAC CAG CCG CTG GCG-3 and 5’-AAA AGT CGA CTC
ACA GGG CTG TTG AGG TG-3’, respectively, with restriction sites in bold type
(Figure 2-4). All constructs containing inserts in the appropriate orientation were verified by automated full-length sequencing (Cleveland Genomics, Cleveland, OH).
73
Figure 2-2 - Construction of mouse RSK2 in pcDNA3. A mouse RSK2 construct was created by PCR amplifying mouse RSK2 from the vector pMT2-HA-RSK2 (containing influenza A virus haemagglutinin (HA)-tagged mouse RSK2). The PCR amplified mouse RSK2 was cloned into the HindIII and KpnI restriction sites of the vector pcDNA3 (Invitrogen) to create the vector RSK2.
74 Figure 2-2
75
Figure 2-3 - Construction of RSK2-EcoRI-REM in pcDNA3. Bases 1938-1940 of the vector RSK2 were mutated from TTC to CAG using Stratagene’s Quick Change Site-
Directed Mutagenesis Kit to remove an EcoRI restriction site at position 1935 of the wild type mouse RSK2 sequence, while maintaining amino acid identity.
76 Figure 2-3
77
Figure 2-4 - Construction of pBABE-RSK2-EcoRI-REM. The RSK2 insert of pRSK2-
EcoRI-REM was subcloned into the EcoRI and SalI sites of pBABE-puro (Morgenstern and Land, 1990) to create pBABE-RSK2-EcoRI-REM.
78 Figure 2-4
79 Antibodies
M2 monoclonal anti-FLAG antibody, polyclonal anti-FLAG antibody, and anti-
FLAG M2 agarose beads were purchased from Sigma Chemical (St. Louis, MO). The
polyclonal 5-HT2A receptor antibody (Ab51) specific to the amino terminus was described previously (Berry et al., 1996). The polyclonal 5-HT2A receptor antibody (ct-
2A) directed against the carboxy terminus was a gift from Dr. Jon Backstrom (Vanderbilt
University) and has been described (Backstrom and Sanders-Bush, 1997). The polyclonal goat RSK2 antibody, monoclonal RSK2 antibody, rabbit polyclonal RSK1 antibody, and polyclonal rabbit Gαq antibodies were obtained from the Santa Cruz
Biotechnology (Santa Cruz, CA). The rabbit polyclonal RSK2 was obtained from Upstate
(Waltham, MA). Polyclonal p42/p44 MAP Kinase antibody and phosphorylated p42/p44
MAP Kinase antibody was purchased from Cell Signaling, Inc. (Beverly, MA).
Polyclonal rabbit GFP antibody was obtained from ABCAM (Cambridge, United
Kingdom). Goat anti-rabbit-HRP, horse anti-goat-HRP, horse anti-mouse-HRP, horse
anti-mouse Texas Red, goat anti-rabbit-Texas Red, and donkey anti-goat Texas Red were
obtained from Vector Laboratories (Burlingame, CA).Goat anti-rabbit-BODIPY-FL and
goat anti-mouse-BODIPY secondary antibodies were obtained from Molecular Probes
(Eugene, OR).
80 2.2 METHODS
Cell Culture and Transfection
Human embryonic kidney 293 (HEK-293) cells, HEK-293-TS cells, RSK2 wild-
type mouse fibroblasts (RSK2 +/+), and RSK2 knockout mouse fibroblasts (RSK2 -/-)
were maintained in Dulbecco-Modified Eagle Medium (DMEM) supplemented with 10%
fetal bovine serum (FBS), 1 mM sodium pyruvate, penicillin (100 U/mL) and
streptomycin (100 mg/mL) (GibcoBRL, Rockville, MD) at 37°C and 5% CO2. HEK-293
cells were transfected in 10 cm dishes at approximately 70% confluency with a total of 6
µg of DNA using Fugene6™ (Roche Molecular Biochemicals; Indianapolis, IN) as
described by the manufacturer. For transfection of receptor alone, the total amount of
DNA transfected was kept constant with the addition of CFP expression vector (pE-CFP-
N2). Transfected cells were used 48 hours post-transfection.
Serum Dialysis
Fetal bovine serum (FBS) was dialyzed to remove excess 5-HT present in the
serum. For this dialysis, 500 mL of Gibco FBS was thawed. Following serum thaw,
Spectra/Por 3500 MWCO dialysis tubing was cut into 22 inch lengths (12 lengths total).
The cut dialysis tubing was soaked in water and rinsed both inside and out with dH2O.
One end of the dialysis tubing was clamped, approximately 40 mL of FBS was pipetted into the dialysis tubing, all air was removed from the dialysis tubing, and the dialysis
81 tubing was clamped at the other end. Dialysis tubes were suspended in a 4 L graduated
cylinder containing a magnetic stir bar by attaching the top dialysis clamp to a serological
pipette via rubber bands. The graduated cylinder was filled up to the top of the dialysis
tubing with approximately 4 L of cold buffer (120 mM NaCl, 10 mM Tris-HCl, pH 7.5),
mixed by stir bar in the bottom, and equilibrated for 24 hours at 4˚C. The buffer was
changed every 24 hours following this for a total of 120 hours of dialysis. After the final
equilibration, dialyzed FBS was decanted into a 500 mL 0.22 µm filter sterilizing unit.
Dialyzed FBS was aliquoted and frozen at -20˚C. This protocol was optimized by Ryan
Strachan. Dr. Betsy Pehek analyzed the dialyzed serum against the undialyzed Gibco-
FBS and dialyzed Gibco-FBS via HPLC-ECD and found that our dialyzed serum (low 5-
HT Dialyzed) contained 0.774 nM 5-HT, while Gibco’s dialyzed FBS contained 25.8 nM
5-HT, and Gibco FBS contained 465 nM 5-HT. Importantly, in the final medium used
(DMEM + 5% low 5-HT dialyzed FBS) the concentration is 0.0387 nM 5-HT.
Amphotrophic Retrovirus Production, Viral Infection, and Polyclonal Cell Line
Production
To produce RSK2 or FLAG-h5-HT2A amphotrophic retroviruses, 100 mm dishes
of HEK293-TS cells grown to 60% confluency in antibiotic-free media were co-
transfected with 4 µg of the vector pBABE-RSK2-EcoRI-REM or pBABE-FLAG-
h5HT2A in addition to 4 µg of the packaging construct pCL-10A1 using Fugene 6
(Roche). Virus-containing supernatants were collected 36 hours following transfection, filtered through a 0.45 µm polyethersulfone filter (PURADISC™ 25 AS, Whatman),
82 frozen on dry ice, and stored at -80oC. RSK2 -/- and/or RSK2 +/+ fibroblasts were infected with the viral supernatant, supplemented with 4 µg / mL sterile polybrene, for 24 hours. Following infection, stable lines were established and maintained through selection in DMEM containing 3 µg/mL puromycin. The expression of RSK2 was verified in the stable lines by Western blot.
Yeast 2-Hybrid Analysis
The i3 loop of the human 5-HT2A receptor was amplified by PCR from the pM05
plasmid containing the human 5-HT2A receptor coding sequence (provided by T.A.
Branchek, Synaptic Pharmaceutical) using the following oligonucleotide primers (EcoRI sites bold): Y2H-i3 loop-FWD: 5’-AAA GAA TTC TTT CTA ACT ATC AAG TCA
CT-3’ and Y2H-i3 loop-REV: 5’-AAA GAA TTC CAC CTT GCA TGC CTT TTG CT-
3’, and Taq DNA polymerase (Boehringer-Mannheim). Following EcoRI digestion and gel purification (Gene-Clean III kit, Bio 101, Vista, CA), these PCR fragments were cloned into the EcoRI site of the pAS2-1 vector (Clontech, Palo Alto, CA) for use as bait, using T4 DNA ligase (New England Biolabs). Proper construction of these bait plasmids was confirmed by sequencing.
For screening, a pre-transformed human brain cDNA library in Saccharomyces cerevisiae strain Y187 (Clontech) was used as the target, and the GAL4-based yeast two- hybrid procedure utilizing yeast mating was done according to the manufacturer’s instructions (Matchmaker kit, Clontech). Positive clones in the library were screened
83 using both nutritional markers and beta-galactosidase expression, and were identified by sequencing of the inserts after rescue of the plasmids from yeast into E. coli. Positive controls were murine p53 and SV40 large T antigen, as provided in the Clontech kit. All controls were done as recommended by the manufacturer to confirm that the bait alone did not activate transcription, and that all strains and plasmids had the appropriate phenotypes. To confirm the results obtained, target and bait plasmids were co- transformed back into yeast strain PJ69-2A using the method of Schiestl & Gietz (Gietz et al., 1995).
In the initial yeast two-hybrid screen of a human brain cDNA library (Clontech),
74 colonies grew on media lacking adenine, histidine, leucine, and tryptophan
(quadruple-drop-out [QDO]) medium and 111 grew on media lacking histidine, leucine, and tryptophan (triple-drop-out [TDO]) medium. Of the 111 growing on TDO, 76
(68.5%) also grew on QDO. About 50% of the colonies grown initially on QDO were also positive for β-galactosidase expression, and about 35% of the colonies grown initially on TDO were positive for β-galactosidase expression. Plasmids from yeast colonies showing the most vigorous growth on QDO and the highest β-galactosidase expression were rescued into E. coli and sequenced.
For identification of the region of the i3 loop that might be involved in interaction with the RSK2 target, stop codons were inserted into four positions of the i3 loop bait plasmid by site-directed mutagenesis (Stratagene Quick-Change kit) using the following primers: C268-STOP-TOP 5’-CTC CAG AAA GAA GCT ACT TTG TGA GTA AGT
84 GAT CTT GGC ACA CGG-3’, C268-STOP-BOT 5’-CCG TGT GCC AAG ATC ACT
TAC TCA CAA AGT AGC TTC TTT CTG GAG-3’, S282-STOP-TOP 5’-GCC AAA
TTA GCT TCT TTC TGA TTC CTC CCT CAG AGT TCT-3’, S282-STOP-BOT 5’-
AGA ACT CTG AGG GAG GAA TCA GAA AGA AGC TAA TTT GGC-3’, Q296-
STOP-TOP 5’-TCT TCA GAA AAG CTC TTC TAG CGG TCG ATC CAT AGG GAG-
3’, Q296-STOP-BOT 5’-CTC CCT ATG GAT CGA CCG CTA GAA GAG CTT TTC
TGA AGA-3’, R310-STOP-TOP 5’-GGG TCC TAC ACA GGC AGG TAG ACT ATG
CAG TCC ATC AGC-3’ and R310-STOP-BOT 5’-GCT GAT GGA CTG CAT AGT
CTA CCT GCC TGT GTA GGA CCC-3’.
The i3 loop bait with a deletion of amino acids 270-280 was constructed through a multi-step PCR amplification. First, the primers i3-270-280-FWD (5’- CAA GTG TCT
GAA GAA CAA CT-3’) and 269-BEG-i3-REV-OVER (5’-GAG GGA GGA AGC
TGA ATA CAC ACA AAG TAG CTT CTT TC-3’) were used to amplify the proximal portion of the i3 loop bait, adding a 16 base pair overhang at the 3’ end (marked in bold).
The primers 281-END-i3-FWD (5’- TTC AGC TTC CTC CCT CAG AG-3’) and i3-
270-280-REV (5’- TTC CCG ACT GGA AAG CGG GC-3’) were used to amplify the distal portion of the i3 loop bait, including a 16 base pair region of overlap with the proximal PCR product (marked in bold). The proximal and distal PCR products were gel purified and used as a template in a second round of PCR with the primers i3-270-280-
FWD and i3-270-280-REV. This PCR product was gel purified, digested with EcoRI, and gel purified a second time. The PCR fragment was cloned into the EcoRI site of the
85 pAS2-1 vector, using T4 DNA ligase. Proper construction and mutagenesis of the above bait plasmids were confirmed by sequencing.
These truncated ‘bait’ constructs and the RSK2 ‘target’ were then co-transformed into yeast strain PJ69-2A using the method of Schiestl & Gietz (Gietz et al., 1995) and interactions with RSK2 were tested by nutritional and β-galactosidase screening as detailed in the Supplemental Material. To compare growth of the various truncation mutants, and therefore i3 loop-RSK2 interactions, cultures of yeast co-transformants were grown overnight in leucine and tryptophan (-L-W) dropout liquid medium. Yeast cells were counted and normalized, and dilutions were prepared of the normalized yeast cultures. One microliter of the diluted cultures was spotted on QDO and -L-W agar plates. Growth was monitored after 72 hours.
Immunocytochemistry, Immunohistochemistry, and Confocal Microscopy
For immunocytochemical studies, 24 hours following transfection, cells were plated onto poly-L-lysine coated coverslips in DMEM supplemented with 5% dialyzed fetal bovine serum, 1 mM sodium pyruvate, penicillin (100 U/mL), and streptomycin
(100 mg/mL) (GibcoBRL, Rockville, MD). Cells were grown for 24 hours in this media and then serum starved in serum-free DMEM for 18 hours. Following serum starvation, cells were treated with vehicle or 10 µM 5-HT for 10 minutes, placed on ice, and then fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 minutes.
Following fixation, cells were permeabilized on ice (0.3% Triton X-100 in PBS) for 20
86 minutes and then incubated with blocking buffer (5% nonfat, dry milk in PBS) for 1 hour
at room temperature. Cells were then incubated for 2 hours at room temperature
o followed by an overnight incubation at 4 C with a 5-HT2A receptor amino terminus- specific antibody (Ab51; 1:3000 dilution) (Berry et al., 1996) and monoclonal anti-RSK2 antibody (1:800 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA) diluted in blocking buffer. Following the overnight incubation, cells were washed twice with PBS and then incubated with a 1:200 dilution of Texas Red-labeled goat anti-mouse antibody and BODIPY-FL-labeled horse anti-rabbit antibody for 1 hour in blocking buffer. Cells were then washed with PBS and mounted for fluorescence confocal microscopic evaluation as previously detailed (Berry et al., 1996).
For immunohistochemical studies, rat brain sections were prepared as previously described (Bubser et al., 2001; Willins et al., 1997b) with the exception that a mouse monoclonal 5-HT2A antibody (Pharmingen, San Diego, CA; 1:1000) and a rabbit polyclonal RSK2 antibody (Upstate, Waltham, MA; 1:100) were used. Secondary antibodies used were 1:50 dilutions of goat anti-rabbit Alexa Fluor ® 488 (Molecular
Probes, Eugene, OR) or goat anti-mouse CY3 (Jackson Immunoresearch Laboratories,
West Grove, PA). Dual-label immunofluorescence confocal microscopy was performed as previously described (Bhatnagar et al., 2004; Bhatnagar et al., 2001; Xia et al., 2003a).
Immunohistochemical studies were performed by Bonnie G. Garcia and Dr. Ariel Y.
Deutch at Vanderbilt Univeristy, Nashville, TN.
87 Rat Brain Synaptic Membrane Preparation
Rat brain synaptic membranes were prepared from rat frontal cortex as previously
described (Roth et al., 1981). Briefly, whole rat brain tissue was cut into pieces using
sterile blades in Buffer H (10 mM HEPES, pH 7.40, 5 mM EDTA, 0.32 M Sucrose and
1× EDTA-free complete protease inhibitor mixture [Roche]) and homogenized using
glass-Teflon homogenizer. The tissue was ground using the pistol with 15 strokes or until there were no more chunks strictly avoiding air bubbles. The homogenized tissue was centrifuged @ 1,000 X g for 10 minutes to remove debris; the supernatant was further centrifuged @ 20,000 X g for 20 minutes the pellet obtained was the crude synaptic membrane preparations. Membrane protein concentrations were determined using an assay kit from Bio-Rad (Hercules, CA) with bovine serum albumin as the standard. Ten micrograms of the crude membrane protein was used for coimmunoprecipitation assays.
Western Blotting and Immunoprecipitation
Co-immunoprecipitation and immunoblotting were performed essentially as previously detailed (Bhatnagar et al., 2004; Gray et al., 2003a; Xia et al., 2003a). For co- immunoprecipitations in HEK-293 cells, cells were transiently transfected with FLAG- h5-HT2A receptors and either RSK2 or CFP. Twenty-four hours following transfection, cells were plated into two 100 mm dishes in DMEM supplemented with 5% dialyzed fetal bovine serum. Cells were serum starved in serum-free DMEM for 18 hours. Cells were then treated with vehicle or 10 µM 5-HT for 5 minutes at 37°C.
88
Cells were placed on ice, washed 2 times with ice-cold Tris-buffered saline (TBS)
(50 mM Tris, pH 7.4, 150 mM NaCl), and incubated with lysis buffer (50 mM HEPES,
pH 7.4, 150 mM NaCl, 1 mM EDTA, 10 mM Na4P2O7, 1.5% w/v CHAPS, 2 mM orthovanadate, and 1X Complete™ EDTA-free protease inhibitor cocktail [Roche;
Indianapolis, IN]) for 15 minutes at 4°C with gentle shaking. Following lysis, cells were harvested by scraping, transferred into microfuge tubes, and spun at 14,000 x g for 20 minutes at 4°C to clarify the lysates. Lysate protein concentrations were determined using an assay kit from Bio-Rad (Hercules, CA) with bovine serum albumin as the standard.
Equivalent amounts of total protein were used for each coimmunoprecipitation.
FLAG-h5-HT2A receptors were immunoprecipitated by incubating equivalent amounts of protein lysate with 50 µL anti-FLAG-M2 agarose beads (Sigma; St. Louis,
MO) at 4°C for 2 hours with constant rotation. Following immunoprecipitation, the anti-
FLAG-M2 agarose beads were washed centrifuged at 5,000 x g for 1 minute at 4°C to recover immunoprecipitated proteins. The immunoprecipitates were washed 3 times with lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 10 mM Na4P2O7,
1.5% w/v CHAPS, 2 mM orthovanadate, and 1X Complete™ EDTA-free protease inhibitor cocktail [Roche; Indianapolis, IN]). The co-immunoprecipitated complexes were eluted with 2X Laemmli sample buffer (50 mM Tris-HCl, pH 6.8; 2% SDS; 10% glycerol; 0.1% bromophenol blue; containing 2% β-Mercaptoethatol). Samples were heated to 65°C for 5 minutes, electrophoresed through 10% SDS-polyacrylamide gels, and transferred to nitrocellulose.
89
Nitrocellulose blots were blocked in blocking buffer (TBS with 0.01% Tween-20
and 5% nonfat dried milk) for 1 hour at room temperature. The blots were then incubated
for 2 hours at room temperature with polyclonal RSK2 antibody (1:1000), Gαq antibody
(1:1000), or polyclonal anti-FLAG (1:1000) in blocking buffer. Following primary antibody incubation, blots were washed three times for 10 minutes each at room temperature with TBS containing 0.05% Tween-20 (TBST) and incubated with either
HRP-anti-rabbit (1:1500) (FLAG and Gαq) or HRP-anti-goat (1:1500) (RSK2) for 1 hour at room temperature in blocking buffer. Following secondary antibody incubation, blots were washed three times for 10 minutes in TBST, washed two times for 10 minutes in
TBS, and visualized with the use of LumiLight horseradish peroxidase substrate (Roche).
Blots were wrapped in plastic-wrap, and were quantified using a Kodak Digital Science
Image Station 440CF (Eastman Kodak, Rochester, NY). For visualization of co-
immunoprecipitation of RSK2, a polyclonal goat RSK2 antibody (1:1000) (Santa Cruz
Biotechnology, Santa Cruz, CA) was used while a rabbit polyclonal FLAG antibody
(1:1000) (Sigma; St. Louis, MO) was used to detect immunoprecipitated FLAG-h5-HT2A receptors. Lysates were also probed for Gαq with a polyclonal rabbit Gαq antibody (Santa
Cruz Biotechnology, Santa Cruz, CA) to verify equivalent protein loading.
A monoclonal mouse RSK2 antibody (4 µg) (Santa Cruz Biotechnology, Santa
Cruz, CA) and protein A/G agarose (Santa Cruz Biotechnology, Santa Cruz, CA) were
used to immunoprecipitate RSK2 from C6 glioma cells and from rat brain synaptic
membrane preparations. The native 5-HT2A receptor was detected by using a polyclonal
90 5-HT2A C-terminus antibody (ct-2A) (Backstrom and Sanders-Bush, 1997) (a gift of Jon
Backstrom, Vanderbilt University). Immunoprecipitated RSK2 was detected with
polyclonal goat RSK2 antibody. Blots were quantified using a chemiluminescence
imaging system (Kodak Digital Science).
Western Blot Analysis of p42/p44 ERK Phosphorylation
RSK2 +/+ and -/- fibroblasts were plated at 120,000 cells/well into 6-well plates
for ERK assays in DMEM supplemented with 5% low 5-HT dialyzed fetal calf serum.
Cells were serum starved for 18 hours prior to experimentation. Cells were then prepared
for measurements of ERK phosphorylation using total ERK for normalization as
previously described (Bhatnagar et al., 2004; Setola et al., 2003; Xia et al., 2003a).
Briefly, cells were treated with 10 µM 5-HT or with 100 nM MDL100,907 as indicated.
Following treatment, the cells were placed on ice, washed 2 times with ice-cold Tris-
buffered saline (TBS) (50 mM Tris, pH 7.4, 150 mM NaCl), and incubated with lysis
buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 10 mM Na4P2O7, 1.5% w/v
CHAPS, 2 mM orthovanadate, and 1X Complete™ EDTA-free protease inhibitor cocktail
[Roche; Indianapolis, IN], pH 7.4) for 15 minutes at 4°C.
Following lysis, cells were harvested by scraping, transferred into microfuge
tubes, and spun at 14,000 x g for 20 minutes at 4°C to clarify the lysates. Lysate protein
concentrations were determined using an assay kit from Bio-Rad (Hercules, CA) with
bovine serum albumin as the standard. Normalized protein lysates were prepared in 1x
91 Laemmli sample buffer. Samples were resolved on 12% SDS-polyacrylamide gels and
electroblotted onto nitrocellulose membranes. The membranes were probed for phospho-
ERK immunoreactivity using a 1:1000 dilution of a polyclonal rabbit phosphor-p42/p44
MAP Kinase antibody (Cell Signaling Technology, Beverly, MA) and a 1:1500 dilution
of horseradish peroxidase-conjugated goat anti-rabbit IgG (Vector Laboratories,
Burlingame, CA). Total ERK immunoreactivity was probed for using a 1:1000 dilution of a polyclonal rabbit p42/p44 MAP Kinase antibody (Cell Signaling Technology,
Beverly, MA). Immunoreactivity was revealed using LumiLight horseradish peroxidase substrate (Roche) and imaged on a Kodak Digital Science Image Station 440CF (Eastman
Kodak, Rochester, NY). Densitometric analysis was performed using Scion Image
software (Scion Corporation, Frederick, MD). Samples were similarly analyzed for total
ERK immunoreactivity, and the resulting values were used to correct phospho-ERK
measurements for slight differences in sample protein content.
Cell Surface Biotinylation
Cell surface biotinylation was performed as described previously (Willins et al.,
1999) with a few modifications. RSK2 -/- and RSK2 +/+ fibroblasts stably expressing
FLAG-h5-HT2A were plated at 300,000 cells/well into 6-well plates in DMEM
supplemented with 5% dialyzed fetal calf serum. Cells were serum starved for 15 hours
prior to experimentation. Following serum starvation, cells were washed 2 times with
warm serum-free DMEM, placed on ice, and rinsed twice with 2 mL of ice-cold
biotinylation buffer (10 mM boric acid, 154 mM NaCl, 7.2 mM KCl, and 1.8 mM CaCl2,
92 pH 8.4). Cells were surface-biotinylated by adding 0.8 mM biotin disulfide N-hydroxy-
succinimide ester (Sigma) in 0.5 mL increments every 5 minutes on ice for a total of 15
minutes. The reaction was quenched with 2 mL of quenching buffer (0.192 M glycine, 25
mM Tris, 1.8 mM CaCl2, and 154 mM NaCl, pH 8.3) and cells lysed in 1 mL of lysis
buffer (20 mM HEPES, 0.1% SDS, 1% Nonidet P-40 [Amresco], 0.5% deoxycholate, and
1× EDTA-free complete protease inhibitor mixture [Roche], pH 7.40) for 15 minutes on ice. Lysates were clarified by centrifugation (14,000 × g for 20 minutes at 4°C), protein concentrations were determined by Bio-Rad protein assay kit (Bio-Rad) and then equivalent amounts of total protein were incubated with 50 µl of streptavidin-agarose for
2 hours at 4°C with constant mixing.
Biotinylated proteins were then recovered by centrifugation, followed by three washes with lysis buffer at 4°C. After the final wash, biotinylated FLAG-h5-HT2A was liberated from the agarose by the addition of 60 µl of SDS sample buffer (10% by volume glycerol, 2% by weight SDS, 0.01 mg/mL bromophenol blue, 2% by volume β-
mercaptoethanol, 62.5 mM Tris, pH 6.8) and heating at 65°C for 4 minutes. Samples
were then electrophoresed on 12% SDS-polyacrylamide gels, transferred to
nitrocellulose, and subjected to immunoblot analysis as described previously (Willins et al., 1999) using polyclonal anti-FLAG antibody (Sigma,USA) to detect FLAG-h-5-HT2A receptor and a 1:1500 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG
(Vector Laboratories, Burlingame, CA). Immunoreactivity was revealed using LumiLight horseradish peroxidase substrate (Roche) and imaged on a Kodak Digital Science Image
Station 440CF (Eastman Kodak, Rochester, NY).
93 [32P]Orthophosphate Metabolic Labeling
RSK2 -/-, RSK2 +/+, RSK2 -/- FLAG-h5-HT2A Stable, and RSK2 +/+ FLAG-h5-
HT2A Stable fibroblasts were plated at 400,000 cells/well into 6 well plates in DMEM
supplemented with 5% dialyzed fetal calf serum. The following day, cells were washed
with phosphate-free DMEM and then incubated in the same medium for 1 hour. Cells
were then labeled with 0.1 mCi/mL [32P]orthophosphate in phosphate-free DMEM for 4 hours and treated with 10 µM 5-HT for varying times. Cells were washed three times with Tris-buffered saline (50 mM Tris, 150 mM NaCl, pH 7.4) and lysed in 32P-Lysis
Buffer (1.5% (w/v) CHAPS, 50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 10 mM
Na4P2O7, 2 mM orthovanadate, and 1 × EDTA-free complete protease inhibitor mixture
[Roche], pH 7.5) for 10 minutes on ice. Cells were harvested by scraping, clarified by
o centrifugation at 13,000 x g for 10 minutes at 4 C, and FLAG-h5-HT2A was
immunoprecipitated as previously described (Bhatnagar et al., 2004; Gray et al., 2003a;
Xia et al., 2003a).
Samples were loaded onto 10% SDS polyacrylamide gels, electrophoresed, and
gels were dried. Autoradiography was used to detect [32P]orthophosphate incorporation.
Samples were also electrophoresed on 12% SDS-polyacrylamide gels, transferred to nitrocellulose, and subjected to immunoblot analysis as described previously (Willins et al., 1999) to demonstrate the immunoprecipitation of FLAG-h5-HT2A receptors. A polyclonal anti-FLAG antibody (Sigma,USA) was used to detect FLAG-h-5-HT2A receptor in conjunction with a 1:1500 dilution of horseradish peroxidase-conjugated goat
94 anti-rabbit IgG (Vector Laboratories, Burlingame, CA). Immunoreactivity was revealed
using LumiLight horseradish peroxidase substrate (Roche) and imaged on a Kodak
Digital Science Image Station 440CF (Eastman Kodak, Rochester, NY).
[γ−32P]-ATP In vitro Kinase Assay
For in vitro kinase assays, HEK-293 cells were transiently transfected with
FLAG-h5-HT2A receptors, FLAG-h5-HT2A receptor mutants as described, or CFP.
Twenty-four hours following transfection, the media was changed to DMEM supplemented with 5% low 5-HT dialyzed fetal bovine serum. The following day, cells were harvested as FLAG-h5-HT2A receptors were immunoprecipitated as described above. Following immunoprecipitation, the anti-FLAG-M2 agarose beads were centrifuged at 5,000 x g for 1 minute at 4°C to recover immunoprecipitated proteins. The immunoprecipitates were washed 3 times with lysis buffer (50 mM HEPES, 150 mM
NaCl, 1 mM EDTA, 10 mM Na4P2O7, 1.5% w/v CHAPS, 2 mM orthovanadate, and 1X
Complete™ EDTA-free protease inhibitor cocktail [Roche; Indianapolis, IN], pH 7.4).
Following the washes with lysis buffer, the immunoprecipitates were washed 2 additional
times with 2.5X RXN Buffer (20 mM MOPS, 1 mM EGTA, pH 7.5). Following the final
2.5X RXN Buffer wash, all of the supernatant was removed from the anti-FLAG-M2 agarose beads, and the immunoprecipitates were stored on ice.
Active RSK2 (RSK2/MAPKAP Kinase 1b, active, Upstate [Waltham, MA], catalog number 14-480) was diluted to a final concentration of 100 ng/µL in Enzyme
95 Dilution Buffer (20 mM MOPS, 1 mM EGTA, 0.01% Brij-35, 5% glycerol, 0.1% BME,
1 mg/mL BSA, pH 7.5). After dilution, 100 µL aliquots of the active RSK2 were
prepared and frozen at -20oC until use. To the immunoprecipitates, 20 µL of 2.5X RXN
Buffer (20 mM MOPS, 1 mM EGTA, pH 7.5) were added, followed by the addition of 5
µL (500 ng) active RSK2. [γ−32P]ATP (stock solution 1 mCi/100 µL, 3000 Ci/mmol,
Perkin-Elmer Life and Analytical Sciences, Boston, MA) was diluted in ATP Dilution
Buffer (75 mM MgCl2, 500 µM ATP, 20 mM MOPS, 25 mM β-glycerol phosphate, 5 mM EGTA, 1 mM NaOVa, 1 mM DTT, pH 7.2) to a final concentration of 1 µCi/µL.
Finally, 20 µL of the diluted [γ−32P]ATP was added to the immunoprecipitates, the immunoprecipitates were mixed by pipette, and were incubated for 1 hour at 30oC in a
water bath.
Following the hour incubation, the immunoprecipitates were eluted with 20 µL
5X Laemmli -sample buffer (50 mM Tris-HCl, pH 6.8; 2% SDS; 10% glycerol; 0.1%
bromophenol blue; containing 2% β-Mercaptoethatol). Samples were heated to 65°C for
5 minutes, loaded onto 10% SDS polyacrylamide gels, electrophoresed, and gels were
dried. Autoradiography was used to detect 32P from [γ−32P]ATP incorporation. Samples were also electrophoresed on 10% SDS-polyacrylamide gels, transferred to nitrocellulose, and subjected to immunoblot analysis as described previously (Willins et al., 1999) to demonstrate the immunoprecipitation of FLAG-h5-HT2A receptors. A polyclonal anti-FLAG antibody (Sigma,USA) was used to detect FLAG-h-5-HT2A receptor in conjunction with a 1:1500 dilution of horseradish peroxidase-conjugated goat
anti-rabbit IgG (Vector Laboratories, Burlingame, CA). Immunoreactivity was revealed
96 using LumiLight horseradish peroxidase substrate (Roche) and imaged on a Kodak
Digital Science Image Station 440CF (Eastman Kodak, Rochester, NY).
Determination of Phosphoinositide (PI) Hydrolysis
RSK2 +/+ and -/- fibroblasts were plated at 50,000 cells/well into 24-well plates
in DMEM supplemented with 5% low 5-HT dialyzed fetal bovine serum. Cell cultures
were grown at 37°C in 5% CO2. Twenty-four hours later, the fibroblasts were incubated
with inositol-free DMEM supplemented with 5% low 5-HT dialyzed fetal bovine serum for 1 hour and then incubated for an additional 18 hours with inositol-free DMEM supplemented with 5% low 5-HT dialyzed fetal bovine serum containing 1 µCi/mL
[3H]inositol. The fibroblasts were then washed with a modified Hanks’-bicarbonate
buffer (138 mM NaCl, 5.3 mM KCl, 1.3 mM CaCl2, 0.49 mM MgCl2, 0.4 mM MgSO4,
0.4 mM KH2PO4, 0.34 mM Na2HPO4, 25 mM NaHCO3, 11 mM Glucose, pH 7.4)
equilibrated to 37°C and 5% CO2 and supplemented with 35 mM LiCl, which inhibits
inositol-1-phosphatase (Figure 2-5) Following the wash in modified Hanks’-bicarbonate
buffer, fibroblasts were incubated for 50 minutes at 37°C and 5% CO2 in the presence or
absence of drugs. For dose-response curves, 5-HT, TRAP, ATP, and bradykinin were
serially diluted in the equilibrated modified Hanks’-bicarbonate buffer prior to addition.
The accumulation of phosphoinositides was terminated by aspiration, followed by
the addition of 1 mL of 10 mM formic acid. Fibroblasts were incubated in the 10 mM
formic acid for at least 30 minutes at room temperature to insure extraction of
97 phosphoinositides. Columns packed with a 1 mL bed of AG® 1-X8 Resin 100-200 mesh
anion-exchange resin (formate form) (Bio-Rad; Hercules, CA) were washed with 10 mL
of water twice. Following the water washes, the entire 1 mL sample volumes were added
to columns, avoiding the transfer of any cells. Columns were washed with 2 mL of
water, then with 10 mL of water, followed by 10 mL of 5 mM sodium borate / 50 mM
sodium formate (Roth et al., 1986). Total phosphoinositides (PIs) were eluted with 10
mL of 0.1 M formic acid / 0.2 M ammonium formate into vials containing 3a70B liquid scintillation cocktail (Research Products International; Elk Grove Village, IL) and radioactivity was measured by liquid scintillation counting. Following use, columns were regenerated with 10 mL of 0.1 M formic acid / 1M ammonium formate and washed with 30 mL of water.
98
Figure 2-5 - Stimulation of phosphoinositide (PI) hydrolysis following Gαq-coupled
GPCR activation. Agonist binding to GPCRs lead to receptor conformational changes
that promote the exchange of GDP for GTP on the G protein α-subunit. This GTP exchange allows the dissociation and activation of receptor-specific heterotrimeric G proteins into α- and βγ- subunits. The α-subunit Gαq subsequently activates phospholipase C (PLC), which catalyzes the hydrolysis of phosphotidylinositol-
4,5,bisphosphate (PIP2) into the second messengers diacylglycerol (DAG) and inositol-
1,4,5-trisphosphate (IP3). Phosphates are sequentially hydrolyzed from IP3 though a
series of phosphatases. The final phosphate is removed by inositol-1-phosphatase to
form inositol, which is recycled to form membrane phospholipids. Li+, added to the experimental samples, inhibits inositol-1-phosphatase (Berridge, 1984) allowing the accumulation of IP, IP2, and IP3. Inositol containing molecules have been labeled with
3 [ H]myo-inositol, therefore, IP, IP2, and IP3 are able to be isolated by anion exchange
chromatography and detected through liquid scintillation counting.
99 Figure 2-5
100 Intracellular Calcium Mobilization
RSK2 +/+ and -/- fibroblasts were plated at 30,000 cells/well into 96-well plates
in DMEM supplemented with 5% low 5-HT dialyzed fetal calf serum. Cells were assayed
for intracellular Ca2+ response to agonist 24 hours after plating. The culture medium was
removed by aspiration and replaced with 30 µL of Calcium Flux Assay Kit for
FlexStation Dye (Molecular Devices, Sunnyvale, CA) dissolved in assay buffer (2.5 mM probenecid, 20 mM HEPES, 138 mM NaCl, 5.3 mM KCl, 1.3 mM CaCl2, 0.49 mM
MgCl2, 0.4 mM MgSO4, 0.4 mM KH2PO4, 0.34 mM Na2HPO4, pH 7.4). Plates were
0 incubated in the dye for 1 hour at 37 C and 5% CO2. Drugs were diluted in assay buffer
as 2X stocks and aliquoted into a 96-well plate. Fluorometric imaging was performed
using a FlexStation II plate reader (Molecular Devices), which transfers 30 µL from the drug plate to the cells and takes fluorescent readings for 2 minutes every second.
Fluorescence is excited at 485 nM and emitted at 525 nM, using a 515 nM cut-off. Drugs are added 20 seconds into the plate read to establish a steady base-line. Fluorescence
data is obtained as relative fluorescence units (RFU). RFU measurements were baseline
subtracted and Calcium response time-courses were plotted using Prism 4.03 software
(GraphPad, San Diego, CA).
Determination of cAMP Production
RSK2 +/+ and -/- fibroblasts were plated at 50,000 cells/well into 24-well plates
in DMEM supplemented with 5% low 5-HT dialyzed fetal bovine serum. Cell cultures
101 were grown at 37°C in 5% CO2. Twenty-four hours later, the cells were washed with 400
µL warm stimulation buffer (DMEM, 15 mM HEPES, pH 7.4, 0.025% ascorbic acid) for
10 minutes at room temperature. Following this incubation, the media was decanted and the cells were placed on ice. To assess β-adrenergic receptor stimulation, 500 µM 3- isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, prepared in stimulation buffer, was first added to all wells to prevent cAMP breakdown. Following
IBMX addition, cells were treated with varying concentrations of isoproterenol for 15 minutes at 37°C, as indicated. For these experiments, 10 µM forskolin treated cells were used to determine maximal cAMP production. The reaction was terminated by aspiration and the addition of 0.5 mL ice-cold 3% trichloroacetic acid. Cell lysates were chilled for at least 2 hours at 4°C.
cAMP was quantified using a competitive binding assay adapted with minor modifications (Nordstedt and Fredholm, 1990). Briefly, TCA extracts (10-40 µL) were added to reaction tubes containing cAMP assay buffer (100 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA). [3H]cAMP (1 nM final concentration) was added to each tube, followed by cAMP-binding proteins (approximately 100 µg of crude extract from bovine adrenal cortex in 500 µL of cAMP assay buffer; prepared as described below). The reaction tubes were incubated on ice for 2 hours then harvested with a Brandel cell harvester onto Whatman GF/C filters. Filters were allowed to dry and radioactivity bound was quantified by liquid scintillation counting. The concentration of cAMP in each sample was estimated from a standard curve ranging from 0.1 to 100 picomoles of cAMP. To make cAMP-binding protein, five bovine adrenal cortices (Pel-Freez
102 Biologicals; Rogers, AR) were partially thawed on ice, chopped into small pieces and
homogenized in 125 mL of PKA purification buffer (100 mM Tris-HCl pH 7.4, 10 mM
EDTA, 250 mM NaCl, 250 mM sucrose, and 0.1% 2-mercaptoethanol). Homogenate was
filtered several times through cheesecloth to remove fatty tissue and stirred for 30
minutes in an ice-water bath. Suspension was spun three times at 30,000 x g at 4°C for 30
minutes, filtering the supernatant through cheesecloth between each spin. The final
supernatant was brought up to 100 mL in PKA purification buffer, split into 1 mL
aliquots and stored at -80°C for up to 2 years. Optimal dilution of the cAMP-binding
protein was determined by testing several dilutions with standard curves of unlabeled cAMP.
Saturation Binding Assays
Saturation binding assays were performed in a total volume of 0.25 mL at room temperature for 1 hour using [3H]ketanserin as the radiolabel. For these binding assays,
20-50 µg of membrane protein harvested with hypotonic buffer (50 mM Tris-HCl pH
7.4) was incubated with the radiolabel as previously described (Choudhary et al., 1992).
Nonspecific binding was defined as radioactivity bound in the presence of 50 µM spiperone or chlorpromazine and represented less than 20% of total binding. Membranes were harvested with a Brandel cell harvester followed by three ice-cold washes onto polyethyleneimine-pretreated (0.3%) Whatman GF/C filters. Filters were dried overnight and then soaked in Ecoscint A liquid scintillation cocktail (National Diagnostics; Atlanta,
GA). Bound radioactivity was quantified by liquid scintillation counting. Membrane
103 protein concentrations were determined using an assay kit from Bio-Rad (Hercules, CA)
with bovine serum albumin as the standard.
Competition Binding Assays
Competition binding assays were performed in a total volume of 0.25 mL at room
temperature for 1 hour using [3H]ketanserin as the radiolabel. For these binding assays,
20-50 µg of membrane protein harvested with hypotonic buffer (50 mM Tris-HCl pH
7.4) was incubated with the radiolabel as previously described (Choudhary et al., 1992).
Nonspecific binding was defined as radioactivity bound in the presence of 50 µM spiperone or chlorpromazine and represented less than 20% of total binding. Membranes were harvested with a Brandel cell harvester followed by three ice-cold washes onto polyethyleneimine-pretreated (0.3%) Whatman GF/C filters. Filters were dried overnight and then soaked in Ecoscint A liquid scintillation cocktail (National Diagnostics; Atlanta,
GA). Bound radioactivity was quantified by liquid scintillation counting. Membrane protein concentrations were determined using an assay kit from Bio-Rad (Hercules, CA) with bovine serum albumin as the standard.
Microarray and Pathway Analysis
Nearly confluent 100 mm dishes of RSK2 +/+ or RSK2 -/- fibroblasts were harvested under RNAse free conditions. Samples were prepared by the Gene Expression
Array Core Facility at Case Western Reserve University (www.geacf.net), following
104 methods recommended by Affymetrix. Briefly, total RNA was extracted using Trizol
(Invitrogen, USA) followed by RNA clean up using Qiagen columns. Qiagen columns
were used to clean up cDNA and/or RNA as required using the manufacturer’s protocol.
This was followed by cDNA synthesis using an oligo-dT primer coupled to T7 RNA
polymerase promoter. Reverse transcription of RNA was performed using Superscript II
reverse transcriptase in a 20 µL reaction at 420C for 1 hour. Second strand synthesis was carried out immediately in presence of E. coli DNA polymerase I, RNAse H and DNA ligase. The reaction mixture was incubated for 2hrs at 16oC and a further 5 minutes in the presence of T4 DNA polymerase. The reaction was terminated by addition of EDTA followed by cDNA clean- up using Qiagen columns and storage overnight at -20oC.
In vitro transcription was used to generate complementary RNA (cRNA) using a
Bioarray High Yield ENZOkit (Affymetrix) followed by clean up of RNA samples.
Purified in vitro transcribed cRNA was subjected to fragmentation at 94oC for 35 minutes using 1X fragmentation buffer (40 mM Tris acetate, pH 8.1, 100 mM KOAc, 30 mM
MgOAc) then placed on ice. Preconditioning for hybridization was performed in 1X hybridization cocktail (100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween20).
Herring sperm and acetylated BSA were added to a final concentration of 0.1 and 0.5 mg/mL, respectively. A 15 µL aliquot of a 20X mixture of in vitro transcripts of bacterial genes BioB BioC BioD and cre were added to the cocktail to give final concentrations of
1.5, 5, 25 and 100 pM respectively. Control oligonucleotide was added to a final concentration of 50 pM. The amount of fragmentation reaction containing 15 µg of cRNA was added to the cocktail and the remaining volume was made up with molecular
105 biology grade water. Preconditioning of the array chip was done in 1X hybridization
buffer for 10-15 minutes at 45oC with rotation (45 rpm). The preconditioning buffer was
then removed from chip chamber. Sample hybridization cocktail was added to the chip
and hybridized overnight (16 hours) at 45oC with rotation (45 rpm). For post hybridization washing and staining, samples were recovered from the chips and stored in their original vials and the hybridization chamber was filled with Buffer A (non stringent:
6X and hybridization was overnight (16 hours) at 45oC with rotation (45 rpm) in sample
hybridization cocktail. For post hybridization washing and staining samples were
recovered from the chips and stored in their original vials. The hybridization chamber
was filled with Buffer A (non stringent: 6X SSPE, 0.01% Tween 20). Modules in the
Fluidics Station 400 were primed according to the Affymetrix’ protocol.
Samples were loaded into the modules and washing and staining was done in
stringent buffer B (10mM MES, 0.1N Na+, 0.01% Tween 20) was also used in the
protocol. The streptavidin-phycoerythrin stain mix was as follows: 50 mM MES, 0.5 M
Na+, 0.025% Tween 20, 2 mg/mL acetylated BSA, 10 µg/mL SAPE). The amplification
(antibody) solution mix was as follows: 50 mM MES, 0.5 M Na+, 0.025% Tween 20, 2 mg/mL acetylated BSA, 0.1 mg/mL normal goat IgG, 3 µg/mL biotinylated antibody. All chips were scanned twice. For data analysis, images obtained were converted into
Microsoft Excel format using MAS5.0 software (Affymetrix). All chips were scaled to mean target intensity of 1500. Affymetrix present (P) and absent (A) calls were used. The
Affymetrix detection algorithm uses probe pair intensities to generate a detection p-value and to assign present (P), marginal (M), or absent (A) calls. Each probe pair in a probe set
106 is considered as having a potential vote in determining the presence or absence of a
measured transcript and this vote is defined by a value called the discrimination score
(R). This R score is calculated by the Affymetrix software for each probe set and
compared to a predefined threshold Tau. Tau was set at the default of 0.015 for this
microarray. Probe pairs with R values lower than Tau vote for the absence of the
transcript and probe pairs with R values higher than Tau vote for the presence of the
transcript. The voting result is summarized as a p-value, with p<0.4 given a present (P) call, p > 0.6 given an absent (A) call, and 0.4 > p > 0.6 given a marginal (M) call. For
comparisons between chips, genes with 2-fold differences in expression compared to
controls were considered to be significantly differentially expressed. For pathway
analysis of RSK2 -/- and RSK2 +/+ fibroblast gene expression patterns, GenMAPP and
MAPPFinder software packages were used (www.GenMAPP.org) (Dahlquist et al., 2002;
Doniger et al., 2003).
RNA Isolation and Quantitative RT-PCR
For RT-PCR studies, RSK2 +/+ and RSK2 -/- fibroblasts were harvested, and total RNA was extracted by the Roche High Pure RNA Isolation kit (Roche, Indianapolis,
IN) according to the manufacturer’s specifications and quantified. Total RNA (5 µg) was reverse transcribed with Superscript II (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Quantitative RT-PCR was performed using the ABI Prism 7700
Sequence Detection System Cycler (Applied Biosystems). Reactions were performed in triplicate and detected by SYBR green dye (Stratagene) using the following primers.
107 RSK2: forward primer, 5’-GTG ACC TCA GCA CTC GGG C-3’; reverse primer, 5’-
GCT CGT TGC TGA TGG ACT GC-3’; β-actin (control): forward primer, 5’-CTT TGC
AGC TCC TTC GTT GC-3’; reverse primer, 5’-ACG ATG GAG GGG AAT ACA GC-
3’. Data were collected during each PCR cycle and analyzed using the Sequence
Detection Software (v. 1.6.3, Applied Biosystems). Results were expressed as the molar ratio of mRNA to mouse β-actin mRNA.
Data Analysis
Phosphoinositide hydrolysis (PI) hydrolysis assays, Calcium Flux assays, and
Adenylate Cyclase (AC) assays were performed in triplicate, repeated at least three times.
PI hydrolysis assays were analyzed by nonlinear regression using Prism 4.03 software
(GraphPad, San Diego, CA). Statistical significance of the data was determined by two-
tailed paired t test, was defined as p<0.05, and was analyzed by GraphPad Prism.
108 CHAPTER 3: The Interaction of p90 Ribosomal S6 Kinase 2
(RSK2) with the 5-HT2A Receptor
3.1 Introduction and Rationale
As described in Chapter 1, 5-HT2A receptors play crucial roles in the modulation
of perception, cognition, and emotion (Jakab and Goldman-Rakic, 1998; Kroeze and
Roth, 1998b; Roth, 1994; Roth et al., 1999a). As such, the elucidation of the regulatory
mechanisms for 5HT2A receptor signaling and trafficking will provide important insight into the therapeutic mechanisms of drugs that target the 5-HT2A receptor and has
implications for the rational design of novel medications (Gray and Roth, 2002; Kroeze et
al., 2002). As stated in Chapter 1, GPCRs can interact with a number of multi-domain
scaffolding proteins (Hall and Lefkowitz, 2002) in addition to a variety of other accessory
or chaperone proteins (Brady and Limbird, 2002). The interaction of GPCRs with these
proteins allows the generation of signaling specificity through the alteration of ligand
recognition, the compartmentalization of signaling complexes, and the regulation of
GPCR trafficking (Brady and Limbird, 2002). Understanding the regulation of 5-HT2A receptors, and other GPCRs, via interactions with scaffolding, accessory, and other proteins will advance our understanding of GPCR signaling and will present novel targets
for drug development and for disease treatment.
109 Within the last decade a number of 5-HT2A receptor interacting proteins (See
Chapter 1) have been identified. These 5-HT2A receptor interacting proteins have been
demonstrated to have a role in the trafficking, function, and regulation of 5-HT2A receptors (Bhatnagar et al., 2004; Gray et al., 2003a; Robertson et al., 2003; Turner and
Raymond, 2005; Xia et al., 2003a). The majority of these proteins interact with the intracellular loops and the C-terminal tail of the 5-HT2A receptor. As the i3 loop is known to be important for G-protein coupling (Hyde and Roth, 1997; Oksenberg et al., 1995), we sought to identify other proteins that could bind to the i3 loop of the 5-HT2A receptor
and to investigate the role of these proteins in 5-HT2A receptor signaling. To address this,
we conducted a yeast two-hybrid screen using the i3 loop of the 5-HT2A receptor as “bait” and a human brain cDNA library as a pool of “target” proteins.
3.2 A Yeast Two-Hybrid Screen Identifies Potential 5-HT2A Receptor-
Interacting Proteins
Transcription factors, such as the Gal4 transcriptional activator, can modulate transcription by binding to an upstream activating sequence (UAS) in the promoter of a gene through a DNA-binding domain (DNA-BD) and subsequently activate transcription through a transcriptional-activation domain (AD). These transcription factors require the function of both the DNA-BD and AD domain in order to activate transcription from a
UAS. Classical yeast two-hybrid screens take advantage of the requirement for both functional DNA-BD and AD domains (Figure 3-1). In these screens, transcription factor domains can be separately fused to a “bait” peptide sequence to create a DNA-BD-“bait”
110 fusion protein, which, although able to bind to an UAS, cannot activate transcription
alone as it lacks an AD. Through the use of cDNA libraries, a “target” peptide can be
fused to the AD to create a peptide library of “target”-AD fusion proteins. These
“target”-AD fusion proteins, although containing a functional AD, are unable to activate
transcription as they are unable to bind to an UAS. If the DNA-BD-“bait” and “target”-
AD fusion proteins are both present and the “bait” and “target” can interact with one
another, the DNA-BD and AD of the transcription factor are brought within close
proximity and the activity of the transcription factor is reconstituted (Figure 3-1). For our
studies, we used the entire third intracellular (i3) loop of the human 5-HT2A receptor as
“bait” and a human brain cDNA library as a pool of “target” proteins in a Gal4-based
yeast two-hybrid screen in order to identify potential 5-HT2A receptor-interacting proteins
(See Chapter 2).
111
Figure 3-1 - A schematic showing the molecular basis of a yeast-2-hybrid screen. In classical yeast two-hybrid screens, a transcription factor (illustrated here by the Gal4 transcriptional activator) (1) can modulate transcription by binding to an upstream activating sequence (UAS) through a DNA-binding domain (DNA-BD) and activate transcription through a transcriptional-activation domain (AD). These transcription factor domains can be separately fused to a “bait” peptide sequence (for example, a
GPCR c-terminal domain) to create a DNA-BD-“bait” fusion protein, which (2) cannot activate transcription alone as it lacks an AD. Through the use of cDNA libraries, a
“target” peptide can be fused to the AD to create a peptide library of “target”-AD fusion proteins. These “target”-AD fusion proteins (3) are unable to activate transcription as they lack a DNA-BD. If the DNA-BD-“bait” and “target”-AD fusion proteins are both present (4) and the “bait” and “target” can interact with one another, the DNA-BD and
AD of the transcription factor are brought within close proximity and the activity of the transcription factor is reconstituted.
112 Figure 3-1
113 For the screen itself, plasmids containing the i3 loop “bait” and human brain cDNA “target” constructs were prepared and transformed into opposite yeast mating strains (See Chapter 2). Following transformation into opposing yeast mating types, the yeast were mated to produce yeast that contain both “bait” and “target” plasmids (Figure
3-2). In addition to containing genes to transcribe DNA-BD-“bait” and “target”-AD proteins, these plasmids also contain genes that allow for growth on nutrient-deficient media (Figure 3-2). For our studies, the i3 loop “bait” plasmid contained a gene required for leucine biosynthesis (Leu) and the human brain cDNA “target” plasmid contained a gene required for tryptophan biosynthesis (Trp). Therefore, co-transformants were selected in media lacking Leu and Trp (-L-W). Following this, the yeast were replica plated onto nutrient deficient media (Figure 3-2). In our study, the yeast strain used,
Y187, contains genes for the synthesis of adenine (Ade) and histidine (His) that are under the transcriptional control of the Gal4 UAS. In addition, yeast strain Y187 also contains the β-galactosidase gene under control of the Gal4 UAS, allowing further confirmation of
“bait”-“target” interaction via β-galactosidase assay.
114
Figure 3-2 - A schematic of a general yeast two-hybrid protocol. Plasmids containing
DNA-BD-“bait” and “target”-AD constructs are prepared and (1) transformed into opposite yeast mating strains. These plasmids also contain genes that allow for growth on nutrient-deficient media, in this example, the “bait” plasmid contains a gene required for leucine biosynthesis (Leu) and the “target” plasmid contains a gene required for tryptophan biosynthesis (Trp). Following transformation into opposing yeast mating types, the yeast are (2) mated to produce yeast that contain both “bait” and “target” plasmids and then (3) replica plated onto nutrient deficient media. In this example, plating the yeast on media lacking leucine and tryptophan is a confirmation of the presence of both the “bait” and “target” plasmids. In addition, the yeast strain transformed by the “bait” and “target” plasmids also contains genes required for histidine
(His) and adenine (Ade) biosynthesis. Therefore, growth of the yeast on media lacking leucine, tryptophan, histidine, and adenine implies that the “bait” and “target” proteins interact. Plasmid isolation (4) and sequencing (5) of these surviving colonies provides a list of potential “bait” interacting proteins.
115 Figure 3-2
116 In our initial yeast two-hybrid screen of a human brain cDNA library, a large
number of putative 5-HT2A receptor interacting proteins were identified based on growth on media lacking leucine, tryptophan, histidine (Triple-Drop-Out, TDO) and on media lacking leucine, tryptophan, histidine, and adenine (Quadruple-Drop-Out, TDO) (Table
3-1). Growth of the yeast on TDO and QDO media implies that the “bait” and “target” proteins interact. To confirm the original yeast two-hybrid results, we re-plated all colonies on QDO media and tested the colonies for β-galactosidase expression (See
Chapter 2) (Table 3-1). Plasmids from yeast colonies showing the most vigorous growth on QDO and the highest β-galactosidase expression were isolated, transformed into E. coli, and sequenced (Figure 3-2). Following sequencing, a number of duplicates, clones containing only untranslated regions of DNA, and clones expressing proteins or fragments not restricted to expression in neuronal tissues or cells were eliminated, leaving a potential pool of 5-HT2A receptor interacting proteins (Table 3-2). One clone,
33.5, which encoded a portion of the C-terminal kinase domain of RSK2, was selected for further study (Table 3-2, Figure 3-3).
117
Table 3-1 - The Results of a Yeast Two-Hybrid Screen Using the i3 Loop of the
5-HT2A Receptor as “Bait” and a Human Brain cDNA as a Pool of “Target”
Proteins
Summary of Yeast Two-Hybrid Results
Number Number of Colonies Number of Colonies Initial of Growing on QDO β-gal Positive Screen Colonies (Total % of Initial Screen) (Total % of Initial Screen)
TDO 111 76 (68.5) 40 (36.0)
QDO 74 74 (100) 40 (54.1)
118
Table 3-2 - A yeast-two-hybrid screen reveals potential 5-HT2A receptor interacting proteins
Clone Identity Gene
amyloid-beta precursor protein intracellular 2.4 AIDA-1A domain associated protein-1a eukaryotic translation initiation factor 3, 5.2 EIF3S5 subunit 5 epsilon neurotrophic tyrosine kinase, receptor, type 3 6.1 NTRK3 isoform c precursor
7.3 melanoma-associated antigen MAAT1
21.1 paraoxonase 2 PON2
29.1 Microtubule associated protein 1A MAP-1A
33.5 ribosomal protein S6 kinase 2 RSK2
39.4 nucleoside-diphosphate kinase 3 NME3
NADH dehydrogenase (ubiquinone) 1 beta 43.5 NDUFB10 subcomplex
47.5 protein phosphatase 5, catalytic subunit PPP5C
47.6 glutamine synthetase GLUL
Shown are the original clone numbers, the identity of the clone, and the gene symbols for
various potential 5-HT2A receptor interacting proteins identified through a yeast two- hybrid screen.
119
Figure 3-3 - RSK2 is identified at a potential 5-HT2A receptor interacting protein.
Figure 3-3A shows the full protein sequence of mouse RSK2 (Genbank Accession number NP_683747). Highlighted in blue is the N-terminal kinase (NTK) domain of
RSK2, highlighted in red is the linker domain, highlighted in purple is the C-terminal kinase domain, and highlighted in yellow is the portion of the full-length RSK2 sequence identified as a 5-HT2A receptor i3 loop binding protein in the yeast two-hybrid screen as
clone 33.5. Figure 3-3B shows the domain structure of RSK2, the residues known to be phosphorylated in RSK2 activation, and the kinases known to phosphorylate those residues.
120 Figure 3-3
121 3.3 Additional Two-Hybrid Analyses Narrow the Region of Interaction Between
the i3 Loop of the 5-HT2A Receptor and the RSK2 “Target” 33.5
To begin the study of the potential interaction between RSK2 and the 5-HT2A receptor, I first wanted to determine the portion of the i3 loop of the 5-HT2A receptor that
interacts with the RSK2 “target.” The site of interaction between RSK2 and the 5-HT2A receptor i3 loop ‘bait’ was identified by constructing serial deletions and domain deletions of the i3 loop “bait”, co-transforming these “bait” constructs and the RSK2
“target” into yeast strain PJ69-2A, and monitoring the interaction with the RSK2 target clone through two-hybrid analyses (See Chapter 2). Figure 3-4 shows that growth of the
yeast on TDO media and on QDO media was lost when the i3 loop was truncated to
amino acid (AA) 268. However, growth still occurred with the truncation of the i3 loop to
AA 282. This was not a “length effect” of the truncation of the i3 loop, because the yeast
also failed to grow on TDO and QDO when transformed with the full i3 loop containing a
deletion of AAs 270-280 and the RSK2 target (Figure 3-4). These findings narrowed the
site of interaction between the RSK2 “target” and the 5-HT2A receptor i3 loop “bait” to
residues 270-280 of the 5-HT2A receptor. Interestingly, this region of the i3 loop contains a putative RSK2 consensus phosphorylation site (See Chapter 1) (AA 275-280; Figure
3-4).
122
Figure 3-4 - Yeast-two hybrid analyses narrow the region of interaction between the
5-HT2A receptor and RSK2. Truncation mutants of the i3 bait and the RSK2 yeast two- hybrid screen target were co-transformed into the yeast strain PJ69-2A and interaction was measured by growth on QDO or TDO and by expression of β-galactosidase activity
(See Chapter 2 for details).
123 Figure 3-4
124 3.4 Full-Length RSK2 and RSK2-GFP Interact with 5-HT2A Receptors In Vitro.
In our initial studies, we constructed a vector to express a FLAG-tagged wild type
(wt) human 5-HT2A receptor (FLAG-h5-HT2A) in a manner similar to that previously described (See Chapter 2) (Xia et al., 2003a), adding a cleavable signal sequence
(MKTIIALSYIFCLVFA) from influenza hemaglutinin and a FLAG tag (DYKDDDDK) to the amino-terminus of the human 5-HT2A receptor. Addition of this signal sequence to
FLAG-tagged GPCRs has been shown to enhance membrane insertion, expression, and function (Guan et al., 1992). The FLAG-h5-HT2A receptor construct was prepared to
facilitate immunoprecipitaion of 5-HT2A receptors using commercially available antibodies. In addition, two different RSK2 constructs were prepared, one expressing wt mouse RSK2 (RSK2), and an N-terminal GFP tagged form of wt mouse RSK2 (RSK2-
GFP). We tagged RSK2 on the N-terminal with GFP as the C-terminal domain of RSK2
was isolated in the yeast two-hybrid screen (Figure 3-3) and we did not want to interfere with any interaction between RSK2 and the 5-HT2A receptor. To verify the expression of
RSK2 and RSK2-GFP, HEK-293 cells were transiently transfected with RSK2 or RSK2-
GFP and Western blots were performed to detect RSK2 in the cell lysates (Figure 3-5).
We found that mouse RSK2 and RSK2-GFP were expressed in HEK-293 cells and that
HEK-293 cells endogenously express RSK2 (Figure 3-5).
125
Figure 3-5 - The expression of RSK2 and RSK2-GFP in HEK-293 cells. To verify the expression of RSK2 and RSK2-GFP, HEK-293 cells were either transiently co- transfected with RSK2 (lanes 3 and 4) or RSK2-GFP (lanes 5 and 6). Untransfected
HEK-293 cells, which endogenously express RSK2, were used as a control (lanes 1 and
2). Cell lysates were prepared, electrophoresed, and Western blotted as described in
Chapter 2. Cell lysates were probed for RSK2 using a polyclonal RSK2 antibody (top blot) and for Gαq using a polyclonal Gαq antibody (bottom blot) as a loading control.
126 Figure 3-5
127 To confirm the interaction of RSK2 with the 5-HT2A receptor, we next performed
co-immunoprecipitation studies to verify that full-length 5-HT2A receptors and full-length
RSK2 interact in vitro. For these studies, HEK-293 cells were transiently transfected with
FLAG-h5-HT2A and RSK2 (Figure 3-6A). As shown in Figure 3-6A, RSK2 was co- immunoprecipitated with FLAG-h5-HT2A receptors (Figure 3-6A, lanes 3 and 4),
indicating that 5-HT2A receptors and RSK2 associate in vitro. The association of FLAG-
h5-HT2A and RSK2 was unaltered by agonist exposure (Figure 3-6A, compare lanes 3
and 4), indicating that 5-HT2A receptors and RSK2 associate in an agonist-independent manner in HEK-293 cells. These studies were also undertaken using RSK2-GFP with identical results (Figure 3-6B).
128
Figure 3-6 - RSK2 and RSK2-GFP interact with human 5-HT2A receptors in vitro.
Figures 3-6A and 3-6B show the co-immunoprecipitation of RSK2 (3-6 A) and RSK2-
GFP (3-6 B) with FLAG-h5-HT2A receptors in transfected HEK-293 cells. For these
experiments, HEK-293 cells were either transiently co-transfected with RSK2 or RSK2-
GFP and CFP (lanes 1 and 2) or with FLAG-h5-HT2A and RSK2 or RSK2-GFP (lanes 3 and 4). Lanes 1 and 3 were treated with vehicle and lanes 2 and 4 were treated with
10µM 5-HT for 5 minutes. FLAG-tagged human 5-HT2A receptors were immunoprecipitated by a monoclonal FLAG antibody conjugated to Sepharose beads.
RSK2 was detected in the immunoprecipitates using a polyclonal RSK2 antibody (Figure
3-6A) and RSK2-GFP was detected in the immunoprecipitates using a polyclonal GFP
antibody (Figure 3-6B). Lysates were probed for the presence of RSK2 with a polyclonal
RSK2 antibody and the presence of FLAG-h5-HT2A receptors was detected using a
polyclonal FLAG antibody. Shown are representative immunoblots from a single
experiment that has been replicated three times with equivalent results.
129 Figure 3-6
130 3.5 Endogenous RSK2 Interacts with Endogenous 5-HT2A Receptors In Vivo.
Having verified that full-length RSK2 and FLAG-h5-HT2A receptors interact in
vitro, we wished to determine if these proteins interact in a native cellular milieu. To this
end, we performed co-immunoprecipitation studies in two different cellular milieus in
which 5-HT2A receptors and RSK2 are constitutively expressed: C6 glioma cells and rat
brain synaptic membranes. In each of these milieus, endogenous RSK2 was
immunoprecipitated to determine whether RSK2 and the 5-HT2A receptor were endogenously associated in vivo (See Chapter 2 for details). To verify the specificity of the interaction, immunoprecipitations were performed using protein-A/G agarose beads in the absence (-) or presence (+) of a monoclonal RSK2 antibody. Figure 3-7A, first panel, shows that 5-HT2A-like immunoreactivity was present in C6 glioma cell immunoprecipitates only when an RSK2 antibody was used in the immunoprecipitation
(compare lanes 1 and 2). The third panel in Figure 3-7A shows the immunoprecipitation of RSK2 in the presence of monoclonal RSK2 antibody and that RSK2 did not immunoprecipitate with protein A/G beads alone, indicating the specificity of the immunoprecipitation. The second and fourth panels in Figure 3-7A show that equivalent levels of 5-HT2A-like immunoreactivity and RSK2 were present in the lysates from which
the immunoprecipitations were performed. These data indicate that RSK2 and 5-HT2A receptors endogenously associate in C6 glioma cells. Identical results were obtained using rat brain synaptic membranes (Figure 3-7B). These results, together with the
previous immunoprecipitation data, demonstrate that RSK2 and 5-HT2A receptors
associate in vitro and in vivo.
131
Figure 3-7 - RSK2 interacts with 5-HT2A receptors in vivo. For these experiments in native cell lines, cell lysates were prepared from either C6 glioma cells (Figure 3-7A), or
from rat brain synaptic membranes (Figure 3-7B), and equivalent amounts of protein
were loaded for all sets (See Chapter 2 for more detail). Endogenous RSK2 was
immunoprecipitated by a monoclonal RSK2 antibody using protein A/G plus agarose as
described in Chapter 2. Figure 3-7A shows the co-immunoprecipitation of native 5-HT2A
receptors with RSK2 in C6 glioma cells. Figure 3-7B shows the co-immunoprecipitation
of native 5-HT2A receptors with RSK2 from rat brain synaptic membrane preparations. In
Figures 3-7A-B the first panel shows that native 5-HT2A receptors are
immunoprecipitated by monoclonal RSK2 antibody (lane 1 [+]) using protein A/G beads
but not with protein A/G beads alone (lane 2 [-]). The second panel shows native 5-HT2A receptors present in the lysates (lanes 1 and 2). The third panel shows robust RSK2 detection in the immunoprecipitate in presence of RSK2 antibody (lane 1 [+]). The fourth panel shows the RSK2 present in the lysates (lanes 1 and 2). RSK2 was detected using a polyclonal RSK2 antibody and endogenous 5-HT2A receptors were detected using a polyclonal 5-HT2A antibody. Shown are representative immunoblots from a single experiment that has been replicated three times with equivalent results.
132 Figure 3-7
133 3.6 5-HT2A Receptors Colocalize with RSK2 and RSK2-GFP in HEK-293 Cells
We performed dual-labeled immunofluorescence confocal microscopy studies to determine the sub-cellular localization of endogenous RSK2 before and following agonist exposure in HEK-293 cells transiently transfected with FLAG-h5-HT2A receptors (See
Chapter 2 for details). As shown in Figure 3-8A-C, FLAG-h5-HT2A receptors and endogenous RSK2 (which is highly expressed in HEK-293 cells) were colocalized on the cell surface of HEK-293 cells. In addition, cells expressing FLAG-h5-HT2A receptors appeared to induce the translocation of RSK2 from a predominantly cytoplasmic location to the cell surface. Agonist exposure (Figure 3-8D-F) resulted in FLAG-h5-HT2A receptor internalization and although RSK2 was found to colocalize with cell surface
FLAG-h5-HT2A receptors, it did not appear to colocalize with internalized FLAG-h-
5HT2A receptors. In addition, co-transfection of RSK2-GFP and FLAG-h5-HT2A receptors (Figure 3-8G-I) demonstrated that RSK2 also appears to localize in 5-HT2A receptor-enriched membrane ‘ruffles’ at the cell surface.
134
Figure 3-8 - RSK2 and RSK2-GFP colocalize with native human 5-HT2A receptors independently of agonist exposure. For these experiments, HEK-293 cells were transiently co-transfected with FLAG-h5-HT2A + RSK2 (A-F) or with FLAG-h5-HT2A +
RSK2-GFP (G-I). At 24 hours following transfection, cells were split in media containing 5% dialyzed serum. At 48 hours following transfection, cells were placed in serum free media for a minimum of 18 hours, and then exposed to vehicle (A-C) or 10
µM 5-HT (D-F, G-I) for 5 minutes. This was followed by dual-label immunofluorescent confocal microscopy (See Chapter 2). Representative images from one of three independent experiments are shown. FLAG-tagged human 5-HT2A receptors are shown in the green channel (A and D) or in the red channel (G). RSK2 is shown in the red channel
(B and E) and RSK2-GFP is shown in the green channel (H). The merged images are in panels C, F, and I. Scale bars are shown in all panels.
135 Figure 3-8
136 3.7 5-HT2A Receptors Colocalize with RSK2 in the Rat Brain Prefrontal Cortex
and Globus Pallidus
Finally, we examined the distribution of RSK2 in rat brain in two regions
enriched in 5-HT2A receptors: the prefrontal cortex and in the globus pallidus.
Immunohistochemistry was performed as detailed in Chapter 2 by our collaborators at
Vanderbilt University, Dr. Ariel Y. Deutch and Bonnie G. Garcia. Figures 3-9A-C show that 5-HT2A receptors and RSK2 were co-localized in the globus pallidus. Figures 3-9D-
F show that 5-HT2A receptors and RSK2 were also co-localized in layer V pyramidal
neurons in the prefrontal cortex. Figures 3-9G-L show higher magnifications of layer V
pyramidal neurons in the prefrontal cortex, and indicate an overlapping and punctate
distribution of 5-HT2A receptors and RSK2. Taken together, these data show that 5-HT2A receptors and RSK2 have overlapping cellular and subcellular distributions in rat brain.
137
Figure 3-9 - RSK2 co-localizes with 5-HT2A receptors in rat brain prefrontal cortex and globus pallidus. Rat brain sections were prepared, stained, and visualized by dual- label immunofluorescent confocal microscopy as described in Chapter 2. Representative images from one of three independent experiments are shown. Globus pallidus sections are shown in panels A-C. Layer V prefrontal cortex sections are shown in panels D-F.
Higher magnifications Layer V prefrontal cortex sections are shown in panels G-L.
Native rat 5-HT2A receptors are shown in the red channel (A, D, G, and J) and native rat
RSK2 is shown in the green channel (B, E, H, and K). The merged images are in panels
C, F, I, L. These experiments were performed by Dr. Ariel Y. Deutch and Bonnie G.
Garcia.
138 Figure 3-9
139 3.8 Discussion
In this chapter, I discussed the interaction of RSK2 with the 5-HT2A receptor.
RSK2 was initially identified as a potential 5-HT2A receptor-interacting protein via a yeast two-hybrid screen that we conducted using the i3 loop of the 5-HT2A receptor as
“bait” and a human brain cDNA library as a pool of potential “targets.” As described in
Chapter 1, RSK2 is a kinase that lies downstream of ERK in the MAPK cascade (Xing
et al., 1996). My rationale for selecting RSK2 to further evaluate as a potential 5-HT2A receptor-interacting protein was three-fold. First, 5-HT2A receptors, and many other
GPCRs activate ERK phosphorylation via the MAPK cascade (Roth et al., 1998b). In
addition, the precise mechanism of MAPK cascade activation by 5-HT2A receptors, or by other GPCRs, has not been fully elucidated. Therefore, it was possible that RSK2 may be involved in the activation of the ERK MAPK cascade by 5-HT2A receptors and I wished
to explore this possibility.
Second, as detailed in Chapter 1, RSK2 belongs to the AGC family of protein
kinases, which include PKC and PKA (Alcorta et al., 1989). As described in Chapter 1,
a number of protein kinases have been discovered to phosphorylate serine and threonine
residues within the intracellular domains of GPCRs following agonist exposure,
including the second messenger-dependent kinases such as PKA and PKC, as well as the
GRKs (Ferguson and Caron, 1998; Ferguson et al., 1998; Lefkowitz, 1993). However, the
regulation of 5-HT2A receptors by phosphorylation is unclear as a high level of basal phosphorylation of 5-HT2A receptors is found in HEK-293 cells when 5-HT2A receptors
140 are over-expressed, agonist exposure does not increase the level of 5-HT2A receptor phosphorylation, and PKC does not play a role in the desensitization of 5-HT2A receptors in HEK-293 cells (Vouret-Craviari et al., 1995). Since RSK2 belongs to the same protein kinase family as PKC and PKA, I was interested in determining what role RSK2 may play in 5-HT2A receptor phosphorylation.
Finally, as detailed in Chapter 1, null mutations in RSK2 result in the X-linked
mental retardation syndrome, Coffin-Lowry Syndrome (CLS) (Trivier et al., 1996). CLS
is characterized by severe mental retardation, pathognomonic craniofacial and skeletal
deformities, growth retardation (Lowry et al., 1971), movement disorders (Stephenson et
al., 2005), cardiovascular disorders, and a schizophrenia-like psychosis in heterozygote
females (Hanauer and Young, 2002; Sivagamasundari et al., 1994). Importantly, 5-HT2A receptors play crucial roles in the modulation of perception, cognition, and emotion, and have been implicated in the etiology of schizophrenia (Jakab and Goldman-Rakic, 1998;
Kroeze and Roth, 1998b; Roth, 1994; Roth et al., 1999a). The presence of a schizophrenia-like psychosis in heterozygote females with CLS indicates a potential dysfunction of 5-HT2A receptors in CLS patients. Hence, the elucidation of the role of
RSK2 in the regulation of 5-HT2A receptors could prove invaluable in understanding the pathogenesis and pathophysiology of schizophrenia.
In this chapter, I employed additional two-hybrid analyses to narrow the region of the i3 loop that interacts with the RSK2 “target” and found that amino acids 270-280 of the i3 loop are required for interaction with the RSK2 “target.” To verify the results of
141 the yeast two-hybrid screen, I demonstrated that RSK2 and FLAG-tagged 5-HT2A receptors co-immunoprecipitate when transfected into HEK-293 cells. Having verified the in vitro interaction of full-length RSK2 and 5-HT2A receptors, I then determined that
endogenous 5-HT2A receptors co-immunoprecipitate with endogenous RSK2 in two systems where these proteins are expressed natively: C6-glioma cells and in rat brain synaptic membrane preparations. Together, these data indicate that 5-HT2A receptors and
RSK2 associate both in vitro and in vivo. In addition, I determined that 5-HT2A receptors and RSK2 co-localize in HEK-293 cells and in the rat brain prefrontal cortex and globus
pallidus. Together, these data indicate that RSK2 and 5-HT2A receptors display overlapping subcellular distributions, further providing support for their interaction. The following chapter will discuss the functional consequences of the interaction between the
5-HT2A receptor and RSK2.
142 CHAPTER 4: The Regulation of 5-HT2A Receptor Signal
Transduction by p90 Ribosomal S6 Kinase 2 (RSK2)
4.1 Introduction and Rationale
As described in Chapter 3, RSK2 was identified as a 5-HT2A receptor interacting
protein through a yeast two-hybrid screen and the interaction of RSK2 with the 5-HT2A receptor was confirmed through co-immunoprecipitation studies in both over-expression
(HEK-293 cells) and in endogenous (C6 glioma and rat brain synaptic membrane) cell systems. These data confirmed the yeast two-hybrid results, indicating that RSK2 and the
5-HT2A receptor associate both in vitro and in vivo. Furthermore, RSK2 and 5-HT2A receptors were demonstrated to display a similar subcellular distribution and colocalize in
HEK-293 cells, in rat brain prefrontal cortex, and in rat brain globus pallidus. Together, these data indicate that 5-HT2A receptors and RSK2 associate in vitro and in vivo.
In order to determine the functional significance of the interaction between the 5-
HT2A receptor and RSK2, we obtained fibroblasts from RSK2 +/+ and RSK2 -/- mice
(Bruning et al., 2000). As described in Chapter 1, previous studies have shown that
RSK2 knock-out mice weigh 10% less and are 14% shorter that RSK2 wild-type mice,
consistent with the role of RSK2 in growth. The mice also exhibit poor learning and
coordination, consistent with what is found in CLS patients (Xing et al., 1996).
Intriguingly, insulin- and exercise-stimulated ERK1/2 phosphorylation was found to be
both increased and prolonged in the skeletal muscle of RSK2 knock-out mice. These
143 RSK2 knock-out mice also showed an increase in the basal level of phosphorylated
ERK1/2 in the skeletal muscle (Dufresne et al., 2001). As 5-HT2A receptors, and many
other GPCRs activate ERK phosphorylation via the MAPK cascade (Roth et al., 1998b),
the role of RSK2 in the activation of the ERK MAPK cascade via 5-HT2A receptors is worth investigating. We wished to determine the role, if any, RSK2 plays in 5-HT2A signaling to phosphoinositide hydrolysis, calcium mobilization, and whether or not RSK2 plays a role in the phosphorylation of 5-HT2A receptors.
4.2 Characterization of RSK2 -/- and RSK2 +/+ Fibroblasts
To begin to study the effects of RSK2 on 5-HT2A receptor signaling, it was
necessary to first characterize the RSK2 -/- and RSK2 +/+ fibroblast cell lines that we
wished to utilize (Bruning et al., 2000). First, we wanted to determine if the RSK2 -/- and
RSK2 +/+ fibroblasts expressed 5-HT2A receptors. Initial experiments indicated that 5-
HT induced an increase in phosphoinositide (PI) accumulation that was abolished by the selective 5-HT2A antagonist MDL100,907 (Sorensen et al., 1993) (Figure 4-1A). These
results indicated that both RSK2 -/- and RSK2 +/+ fibroblasts express functional 5-HT2A receptors and implied that other member of the 5-HT2 receptor class, the 5-HT2B and 5-
HT2C receptors, were not present as MDL100,907 is highly selective (5-HT2A Ki = 0.85 nM, 5-HT2B Ki = 261 nM, 5-HT2C Ki = 112 nM) for 5-HT2A receptors
(http://pdsp.cwru.edu/).
144 We next wanted to determine if equivalent levels of 5-HT2A receptor mRNA were
present in the RSK2 -/- and RSK2 +/+ fibroblasts via quantitative RT-PCR studies (See
Chapter 2 for details). These studies disclosed that both cell lines expressed equivalent
amounts of 5-HT2A mRNA, normalized to β-actin mRNA levels (Figure 4-1B). Finally,
3 we also measured 5-HT2A receptor expression via [ H]ketanserin saturation binding
assays (See Chapter 2 for details) and determined that RSK2 -/- fibroblasts express
264.9 ± 44.7 fmol/mg (Kd = 13.01 ± 3.71 nM) and RSK2 +/+ fibroblasts express 289.2 ±
109.4 fmol/mg (Kd = 18.93 ± 10.86 nM) of the 5-HT2A receptor. Together, these results indicated that both cell lines expressed equivalent amounts of 5-HT2A receptors. We now had a model system to study the effects of RSK2 on 5-HT2A receptor signaling as we
would be able to make use of a system where 5-HT2A receptors were endogenously expressed in the presence and absence of RSK2.
145
Figure 4-1 - RSK2 knock-out augments 5-HT2A receptor signaling without altering
5-HT2A receptor expression. Figure 4-1A shows the results of quantitative RT-PCR for
the 5-HT2A receptor normalized to β-actin levels, indicating that both the RSK2 +/+ and -
/- fibroblasts express similar levels of 5-HT2A mRNA. For Figure 4-1B, RSK2 +/+ and -/- fibroblasts were plated and treated as described in Chapter 2 and maximal PI hydrolysis stimulated by 10 µM 5-HT was measured in the presence and in the absence of 100 nM
MDL100,907. These data are normalized with RSK2 +/+ fibroblast maximal signaling equal to 100%. RFU = relative fluorescence units; NS = not statistically significant; * = statistically significant p<0.05
146 Figure 4-1
147 4.3 RSK2 Knock-out Augments 5-HT2A Receptor Signaling to Phosphoinositide
Hydrolysis and to Calcium Mobilization
As previously described, RSK2 -/- and RSK2 +/+ fibroblasts displayed a 5-HT- induced increase in phosphoinositide (PI) accumulation that was abolished by the selective 5-HT2A antagonist MDL100,907. Intriguingly, in addition to disclosing that functional 5-HT2A receptors are expressed in both the RSK2 -/- and RSK2 +/+
fibroblasts, these studies also show that 5-HT2A-mediated PI accumulation was augmented in the RSK2 -/- fibroblasts (Figure 4-1B). To confirm these results, we expanded upon our original PI hydrolysis studies, conducting a full 5-HT-dose response; we found an augmentation of maximal phosphoinositide hydrolysis, confirming our previous results (Figure 4-2A). We also conducted calcium mobilization studies and found an increase in maximal calcium mobilization in the absence of RSK2 (Figures 4-
2B-C).
For these phosphoinositide hydrolysis studies (Figure 4-2A), RSK2 -/- fibroblasts displayed a relative agonist efficacy of 2.72 ± 0.19 and an EC50 of 678 nM for 5-HT and
RSK2 +/+ fibroblasts displayed a relative agonist efficacy of 0.99 ± 0.02 and an EC50 of
668 nM for 5-HT. Therefore, in addition to confirming our initial studies, these data
indicate that although there is an augmentation in efficacy in the absence of RSK2, there
is no change in the potency of 5-HT for PI hydrolysis. Figure 4-2B shows a
representative tracing of calcium mobilization in RSK2 -/- and RSK2 +/+ fibroblasts,
where 10 µM 5-HT is added at the 20 second time-point. This demonstrates the
148 augmentation of calcium mobilization in the absence of RSK2, consistent with the PI
hydrolysis data (Figure 4-2A-C). For the calcium mobilization studies, we found that
RSK2 -/- fibroblasts displayed a relative agonist efficacy of 1.90 ± 0.04 and an EC50 of
101 nM for 5-HT and RSK2 +/+ fibroblasts displayed a relative agonist efficacy of 1.06
± 0.04 and an EC50 of 260 nM for 5-HT. Therefore, both PI hydrolysis and calcium mobilization studies indicate an augmentation of 5-HT2A receptor signaling in the
absence of RSK2. It should be noted that although no change in EC50 was demonstrated
for 5-HT in PI hydrolysis studies, a small, 2.5-fold increase in 5-HT potency was
demonstrated in the absence of RSK2 for calcium mobilization studies. Such a small
change in potency is noteworthy, but will require further study.
149
Figure 4-2 - RSK2 knock-out augments the signaling of 5-HT2A receptors as
measured by phosphoinositide hydrolysis or by calcium mobilization. For Figure 4-
2A, RSK2 +/+ fibroblasts and RSK2 -/- fibroblasts were plated onto 24-well plates.
Phosphoinositide accumulation upon agonist exposure was measured as outlined in the
Chapter 2. Figure 4-2A shows the 5-HT mediated dose response for phosphoinositide
hydrolysis for RSK2 +/+ fibroblasts and RSK2 -/- fibroblasts in dpm/mg of total protein normalized to RSK2 +/+ fibroblast signaling. For Figures 4-2B-C, RSK2 +/+ fibroblasts and RSK2 -/- fibroblasts were plated onto 96-well plates. Cells were serum-starved, treated with agonist, and calcium mobilization was measured as described in Chapter 2.
Figure 4-2 B shows a representative tracing of the changes in fluorescence of RSK2 -/- and RSK2 +/+ fibroblasts upon the addition of 10 µM 5-HT. Figure 4-2C shows the 5-HT mediated dose response for calcium mobilization for RSK2 +/+ fibroblasts and RSK2 -/- fibroblasts in RFU normalized to RSK2 +/+ fibroblast signaling.
150 Figure 4-2
151 There are a number of possible explanations for this phenotype that do not
involve a direct effect of RSK2. First, the absence of RSK2 may lead to an alteration in the gene expression of genes involved in serotonergic signaling. Second, 5-HT2A receptor signaling could be enhanced by an increase in 5-HT2A receptor surface expression in the absence of RSK2. Finally, an augmentation of 5-HT2A receptor-mediated PI hydrolysis could occur via the direct potentiation of G-protein activity in the absence of RSK2. We decided to evaluate each of these possibilities individually.
4.4 Microarray Analysis Reveals no Global Alterations in Gene Expression
Occur in the Absence of RSK2
Microarray studies were then conducted to determine if global changes in the expression of genes involved in serotonergic signaling can account for the augmented signaling seen in RSK2 -/- fibroblasts. No global alterations in gene expression were measured and, in fact, similar numbers of genes were expressed (“present” calls by the
Affymetrix software, see Chapter 2 for more detail; Microarray data is discussed in detail in Chapter 6) in the RSK2 +/+ and RSK2 -/- fibroblasts. Similar numbers of genes were up-regulated and down-regulated when RSK2 -/- and RSK2 +/+ fibroblasts were compared. Additionally, 97% of the expressed genes showed less than a 2-fold change between RSK2 +/+ and -/- lines (See Chapter 5 for details). Taken together, these findings indicate that global changes in gene expression do not occur in RSK2 -/-
cells.
152 We also performed detailed analyses of the microarray data. No systematic
changes in gene expression were seen for any measured members of various GPCR
signal transduction cascades (Figure 4-3). In addition, we found no alterations in the
expression patterns of members of the ERK MAPK cascade (Figure 4-4). These
microarray studies allowed us to determine if other 5-HT receptors were expressed and if
alterations in expression of these receptors might, in some way, explain the changes we
measured in 5-HT signaling. Importantly, other receptors in the 5-HT2 family, 5-HT2B and 5-HT2C, were not expressed. These data indicate that the augmented 5-HT2A signaling (e.g., PI accumulation, in RSK2 -/- fibroblasts) was likely not due to alterations in gene expression. Since microarray studies only measure mRNA levels, these studies do not address the possibility that protein levels might be altered and further studies are necessary to investigate this possibility. A complete discussion of the microarray data appears in Chapter 6; only microarray data salient to the current study will be discussed in this chapter.
153
Figure 4-3 - RSK2 +/+ and -/- fibroblasts do not show differences in GPCR signaling
pathway gene expression. Figure 4-3 shows the microarray data of GPCR signaling
pathways overlaid with gene-expression color criterion and fold-changes from the
programs GenMAPP and MAPPFinder. Gray colored genes are those expressed in RSK2
-/- and RSK2 +/+ fibroblasts that show no change in gene expression. White colored genes are not expressed in either RSK2 -/- or RSK2 +/+ fibroblasts. Green colored genes are those genes in the RSK2 -/- fibroblasts that show greater than a 2-fold increase in expression over the RSK2 +/+ fibroblasts. Red colored genes are those genes in the
RSK2 -/- fibroblasts that show greater than a 2-fold decrease in expression compared the
RSK2 +/+ fibroblasts.
154 Figure 4-3
155
Figure 4-4 - RSK2 +/+ and -/- fibroblasts do not show differences in MAPK cascade gene expression. Figure 4-4 shows the microarray data of MAPK signaling cascades overlaid with gene-expression color criterion and fold-changes from the programs
GenMAPP and MAPPFinder. Gray colored genes are those expressed in RSK2 -/- and
RSK2 +/+ fibroblasts that show no change in gene expression. White colored genes are not expressed in either RSK2 -/- or RSK2 +/+ fibroblasts. Green colored genes are those genes in the RSK2 -/- fibroblasts that show greater than a 2-fold increase in expression over the RSK2 +/+ fibroblasts. Red colored genes are those genes in the RSK2 -/- fibroblasts that show greater than a 2-fold decrease in expression compared the RSK2 +/+ fibroblasts.
156 Figure 4-4
157 4.5 RSK2 Knock-out does not Alter the Surface-Expression of 5-HT2A Receptors
We next wished to evaluate whether alterations in 5-HT2A receptor expression could account for the augmentation of PI hydrolysis found in the absence of RSK2.
Unfortunately, levels of endogenous 5-HT2A receptor expression were too low to quantify surface expression by conventional biochemical techniques such as surface biotinylation followed by Western blot analysis. Accordingly, we prepared stable lines over-expressing
FLAG-5-HT2A receptors and subsequently performed surface biotinylation assays (See
Chapter 2 for more detail). We found that these stable lines of RSK2 -/- fibroblasts which over-express FLAG-5-HT2A receptors also displayed enhanced 5-HT2A-mediated maximal PI accumulation (Figure 4-5A). Importantly, not only do RSK2 -/- fibroblasts not express higher cell surface levels of 5-HT2A receptors, they actually demonstrate a
lower cell surface expression of FLAG-5-HT2A receptors as determined by cell surface biotinylation (Figure 4-5B-C). These data imply that the alterations in signaling are not due to differences in 5-HT2A receptor surface expression.
158
Figure 4-5 - RSK2 knock-out does not alter FLAG-5-HT2A receptor cell surface expression but augments FLAG-5-HT2A receptor signaling. For Figure 4-5A, RSK2
+/+ FLAG-5-HT2A Stable line and RSK2 -/- FLAG-5-HT2A Stable line fibroblasts were
plated onto 24-well plates. Phosphoinositide accumulation upon agonist exposure was
measured as outlined in Chapter 2 and normalized to receptor expression, with RSK2 +/+
FLAG-5-HT2A Stable line signaling equal to 100%. Figure 4-5B shows a representative immunoblot of cell surface FLAG-5-HT2A receptors in the RSK2 +/+ FLAG-5-HT2A
Stable line and in the RSK2 -/- FLAG-5-HT2A Stable line as determined by cell surface
biotinylation as outlined in Chapter 2. Figure 4-5C shows the quantification of the net
pixel intensities for surface-biotinylated receptor normalized to RSK2 +/+ FLAG-5-HT2A
Stable receptor expression. * = Statistically significant p<0.05
159 Figure 4-5
160 4.6 RSK2 Does Not Alter the Signaling of Constitutively Active Gαq
Since deletion of RSK2 augments 5-HT2A receptor signaling, we wondered if this
occurs via the direct potentiation of G-protein activity. To examine this possibility, we
measured the basal signaling of a constitutively active form of Gαq, Gαq(Q209L), in
RSK2 -/- and RSK2 +/+ fibroblasts. Gαq(Q209L) directly activates phospholipase C and
elevates phosphoinositide levels independently of GPCRs (Booden et al., 2002).
Accordingly, RSK2 -/- and RSK2 +/+ fibroblasts were transfected with either wild type
Gαq or Gαq(Q209L) and basal phosphoinositide hydrolysis was measured. We found that deleting RSK2 has no effect on Gαq(Q209L) signaling compared to wild type Gαq basal signaling (Figure 4-6). These data imply that alterations in 5-HT2A receptor signaling demonstrated in RSK2 -/- fibroblasts occur upstream of Gαq, at the level of receptor-
effector coupling.
161
Figure 4-6 - RSK2 knock-out does not alter the signaling of a constitutively active
Gαq. For Figure 4-6, RSK2 +/+ and -/- fibroblasts were transfected with wild type Gαq or the constitutively active Gαq mutant Gαq(Q209L), plated, and treated as described in
Chapter 2, and basal PI hydrolysis was measured. These data are presented as the Fold
Induction of PI hydrolysis of the Gαq(Q209L) constitutively active mutant over that of the
wild type Gαq, with normalization to Gαq expression as determined by Western blot
analyses. NS = not statistically significant
162 Figure 4-6
163 4.7 RSK2 Modulates 5-HT2A Receptor-Mediated p42/44 ERK Phosphorylation
As previously described, we established that the effects of RSK2 on 5-HT2A receptor signaling to PI hydrolysis were not due to alterations in sertonergic signaling gene expression, alterations in the cell surface expression of 5-HT2A receptors, or to a direct potentiation of Gαq signaling. We next wanted to determine if there were alterations in 5-HT2A-mediated p42/44 ERK phosphorylation in response to 5-HT. As
previously discussed, we wanted to determine if the absence of RSK2 has effects on 5-
HT2A-mediated p42/44 ERK phosphorylation, as earlier studies revealed that insulin-
stimulated p42/44 ERK phosphorylation was altered in RSK2 knock-out mice (Dufresne
et al., 2001). As shown in Figure 4-7A, RSK2 -/- fibroblasts shows an increase in both
basal (0 minute) and 5-HT stimulated (5, 10, 15, and 30 minute) p42/44 ERK
phosphorylation compared to RSK2 +/+ fibroblasts. Significantly, we found that the
elevated basal levels of p42/44 ERK phosphorylation were attenuated by treatment with
100 nM MDL100,907 (Figure 4-7A, compare lanes M and 0), a highly selective 5-HT2A antagonist (Sorensen et al., 1993).
Statistical analyses of the Western blot data indicated a significant potentiation in p42/44 ERK phosphorylation after 0, 5, 10, and 15 minutes (p<0.05) of 5-HT exposure in the RSK2 -/- fibroblasts (Figure 4-7B). In addition, we compared phosphorylated p42/44
ERK levels in the RSK -/- fibroblasts to RSK +/+ fibroblasts (after normalization to total p42/44 ERK) in untreated and MDL100,907 treated samples (Figure 4-7C). These data show that the levels of basal p42/44 ERK phosphorylation were considerably elevated in
164 untreated RSK -/- fibroblasts compared to levels in RSK +/+ fibroblasts (p<0.05) and that
basal p42/44 ERK phosphorylation in both cell lines was significantly attenuated by the
5-HT2A-selective antagonist MDL100,907. These results imply that the increase in basal
phosphorylation seen in the RSK2 -/- fibroblasts may be attributable, in part, to an augmented constitutive activity of 5-HT2A receptors.
165
Figure 4-7 - RSK2 knock-out potentiates basal and agonist-stimulated p42/44 ERK
phosphorylation. Figure 4-7A shows a representative immunoblot from a single
experiment that has been repeated four independent times with equivalent results. RSK2
+/+ fibroblasts and RSK2 -/- fibroblasts were exposed to 100 nM MDL100,907 (M) for
10 minutes, with vehicle (0 minute lane) or to 10 µM 5-HT (5, 10, 15 and 30 minute lanes). The top immunoblot shows phosphorylated p42/44 ERK levels with a short exposure time so that the varying time points are not over-exposed. The second blot from the top shows a longer exposure of the phosphorylated p42/44 ERK immunoblot to place emphasis on the MDL100,907 (M) and vehicle treated (0) lanes. The middle immunoblot shows total p42/44 ERK levels. The fourth immunoblot from the top shows that RSK2 is absent in the RSK2 -/- fibroblasts. The bottom immunoblot shows RSK1 levels, and that
RSK1 expression is decreased but not absent in the RSK2 -/- fibroblasts. Figure 4-7B shows a quantification of the net pixel intensities of phosphorylated p42/44 ERK normalized to total p42/44 ERK as the fold over RSK2 +/+ fibroblast basal for the entire time course. Figure 4-7C shows a quantification of the net pixel intensities of phosphorylated p42/44 ERK normalized to total p42/44 ERK as the fold over RSK2 +/+ fibroblast basal comparing the 0 minute and 100 nM MDL100,907 treated time points of
RSK2 +/+ fibroblasts and RSK2 -/- fibroblasts. * = Statistically significant p<0.05; NS =
Not statistically significant
166 Figure 4-7
167 4.8 RSK2 Can Directly Phosphorylate 5-HT2A Receptors
Having found alterations in 5-HT2A receptor signaling to PI hydrolysis, calcium mobilization, and to p42/44 ERK phosphorylation, we next wanted to determine if RSK2 can phosphorylate 5-HT2A receptors and, if so, if the kinase activity of RSK2 is essential for the signaling phenotype we have discovered. To address these possibilities, we first
performed in vitro kinase assays wherein purified, active, homogeneous RSK2 was
incubated with affinity-purified 5-HT2A receptors. For these studies, FLAG-5-HT2A receptors were over-expressed in HEK-293T cells, solubilized and affinity purified using anti-FLAG agarose (See Chapter 2 for details). The affinity-purified FLAG-5-HT2A was then subjected to direct phosphorylation by active RSK2. As shown in Figure 4-8A, active RSK2 robustly phosphorylated a band with an apparent molecular weight (MW) equivalent to that of affinity-purified 5-HT2A receptors (Figure 4-8B). Figure 4-8A also
shows that RSK2 is autophosphorylated, as has been previously described (Frodin and
Gammeltoft, 1999), with Figure 4-8C showing a Western blot of the active RSK2 probed for RSK2. Additionally, no apparent phosphorylation of a band with a MW identical to that of FLAG-5-HT2A was seen in preparations from HEK-293T cells transfected with
CFP (Figure 4-7 A, lane 2) nor in samples wherein RSK2 was omitted (Figure 4-7 A,
lanes 1 and 4). Together, these data demonstrate that RSK2 can phosphorylate the 5-HT2A receptor.
168
Figure 4-8 - RSK2 phosphorylates the 5-HT2A receptor. Figure 4-8A shows the
phosphorylation of the 5-HT2A receptor by active RSK2 in an in vitro kinase assay. For the in vitro kinase assay, HEK-293T cells were transfected with either CFP or FLAG-5-
HT2A, subjected to FLAG immunoprecipitation, and subsequently used in an in vitro kinase assay as described in Materials and Methods. Background 32P incorporation in the
absence of active RSK2 is shown in lanes 1 and 2. Lanes 3 and 4 show the incorporation
of 32P into the FLAG immunoprecipitates in the presence of active RSK2. Lane 5 shows
incorporation of 32P into the active RSK2 itself in the absence of FLAG
immunoprecipitated proteins. Shown is a representative autoradiogram from a single
experiment that has been replicated four times with equivalent results. Figure 4-8B shows
a Western blot of CFP and FLAG-5-HT2A transfected immunoprecipitates probed for
FLAG to show the apparent MW of the 5-HT2A receptor. Figure 4-8C shows a Western
blot of the active, purified RSK2 probed for RSK2 to show the apparent MW of RSK2.
169 Figure 4-8
170 4.9 The Kinase Activity of RSK2 is Essential for the 5-HT2A Receptor
Phosphoinositide Hydrolysis Phenotype
Because 5-HT2A receptors have more than 30 potential phosphorylation sites
(Gray et al., 2003b), it is was not possible at present to determine which of these represent the site(s) of RSK2 phosphorylation. Therefore, we decided instead to perform a potentially more illuminating study wherein we attempted to rescue the knock-out phenotype with either wild-type RSK2 or ‘kinase-dead’ RSK2 mutants. We made stable lines in the RSK2 -/- fibroblast background that express either an N-terminal kinase dead
RSK2 (K100A) or a C-terminal kinase dead RSK2 (K451A). These mutations remove a conserved lysine residue that is essential for ATP binding to protein kinases. However, after establishing stable lines expressing the wild-type RSK2, RSK2-K100A, and RSK2-
K451A, only with the wild-type RSK2 and the RSK2-K451A kinase dead mutant could we obtain wild-type levels of expression (Figure 4-9A). Therefore, we did not pursue functional studies with the RSK2-K100A stable cell line. We measured 5-HT2A signaling via PI hydrolysis and found that wild-type RSK2 but not the RSK2-K451A ‘kinase-dead’ mutant could rescue the signaling phenotype (Figure 4-9B, Table 4-1). These results demonstrate that the kinase activity of RSK2 is essential for the 5-HT2A receptor PI
hydrolysis phenotype we have discovered.
171
Figure 4-9 - RSK2 kinase activity is essential for exerting a “tonic brake” on 5-HT2A receptor signaling. Figure 4-9A shows the expression of RSK2 in the RSK2 -/- fibroblasts, in the RSK2 +/+ fibroblasts, in the RSK2 -/- RSK2-transfected stable line, in the RSK2 -/- RSK2-K100A-transfected stable line, and in the RSK2 -/- RSK2-K451A- transfected stable line. For Figure 4-9B, cells were plated and treated as described in
Chapter 2 and PI hydrolysis was measured. Figure 4-9B shows the 5-HT mediated dose response for phosphoinositide hydrolysis, normalized to dpm/mg total protein where
RSK2 +/+ fibroblast maximal signaling is set to 100%.
172 Figure 4-9
173
Table 4-1 - RSK2 knock-out augments 5-HT2A receptor signaling and the re-
introduction of wild-type, but not ‘kinase-dead’ RSK2 reverts the phosphoinositide
hydrolysis phenotype
Agonist Potency Relative Agonist Cell Line EC50 Efficacy (pEC50 ± SEM) Emax ± SEM
RSK2 -/- Fibroblasts 498 nM (6.30 ± 0.13) 1.72 ± 0.10*
RSK2 +/+ Fibroblasts 423 nM (6.37 ± 0.06) 0.97 ± 0.02
RSK2 -/- RSK2 Stable 368 nM (6.43 ± 0.10) 1.03 ± 0.04
RSK2 -/- RSK2-K451A Stable 494 nM (6.31 ± 0.06) 1.54 ± 0.05*
Agonist potencies (EC50) and efficacies (Emax) were determined for agonist-mediated activation of phosphoinositide hydrolysis as described in Chapter 2. pEC50 values are
represented as –log of EC50 in M. The results represent the average of four independent
experiments. *Statistically different from RSK2 +/+ fibroblasts, p<0.05
174 4.10 The Kinase Activity of RSK2 is not Essential for an Interaction with the 5-
HT2A Receptor
We also performed additional control studies to determine if the kinase-dead
mutants are able to interact with 5-HT2A receptors. These controls were essential because
it was conceivable that RSK2 could modulate 5-HT2A signaling via direct interaction with
the 5-HT2A receptor and not via phosphorylation. For these studies we utilized epitope-
tagged RSK2 (RSK2-GFP) expressed in HEK-293T cells to avoid confusion with
endogenous RSK2. As is shown in Figure 4-10, various GFP-tagged kinase-dead RSK2
mutants interact with 5-HT2A receptors in a manner equivalent to GFP-tagged wild-type
RSK2. Taken together, these results demonstrate that a direct physical interaction
between 5-HT2A receptors and RSK2 is insufficient to modulate signaling, since kinase- dead mutants can interact but not rescue the knock-out phenotype. Given that RSK2 can directly phosphorylate 5-HT2A receptors in vitro, the most logical explanation for our
results is that RSK2 modulates 5-HT2A signaling via receptor phosphorylation, although
further studies are needed to directly address this possibility.
175
Figure 4-10 - N-terminal and C-terminal kinase dead RSK2 mutants interact with
human 5-HT2A receptors in vitro. Figure 4-10 shows the co-immunoprecipitation of
RSK2-GFP, RSK2-K100A-GFP, and RSK2-K451A-GFP with FLAG-5-HT2A receptors in transfected HEK-293 cells. For these experiments, HEK-293 cells were either transiently co-transfected with FLAG-5-HT2A and CFP (lane 1), FLAG-5-HT2A and
RSK2-GFP (lane 2), FLAG-5-HT2A and RSK2-K100A-GFP (lane 3), or FLAG-5-HT2A and RSK2-K451A-GFP (lane 4). FLAG-tagged human 5-HT2A receptors were
immunoprecipitated by a monoclonal FLAG antibody conjugated to Sepharose beads.
The top panel shows the immunoprecipitates probed for GFP. The second panel from the
top shows the immunoprecipitates probed for FLAG. The third panel from the top shows
the protein lysates probed for RSK2. The bottom panel shows the protein lysates probed
for Gαq. Shown are representative immunoblots from a single experiment that has been
replicated three times with equivalent results.
176 Figure 4-10
177 4.11 Discussion
The main findings of this chapter are that (1) RSK2 exerts a ‘tonic brake’ on the
signaling of 5-HT2A receptors, constitutively attenuating signaling; (2) RSK2 can directly phosphorylate 5-HT2A receptors; and (3) that the kinase activity of RSK2 is essential for the effects of RSK2 on 5-HT2A receptor signaling. The alterations in 5-HT2A receptor
signaling must occur upstream of Gαq signaling at the level of receptor-effector coupling as there are no alterations in the signaling of a constitutively active form of Gαq,
Gαq(Q209L), signaling in the absence of RSK2. In addition, 5-HT2A receptor signaling differences are not due to alterations in the cell surface expression of 5-HT2A receptors.
Importantly, the re-introduction of wild-type but not ‘kinase-dead’ RSK2 into RSK2
knock-out fibroblasts rescued the wild–type 5-HT2A receptor signaling phenotype. Taken
together these findings describe an entirely novel mode of GPCR regulation. Given that
RSK2 can directly phosphorylate 5-HT2A receptors in vitro, the most logical explanation for our results is that RSK2 modulates 5-HT2A signaling via receptor phosphorylation,
although further studies are needed to directly address this possibility.
Previous studies with RSK2 knock-out mice have revealed an increase in basal,
insulin-, and exercise-stimulated phosphorylation of p42/44 ERK (Dufresne et al., 2001).
Dufresne et al (2001) hypothesized that the augmented basal p42/44 ERK
phosphorylation could be due to either diminished feedback inhibition of RSK2 on the
ERK-MAP kinase cascade or altered expression of one of the ERK phosphatases
(Dufresne et al., 2001). Instead, we suggest an alternative explanation that the increase in
178 basal p42/44 ERK phosphorylation induced RSK2 knock-out might be due to the removal
of a “tonic brake” on 5-HT2A receptor, and perhaps other GPCR signaling. In support of
this idea, we found that pre-treatment of RSK2 -/- fibroblasts with the 5-HT2A receptor- selective antagonist MDL100,907 lowered basal p42/44 ERK phosphorylation to levels similar to those seen in the RSK2 +/+ fibroblasts. Because the microarray data do not demonstrate alterations in the gene expression patterns or levels of the ERK phosphatases or MAPK cascade members it is unlikely that changes in the expression of various phosphatases or other kinases can explain these findings (Table 4-2 and Figure 4-4). It should be noted, of course, that microarray studies do not rule out the potential for alterations in the expression of these proteins. Regardless, these data argue for a novel role for RSK2 in maintaining the appropriate basal ERK-MAP kinase cascade activity through suppressing basal and receptor-stimulated activity of 5-HT2A receptors, and
perhaps other GPCRs.
179 Table 4-2 - Microarray analysis of mouse RSK2 +/+ and RSK2 -/- fibroblasts reveals that phosphatase expression patterns are similar
RSK2 -/- RSK2 -/- RSK2 -/- GENE GENE GENE Fibroblasts Fibroblasts Fibroblasts SYMBOL SYMBOL SYMBOL Expression Expression Expression
Acp1 = Ppm1b1 = Ppp5c = Acp2 = Ppm1b2 = Pppap2b = Akp2 = Ppm1d = Pps = CaM-Prp = Ppm1g = Ptp4a1 = dis2m2 = Ppp1c = Ptp4a2 = Dusp10 = Ppp1ca = Ptp4a3 = Dusp12 = Ppp1cb = Ptpn1 = Dusp14 = Ppp1cc = Ptpn12 = Dusp6 UP Ppp1r11 = Ptpn13 = Esp UP Ppp1r14b = Ptpn14 = Impa1 = Ppp1r2 = Ptpn16 = Impa2 = Ppp1r3c = Ptpn2 = Inpp1 = Ppp1r7 = Ptpn21 = Inpp5b = Ppp2ca = Ptpn9 = Inpp5e = Ppp2cb = Ptpra = Inppl1 = Ppp2r1a = Ptpre DOWN Minpp1 = Ppp2r3a = Ptprf = Mtmr4 = Ppp2r4 = Ptprg = P19-Ptp = Ppp2r5c = Ptprj UP Pp2ca1b = Ppp3c = Ptprk UP Ppap2a = Ppp3ca = Ptprm = Ppap2c = Ppp3cb = Ptprn = Ppm1a = Ppp3cc = Ptprs = Ppm1b = Ppp4c = Spph1 UP
Shown are the gene symbols for various expressed phosphatase mRNAs identified by microarray analysis of mouse RSK2 +/+ and -/- fibroblast mRNA (See Chapter 2 for details). Differences in the microarray data of the RSK2 -/- fibroblasts from the RSK2
+/+ fibroblasts are as indicated: =; equivalent expression, UP; RSK2 -/- expression more than 2-fold greater than +/+ fibroblasts, DOWN; RSK2 -/- expression more than 2-fold less than RSK2 +/+ fibroblasts.
180 As described in Chapter 1, the kinases that participate in the phosphorylation of
the 5-HT2A receptor have proven elusive, despite the presence of predicted phosphorylation sites and the identification of serine and threonine residues involved in desensitization (Gray et al., 2003b). To date, no agonist directed phosphorylation of the
5-HT2A receptor has been reported despite continued study (Gray et al., 2003b; Gray and
Roth, 2001; Vouret-Craviari et al., 1995). Indeed, the association of RSK2 with the 5-
HT2A receptor appears to be agonist independent, as agonist stimulation has no effect on the co-immunoprecipitation of RSK2 with epitope tagged 5-HT2A receptors as demonstrated in Chapter 3. It is therefore possible that RSK2 constitutively phosphorylates the 5-HT2A receptor, a hypothesis in keeping with our findings showing that RSK2 can directly phosphorylate 5-HT2A receptors in vitro. Hypothetically, since 5-
HT efficacy is increased in RSK2 knock-out cells, the possibility exists that constitutive
phosphorylation of the 5-HT2A receptor may interfere with G protein-coupling efficiency, leading to a “pre-desensitized” state of the receptor.
Since 5-HT2A receptors have more than 30 potential phosphorylation sites (Gray et al., 2003b) it was not possible within these current studies to determine which of these represent the site(s) of RSK2 phosphorylation. In addition, since 5-HT2A receptors show
high levels of constitutive phosphorylation (presumably due to multiple kinases (Gray et
al., 2003b)), it has also been impossible to measure any alteration in basal 5-HT2A phosphorylation in RSK2 -/- cells. Therefore, we are currently investigating whether the alterations in 5-HT2A receptor signaling are due to differences in RSK2-mediated receptor phosphorylation through a combination of mutagenesis, in vitro kinase assays, and
181 functional studies. Determination of the site(s) of 5-HT2A receptor phosphorylation by
RSK2 and other kinases will further the understanding of 5-HT2A receptor regulation and may provide additional targets for the design of novel therapeutic drugs.
Individuals with Coffin-Lowry Syndrome have an increased risk of cardiac abnormalities, including mitral, tricuspid, and aortic valve abnormalities, pulmonary and aortic root dilation, and cardiomyopathy, suggesting an indispensable role for RSK2 in cardiovascular function (Hunter, 2002). Interestingly, as described in Chapter 1, the 5-
HT2A receptor is highly expressed in vascular smooth muscle, pulmonary artery, and cardiac tissues. Since RSK2 deletion augments 5-HT2A receptor signaling, our findings imply that the cardiovascular abnormalities in patients with Coffin-Lowry Syndrome may be due to a hyperactivation of 5-HT2A receptor signaling.
In summary, RSK2 has emerged as a novel regulator of 5-HT2A receptors, exerting a “tonic brake” on signaling. Inactivating mutations of RSK2 lead to mental retardation, skeletal muscle deformities, and psychosis. Intriguingly, GPCR signaling has been implicated in signaling pathways that regulate brain, bone, and skeletal muscle development (Cheung et al., 2003; Chinni et al., 1999; de Niese et al., 2002; Pagel et al.,
2003; Pierce et al., 2002). Therefore, it will prove interesting to determine the role, if any, of RSK2 in the regulation of other GPCRs. The following chapter will summarize the initial characterization of the effects of RSK2 on the signaling of PAR-1 thrombin, P2Y- purinergic, bradykinin-B, and β1-adrenergic receptors in the RSK2 -/- and RSK2 +/+ fibroblasts. Given the phenotype of Coffin-Lowry Syndrome, as described in Chapter 1,
182 it is likely that the selective alteration of 5-HT2A receptor, and perhaps other GPCR signaling induced by genetic deletion or inactivation of RSK2 plays a role in the pathogenesis of Coffin-Lowry Syndrome, and may be involved in other disorders manifested by dysmorphogenesis, psychosis, and/or mental retardation.
183 CHAPTER 5: The Role of p90 Ribosomal S6 Kinase 2 (RSK2)
in G Protein-Coupled Receptor (GPCR) Signal Transduction
5.1 Introduction and Rationale
As discussed in Chapters 3-4, we discovered that RSK2 interacts with the 5-
HT2A receptor and plays a novel role in regulating 5-HT2A receptor signal transduction.
Due to our discovery, we wanted to initially characterize what role, if any, RSK2 plays in the regulation of other GPCRs. As described in Chapter 4, we conducted a microarray study to evaluate the expressed genes in both RSK2 -/- and RSK2 +/+ fibroblasts. We conducted these microarray studies in order to demonstrate whether alterations in 5-HT2A receptor signaling are due to aberrant gene expression in serotonergic signaling genes, in members of the MAPK cascade, or in phosphatase expression (Chapter 4). These microarray data also provide a wealth of other information (See Chapter 6 for details), including the identification of GPCRs expressed in both RSK2 -/- and RSK2 +/+ fibroblasts (Table 5-1). These data allowed us to examine the role of RSK2 in the signaling of other GPCRs expressed in RSK2 -/- and RSK2 +/+ fibroblasts. We initially focused on other Gαq-coupled GPCRs including the PAR-1-thrombinergic, P2Y- purinergic, and Bradykinin-B receptors. We evaluated these receptors for agonist-induced
PI hydrolysis and calcium mobilization.
184
Table 5-1 - Microarray analysis of RSK2 +/+ and RSK2 -/- fibroblasts reveals the expression of selected GPCRs
GENE GENE TITLE SYMBOL 5-hydroxytryptamine (serotonin) receptor 1A Htr1a angiotensin II receptor, type 2 Agtr2 arginine vasopressin receptor 1A Avpr1a beta 1 adrenergic receptor Adrb1 bradykinin receptor, beta Bdkrb chemokine (C-X-C) receptor 3 Cmkar3 coagulation factor II (thrombin) receptor F2r coagulation factor II (thrombin) receptor-like 2 F2rl2 endothelial differentiation sphingolipid GPCR 1 Edg1 endothelial differentiation, lysophosphatidic acid GPCR 2 Edg2 endothelial differentiation, sphingolipid GPCR 3 Edg3 endothelin receptor type B Ednrb G protein-coupled receptor 19 Gpr19 G protein-coupled receptor 56 Gpr56 G protein-coupled receptor 73 Gpr73 G protein-coupled receptor 85 Gpr85 gastrin releasing peptide receptor Grpr G-protein coupled receptor 88 Gpr88 melanocortin 2 receptor Mc2r melanocortin 3 receptor Mc3r neuropeptide Y receptor Y1 Npy1r neuropeptide Y receptor Y5 Npy5r prostaglandin E receptor 1 (subtype EP1) Ptger1 prostaglandin E receptor 4 (subtype EP4) Ptger4 prostaglandin F receptor Ptgfr prostaglandin I receptor (IP) Ptgir
Shown are the Gene Title and Gene symbols for various GPCRs identified by microarray analysis of mouse RSK2 +/+ and -/- fibroblast mRNA as being present in both cell lines
(See Appendix for Details)
185 5.2 RSK2 Attenuates Gαq-Coupled GPCR Signaling to Phosphoinositide
Hydrolysis
As previously described, microarray studies were used to identify GPCRs expressed in both RSK2 +/+ and -/- cell lines (Table 5-1). Knowledge of the expressed
GPCRs allowed us to examine three other Gαq-coupled GPCRs that were expressed in both cell lines: PAR-1 thrombin receptors, bradykinin-B receptors, and P2Y-purinergic receptors. Agonist-stimulated PI hydrolysis was tested for each of these receptors, using the following agonists: thrombin receptor activating peptide (TRAP) (PAR-1 thrombin receptors), bradykinin (bradykinin-B receptors), and ATP (P2Y-purinergic receptors)
(Figure 5-1). For these studies, we also made use of the wild-type RSK2 stable line that we had created in the RSK2 -/- fibroblast background (Chapter 4) to determine if the signaling differences between RSK2 +/+ fibroblasts and RSK2 -/- fibroblasts were due strictly to the absence of RSK2 in the knock-out cells and not mouse strain background differences nor ‘phenotypic drift’ among cell lines (Figure 5-1).
Upon measuring agonist-mediated PI hydrolysis (See Chapter 2 for details), we found that RSK2 -/- fibroblasts show an augmentation of PAR-1-thrombinergic, bradykinin-B, and P2Y-purinergic receptor-mediated signaling compared to RSK2 +/+ fibroblasts (Figure 5-1 and Table 5-2), similar that found for 5-HT2A receptors (Chapter
4). Agonist potency was left-shifted in RSK2 -/- fibroblasts and in RSK2 -/- RSK2 stable
lines for PAR-1 thrombin and Bradykinin B receptors (Table 5-2). However, for the
P2Y-purinergic receptor, agonist potency was not significantly altered (Table 5-2). Re-
186 introduction of RSK2 into the RSK2 -/- background at least partially reversed the PI hydrolysis phenotype for PAR-1-thrombinergic, bradykinin-B, and P2Y-purinergic receptor-mediated signaling (Figure 5-1). Thus, deletion of RSK2 augments signaling to phosphoinositide hydrolysis for all tested Gαq-coupled GPCRs while re-introduction of
RSK2 diminishes Gαq-mediated signaling.
187
Figure 5-1 - RSK2 exerts a “tonic brake” on Gαq-coupled GPCR signaling. For these experiments, RSK2 +/+ and -/- fibroblasts were plated and treated as described in
Chapter 2 and PI hydrolysis was measured. Figures 5-1A-C show the sigmoidal dose responses to various agonists, normalized to dpm/mg total protein where RSK2 +/+ fibroblast maximal signaling is set to 100%. Figure 5-1A shows the TRAP mediated dose response for phosphoinositide hydrolysis for RSK2 -/- fibroblasts, RSK2 +/+ fibroblasts, and the RSK2 -/- RSK2 stable line. Figure 5-1B shows the bradykinin mediated dose response for phosphoinositide hydrolysis for RSK2 -/- fibroblasts, RSK2 +/+ fibroblasts, and the RSK2 -/- RSK2 stable line. Figure 5-1C shows the ATP mediated dose response for phosphoinositide hydrolysis for RSK2 -/- fibroblasts, RSK2 +/+ fibroblasts, and the
RSK2 -/- RSK2 stable line.
188 Figure 5-1
189 Table 5-2 - RSK2 knock-out augments Gαq-coupled GPCR signaling to phosphoinositide hydrolysis
Agonist Potency Relative Agonist Efficacy PAR-1 EC50 (pEC50 ± SEM) Emax ± SEM
RSK2 -/- Fibroblasts 8.6 µM (5.07 ± 0.10)* 11.77 ± 0.70*
RSK2 +/+ Fibroblasts 16.9 µM (4.78 ± 0.09) 1.16 ± 0.08
RSK2 -/- RSK2 Stable 6.3 µM (5.21 ± 0.06)* 8.37 ± 0.24*
Agonist Potency Relative Agonist Efficacy P2Y EC50 (pEC50 ± SEM) Emax ± SEM
RSK2 -/- Fibroblasts 8.6 µM (5.06 ± 0.24) 3.22 ± 0.34*
RSK2 +/+ Fibroblasts 6.0 µM (5.22 ± 0.09) 0.98 ± 0.04
RSK2 -/- RSK2 Stable 6.2 µM (5.21 ± 0.22) 1.83 ± 0.16*
Agonist Potency Relative Agonist Efficacy Bradykinin B EC50 (pEC50 ± SEM) Emax ± SEM
RSK2 -/- Fibroblasts 0.19 nM (9.72 ± 0.40)* 3.83 ± 0.40*
RSK2 +/+ Fibroblasts 1.31 nM (8.88 ± 0.11) 0.91 ± 0.11
RSK2 -/- RSK2 Stable 0.26 nM (9.59 ± 0.27)* 1.06 ± 0.11
Agonist potencies (EC50) and efficacies (Emax) were determined for agonist-mediated activation of phosphoinositide hydrolysis as described in Chapter 2. pEC50 values are
represented as –log of EC50 in M. The results represent the average of four independent
experiments. *Statistically different from RSK2 +/+ fibroblasts, p<0.05
190 5.3 RSK2 Alters Gαq-Coupled GPCR Signaling as Measured by Calcium
Mobilization
We next wanted to examine the effects of RSK2 knock-out on agonist-mediated calcium mobilization for Gαq-coupled GPCRs. Similar to the PI hydrolysis studies, we tested PAR-1-thrombinergic, Bradykinin-B, and P2Y-purinergic receptor-mediated signaling. As shown in Figure 5-2 and Table 5-3, the effects of RSK2 on PAR-1-
thrombinergic, P2Y-purinergic, and Bradykinin-B receptor signaling to calcium
mobilization were far more variable than the effects on phosphoinositide hydrolysis.
PAR-1 thrombin receptors and P2Y-purinergic receptors showed an augmentation of
maximal calcium mobilization that was partially reverted (PAR-1) or fully reverted
(P2Y) by the re-introduction of RSK2 into the RSK2 -/- background (Figure 5-2, Table
5-3). In addition, both PAR-1 and P2Y receptors displayed a left-shift in agonist potency
in the absence of RSK2 that was partially reverted by the re-introduction of RSK2 into
the RSK2 -/- background (Table 5-3). However, Bradykinin-B receptors demonstrated an
attenuation of maximal calcium mobilization in the absence of RSK2; this attenuation
was reverted by the re-introduction of RSK2 into the RSK2 -/- background (Figure 5-2,
Table 5-3). The Bradykinin-B receptors did not demonstrate any alteration in agonist
potency (Table 5-3).
191
Figure 5-2 - RSK2 knock-out alters the signaling of PAR-1-thrombinergic, P2Y-
purinergic, and Bradykinin-B receptors as measured by calcium mobilization. For these studies, RSK2 +/+ fibroblasts, RSK2 -/- fibroblasts, and RSK2 -/- RSK2 stable line fibroblasts were plated onto 96-well plates. Cells were serum-starved, treated with agonist, and calcium mobilization was measured as described in Chapter 2. Figure 5-2A shows the TRAP-mediated dose response for PAR-1 receptor calcium mobilization in
RSK2 -/-, RSK2 +/+, and RSK2 -/- RSK2 stable fibroblasts given in RFU normalized to
RSK2 +/+ fibroblast signaling. Figure 5-2B shows a representative tracing of the changes in fluorescence of RSK2 -/-, RSK2 +/+, and RSK2 -/- RSK2 stable fibroblasts upon the addition of EC50 concentrations of TRAP. Figure 5-2C shows the ATP-mediated dose response for P2Y receptor calcium mobilization in RSK2 -/-, RSK2 +/+, and RSK2
-/- RSK2 stable fibroblasts given in RFU normalized to RSK2 +/+ fibroblast signaling.
Figure 5-2D shows a representative tracing of the changes in fluorescence of RSK2 -/-,
RSK2 +/+, and RSK2 -/- RSK2 stable fibroblasts upon the addition of EC50 concentrations of ATP. Figure 5-2E shows the bradykinin-mediated dose response for
Bradykinin-B receptor calcium mobilization in RSK2 -/-, RSK2 +/+, and RSK2 -/- RSK2 stable fibroblasts given in RFU normalized to RSK2 +/+ fibroblast signaling. Figure 5-
2F shows a representative tracing of the changes in fluorescence of RSK2 -/-, RSK2 +/+, and RSK2 -/- RSK2 stable fibroblasts upon the addition of EC50 concentrations of
bradykinin.
192 Figure 5-2
193 Table 5-3 - RSK2 knock-out alters Gαq-coupled GPCR signaling to calcium
mobilization
Agonist Potency Relative Agonist PAR-1 EC50 (pEC50 ± SEM) Efficacy Emax ± SEM
RSK2 -/- Fibroblasts 371 nM (6.43 ± 0.05)* 1.33 ± 0.02*
RSK2 +/+ Fibroblasts 922 nM (6.04 ± 0.06) 1.00 ± 0.03
RSK2 -/- RSK2 Stable 630 nM (6.20 ± 0.11) 1.26 ± 0.05* Agonist Potency Relative Agonist P2Y EC50 (pEC50 ± SEM) Efficacy Emax ± SEM
RSK2 -/- Fibroblasts 28.4 µM (4.55 ± 0.05)* 1.17 ± 0.03*
RSK2 +/+ Fibroblasts 80.6 µM (4.09 ± 0.05) 1.07 ± 0.03
RSK2 -/- RSK2 Stable 69.8 µM (4.16 ± 0.07) 0.76 ± 0.03* Agonist Potency Relative Agonist Bradykinin B EC50 (pEC50 ± SEM) Efficacy Emax ± SEM
RSK2 -/- Fibroblasts 8.1 nM (8.09 ± 0.05) 0.74 ± 0.03*
RSK2 +/+ Fibroblasts 6.4 nM (8.20 ± 0.05) 1.09 ± 0.04
RSK2 -/- RSK2 Stable 9.3 nM (8.03 ± 0.07) 1.06 ± 0.06
Agonist potencies (EC50) and efficacies (Emax) were determined for agonist-mediated activation of phosphoinositide hydrolysis as described in Chapter 2. pEC50 values are
represented as –log of EC50 in M. The results represent the average of four independent
experiments. *Statistically different from RSK2 +/+ fibroblasts, p<0.05
194 5.4 RSK2 Knock-out Augments the Signaling of β1-Adrenergic Receptors
As shown in Chapter 4, the absence of RSK2 was found to augment 5-HT2A receptor signaling to PI hydrolysis and to calcium mobilization. Initial evaluation of other
Gαq-coupled GPCR signaling demonstrated that PAR-1, P2Y, and Bradykinin-B receptor
signaling to PI hydrolysis were augmented in the absence of RSK2. In addition, PAR-1
and P2Y receptor signaling to calcium mobilization were also demonstrated to be
augmented in the absence of RSK2. Therefore, RSK2 appeared to emerge as a general
regulator of Gαq-coupled GPCR signal transduction. To determine if the effects on GPCR
signaling were restricted to Gαq-coupled GPCRs, we performed adenylate cyclase assays for the β1-adrenergic receptor, a Gαs-coupled GPCR, which is expressed in RSK2 -/- and
RSK2 +/+ fibroblasts (Table 5-1). We found that isoproterenol-stimulated cAMP
production was also augmented in the RSK2 -/- fibroblasts compared to the RSK2 +/+
fibroblasts (Figure 5-3). The isoproterenol-stimulated RSK2 -/- fibroblasts demonstrated
an Emax of 55.8 ± 3.5 % of the forskolin response in this cell line, whereas isoproterenol- stimulated RSK2 +/+ fibroblasts demonstrated an Emax of 33.4 ± 1.7 % of the forskolin
response in this cell line (Figure 5-3). These data indicate that the effects of RSK2 on
GPCR signaling are not limited to Gαq-coupled GPCRs.
195
Figure 5-3 - RSK2 knock-out augments β1-adrenergic signaling. Figure 5-3 shows the isoproterenol-stimulated dose response of cAMP production for RSK2 -/- and RSK2 +/+ fibroblasts. Data are presented as the % of the forskolin response (defined as 100% response) in the RSK2 -/- and RSK2 +/+ fibroblasts.
196 Figure 5-3
197 5.5 Discussion
In summary, the findings of this chapter are that (1) RSK2 exerts a ‘tonic brake’
on the PI hydrolysis signaling of all Gαq-coupled GPCRs thus far examined; (2) RSK2 exerts a ‘tonic brake’ on the calcium signaling of the PAR-1-thrombinergic and P2Y- purinergic receptors; (3) RSK2 exerts a ‘tonic brake’ on β1-adrenergic receptor signaling to cAMP production; and (4) RSK2 knock-out has variable effects on GPCR agonist potency. Taken together these findings describe an entirely novel mode of GPCR regulation. The studies in this chapter only provide an initial characterization of the role of RSK2 in PAR-1-thrombinergic, P2Y-purinergic, Bradykinin-B, and β1-adrenergic receptor signaling. Additional studies will be necessary to further characterize these effects.
The signaling of Gαq-coupled GPCRs followed one of three trends in the absence of RSK2 (Table 5-4). The first trend is that demonstrated by the 5-HT2A receptor and by
the P2Y-purinergic receptor. These receptors demonstrated an increase in PI hydrolysis
and calcium mobilization Emax that was reverted by the addition of RSK2 back into the
RSK2 -/- background (Table 5-4). The 5-HT2A and P2Y receptors also demonstrated no change in PI hydrolysis agonist potency, but they show and a left-shift in agonist potency
for calcium mobilization that was reverted by the addition of RSK2 back into the RSK2 -
/- background (Table 5-4). The second trend was shown by the PAR-1 thrombin
receptor, which differed from the 5-HT2A and P2Y receptors by demonstrating a left-shift
in both PI hydrolysis and calcium mobilization agonist potency with only the calcium
198 mobilization potency showing a phenotype reversion by the addition of RSK2 back into the RSK2 -/- background (Table 5-4). Bradykinin-B receptors, on the other hand, displayed a number of discrepancies. These receptors showed a left-shift in potency and an increase in PI hydrolysis Emax that was reverted by re-addition of RSK2. However, the calcium signaling showed a reverse phenotype, where the Emax was decreased in the absence of RSK2 and this decrease was reverted by the re-addition of RSK2. Since three different patterns were discovered among the Gαq-coupled GPCRs tested, there must be differences in the role of RSK2 in the signaling of these GPCRs.
199
Table 5-4 – A summary of Gαq-coupled GPCR functional studies using endogenous
GPCR ligands
PI PI Ca2+ Ca2+ Receptor Hydrolysis Hydrolysis Mobilization Mobilization EC50 Emax EC50 Emax
5-HT2A NC ↑ R ← R ↑ R
PAR-1 ← NR ↑ R ← R ↑ R
P2Y NC ↑ R ← R ↑ R
Bradykinin B ← R ↑ R NC ↓ R
A summary of the trends for EC50 and Emax values for both phosphoinositide (PI) hydrolysis and calcium mobilization studies. NC = no change among RSK2 -/-, RSK2
+/+, and RSK2 -/- RSK2 stable fibroblasts; ↑ = RSK2 -/- fibroblasts show an increase in
Emax compared to RSK2 +/+ fibroblasts; ↓ = RSK2 -/- fibroblasts show an decrease in
Emax compared to RSK2 +/+ fibroblasts; ← left shift in agonist potency in RSK2 -/- fibroblasts compared to RSK2 +/+ fibroblasts; R = RSK2 -/- RSK2 stable fibroblasts revert the phenotype toward RSK2 +/+ fibroblasts; NR = RSK2 -/- RSK2 stable fibroblasts do not revert the phenotype toward RSK2 +/+ fibroblasts.
200 We have previously shown that active RSK2 can phosphorylate the 5-HT2A receptor and that the kinase activity of RSK2 is necessary to rescue the 5-HT2A receptor
PI hydrolysis phenotype (Chapter 4). In these studies, we measured 5-HT2A receptor expression, the cell surface expression of 5-HT2A receptors, and performed studies using
a constitutively active form of Gαq, Gαq(Q209L) (Chapter 4). Together, these studies
demonstrated that the alterations in 5-HT2A receptor signaling were not due to alterations in receptor expression or cell surface expression of 5-HT2A receptors. In addition, the
Gαq(Q209L) studies demonstrated that alterations in signaling occur upstream of Gαq, at
the level of the receptor (Chapter 4). As described above, the GPCRs screened show
variations in the phenotype for PI hydrolysis and for calcium mobilization. Therefore, in
order to determine the role of RSK2 in PAR-1, P2Y, Bradykinin-B, and β1-adrenergic
receptor signaling, it will be necessary to conduct additional studies with these GPCRs,
similar to those conducted with the 5-HT2A receptor in order to rule-out non-specific effects of RSK2 knock-out.
As discussed in Chapter 1, individuals with Coffin-Lowry Syndrome have an increased risk of cardiac abnormalities and cardiomyopathy, suggesting an indispensable role for RSK2 in cardiovascular function (Hunter, 2002). PAR-1 thrombin receptors are highly expressed in the heart and have many cardiovascular functions (Steinberg, 2005).
Since RSK2 deletion augments PAR-1 signaling, our findings imply that the cardiovascular abnormalities in patients with Coffin-Lowry Syndrome may be due to a hyperactivation of PAR-1 receptor signaling. As such, further studies into the role of
RSK2 in PAR-1 receptor regulation are warranted. In addition, as stated in Chapter 4,
201 GPCR signaling has also been implicated in signaling pathways that regulate brain, bone,
and skeletal muscle development (Cheung et al., 2003; Chinni et al., 1999; de Niese et al., 2002; Pagel et al., 2003; Pierce et al., 2002). The initial studies described in the chapter point to a novel role or RSK2 in the regulation of GPCR activity; a role that has only begun to be appreciated. It is likely that the selective alteration of GPCR signaling induced by genetic deletion or inactivation of RSK2 plays a role in the pathogenesis of
Coffin-Lowry Syndrome. Understanding the role of RSK2 in these processes can advance our understanding of the pathology of Coffin-Lowry Syndrome.
202 CHAPTER 6: Microarray and Pathway Analysis of Genes
Expressed in RSK2 -/- and in RSK2 +/+ Fibroblasts
6.1 Introduction and Rationale
As discussed in Chapter 3, RSK2 associates with 5-HT2A receptors in vivo and in
vitro. The examination of RSK2 knock-out fibroblasts lead to the discovery that 5-HT2A receptors display augmented signaling. These alterations in 5-HT2A receptor signaling include an augmentation of PI hydrolysis, augmented Ca2+ mobilization, and an augmentation in both basal and 5-HT-stimulated p42/p44 ERK phosphorylation
(Chapter 4). This 5-HT2A receptor phosphoinositide hydrolysis phenotype was reverted
by reintroducing wild-type but not ‘kinase-dead’ RSK2 into the RSK2 knock-out
fibroblasts. The augmentation of signaling was determined to occur upstream of G-
protein signaling as demonstrated by the lack of an effect on the signaling of
Gαq(Q209L), a constitutively active form of Gαq, in the absence of RSK2. Finally, we
determined that purified active RSK2 phosphorylates the 5-HT2A receptor in vitro and
that the kinase activity of RSK2 is not necessary for the interaction of RSK2 with the 5-
HT2A receptor.
We also demonstrated that RSK2 knock-out augments adenosine 5’-triphosphate
(ATP)-stimulated P2Y-purinergic receptor, bradykinin-stimulated bradykinin-B receptor,
and thrombin receptor activating peptide (TRAP)-stimulated PAR-1 thrombin receptor
signaling, as measured by PI hydrolysis (Chapter 5). The augmentation of GPCR
203 signaling in the absence of RSK2 was not restricted to Gαq-coupled GPCRs.
Isoproterenol-stimulated β1-adrenergic receptor signaling, a Gαs-coupled GPCR, was augmented as well in the RSK2 knock-out fibroblasts (Chapter 5). Together, our
findings demonstrate that RSK2 knock-out appears to remove a “tonic brake” on GPCR
signaling.
As previously discussed in Chapter 4, quantitative RT-PCR studies had revealed
that 5-HT2A receptor mRNA expression was unaltered and that no change in the cell surface expression of the 5-HT2A receptors were measured by cell surface-biotinylation
studies. In addition, saturation binding studies indicated similar levels of expression of
the 5-HT2A receptor in the absence and presence of RSK2. To further evaluate the effects
of RSK2 knock-out and to gain further insight into the augmentation of 5-HT2A, P2Y,
Bradykinin-B, and PAR-1 receptor signaling, we performed microarray analysis and
pathway analysis on RSK2 +/+ fibroblasts and RSK2 -/- fibroblasts. The microarrray analysis revealed that RSK2 -/- fibroblasts do not exhibit global changes in gene expression compared to RSK2 +/+ fibroblasts (Figure 6-1), although there were selected
gene expression changes.
204
Figure 6-1 - RSK2 +/+ and -/- fibroblasts have few differences in global gene expression. Figure 6-1 shows the full microarray data of expressed gene probes plotted as RSK +/+ Fibroblast mean signal vs. RSK2 -/- Fibroblast mean signal. The microarray data demonstrated the expression of 13,320 gene probes in both RSK2 -/- and RSK2 +/+ fibroblasts. A linear regression of the data set is shown in blue as are theoretical lines showing the cut-off for greater than a 2-fold change in gene expression (red) and for greater than 10-fold change in gene expression (grey).
205 Figure 6-1
206 6.2 Gene Expression Profiles of Serotonin Receptors and GPCR Signaling
Proteins
It has been previously discussed in Chapter 4 that the microarray data revealed
that there were no alterations in gene expression of the proteins involved in Gαq-mediated signaling (Chapter 4, Figure 4-3). Therefore, differences in the signaling of Gαq-coupled
GPCRs, as shown in Chapters 3-5, are not due to an alteration of the expression of signaling cascade members downstream of receptor / effector coupling. Alternatively, a few proteins involved in other G-protein signaling pathways displayed alterations in gene expression. The Gβ-subunit Gnb4, the Gαi-subunit Gnai1, and the cAMP phosphodiesterase Pde8a all show down-regulation in the absence of RSK2. For these gene probes, Gnb4 displayed a 4.91-fold lower expression, Gnai1 displayed a 6.70-fold lower expression, and Pde8a displayed a 2.32-fold lower expression in the RSK2 -/- fibroblasts.
Importantly, no other serotonin receptors, specifically those in the 5-HT2 family, were found to be expressed in either the RSK2 -/- or RSK2 +/+ fibroblasts (Figure 6-2).
In addition, only one monoamine GPCR, the β1-adrenergic receptor, was shown to be
expressed (gene symbol Adrb1). It should be noted that the Affymetrix Mouse
Expression 400A chip used for these microarray studies does not contain a probe set for
the 5-HT2A receptor (gene symbol HTR2A), but that quantitative RT-PCR studies and saturation binding studies revealed equivalent expression of 5-HT2A receptors as
demonstrated in Chapter 4. On the other hand, the Affymetrix Mouse Expression 400A
chip does have probe sets for the 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2B, 5-HT2C, 5-
207 HT5A, 5-HT5B, 5-HT6, and 5-HT7 receptors, none of which are expressed in either RSK2 -
/- or RSK2 +/+ fibroblasts.
208
Figure 6-2 - RSK2 +/+ and -/- fibroblasts express few monoamine GPCRs. Figure 6-2
shows the microarray data of monoamine GPCRs overlaid with gene-expression color
criterion and fold-changes from the programs GenMAPP and MAPPFinder. Gray colored
genes are those expressed in RSK2 -/- and RSK2 +/+ fibroblasts that show no change in gene expression. White colored genes are not expressed in either RSK2 -/- or RSK2 +/+ fibroblasts. Green colored genes are those genes in the RSK2 -/- fibroblasts that show greater than a 2-fold increase in expression over the RSK2 +/+ fibroblasts. Red colored genes are those genes in the RSK2 -/- fibroblasts that show greater than a 2-fold decrease in expression compared the RSK2 +/+ fibroblasts.
209 Figure 6-2
210 6.3 Gene Expression Profiles of Mitogen-Activated Protein Kinase Cascade
Members
As noted in Chapter 4, an increase in both the basal and 5-HT-stimulated p42/p44 ERK phosphorylation was demonstrated in the absence of RSK2. As stated in
Chapter 4, no alterations were found in the expression patterns of members of the ERK
MAPK cascade (Chapter 4, Figure 4-4). Therefore, the increase in ERK phosphorylation that is displayed in the absence of RSK2 is not due to alterations in the expression of ERK cascade members. Taken together, these microarray data imply that the changes in 5-HT2A receptor signaling are a direct consequence of the absence of
RSK2 and not due to an extraneous dysregulation of the gene expression of proteins associated with 5-HT2A receptor signaling. However, the protein levels of members of
the MAPK cascade were not directly measured so it is unknown if alterations in
expression occur at the protein level.
6.4 Gene Expression Profile of the p90 Ribosomal S6 Kinases (RSKs)
The basis of our study, as discussed in Chapters 3-5, is that the augmentation of
signal transduction seen with 5-HT2A receptors, P2Y-purinergic receptors, PAR-1
thrombin receptors, bradykinin-B receptors, and β1-adrenergic receptors is a
consequence of RSK2 knock-out in the RSK2 -/- fibroblasts. By Western analysis, RSK2
is absent in the RSK2 -/- fibroblasts, as has been demonstrated in Chapter 4. The RSK2 -
/- mice were created by the introduction of a neomycin-resistance cassette into the N-
211 terminal domain of the RSK2 gene, causing the insertion of multiple stop codons
(Dufresne et al., 2001). The RSK2 -/- fibroblasts we obtained were created from these mice (Bruning et al., 2000) and we wanted to ascertain whether the loss in RSK2 protein expression was indeed a consequence of the targeted disruption of the RSK2 gene. In addition, we wanted to compare the expression of the other RSK isoforms, RSK1 and
RSK3, in the RSK2 -/- and RSK2 +/+ fibroblasts. We found that RSK2 (Gene Symbol
Rps6ka3) mRNA was down-regulated 6-fold in the RSK2 -/- fibroblasts. Additionally
RSK1 (Gene Symbol Rps6ka1) and RSK3 (Gene Symbol Rps6ka2) mRNA levels were also down-regulated to a slight extent in the RSK2 -/- fibroblasts (1.3-fold and 1.8-fold respectively), implying that the RSK kinases may be coordinately regulated in their gene expression or that these kinases may play a feedback role on the expression of other members of the RSK family of protein kinases. The slight down-regulation of RSK1 in the absence of RSK2 has been confirmed via Western blot as shown in Chapter 4.
The purpose of the following portion of this chapter is to systematically discuss genes and pathways dysregulated in their expression patterns between the RSK2 -/- and
RSK2 +/+ fibroblasts. The focus of these studies will be on pathways where there are changes in gene expression in the absence of RSK2. Genes and pathways discussed will include: GPCRs, enzymes involved in biogenic amine synthesis, cell cycle control pathways, and insulin signaling pathways. The gene expression changes in these pathways can provide insight into RSK2’s function in regulating these pathways in the cell. In addition, a variety of the changes in gene expression seen in these pathways would have been predicted given the known targets of RSK2, which will be discussed
212 where applicable. In addition to the genes in these pathways, there are a number of genes
whose expression is greatly altered between the RSK2 -/- and RSK2 +/+ fibroblasts.
Down-regulated genes include: secreted phosphoprotein-1 (Spp1), (general receptor for
phosphoinositides)-associated scaffold protein (GRASP), and Apolipoprotein D (ApoD),
among others, whereas up-regulated genes include: bone-morphogenic protein 4 (Bmp4)
and lysozyme, among others (see appendix for full expression data). The role of RSK2
in regulating the expression of such genes will require additional investigation.
6.5 Gene expression profile of the G Protein-Coupled Receptors
The GPCRs, as described in Chapter 1, are classified into several classes based upon homology and conserved features: The rhodopsin superfamily (Class A), the secretin receptor superfamily (Class B), and the metabotropic glutamate receptor superfamily (Class C). There are a number of GPCRs that are expressed in RSK2 -/- and
RSK2 +/+ fibroblasts, including GPCRs from Class A, B, and C (Table 6-1). Of the expressed GPCRs, there are a number of GPCRs that show alterations in gene expression in the absence of RSK2. These GPCRs will be discussed in the following section in context of their function and whether or not there is any known relation to the function of
RSK2. Class A GPCRs including the adenosine A2B receptor, endothelin receptor type
A, proteinase activated receptor 1, melanocortin-2 receptor, and prostaglandin E receptor
4, subtype EP4 will be discussed. In addition, the Class B GPCRs CD97 antigen, calcitonin gene-related peptide type 1 receptor and parathyroid hormone receptor 1 will
213 be discussed. No alterations were found in Class C GPCR expression, and as such, they will not be discussed (Table 6-1).
214 Table 6-1 – Microarray analyses reveal dysregulation of GPCR expression
CLASS A GPCRs GENE GENE RSK2 -/- SYMBOL INFORMATION EXPRESSION
Adrb1 Beta-1 adrenergic receptor = Adora2b Adenosine A2b receptor ↑ (2.73) Agtr2 Type-2 angiotensin II receptor (AT2) = Cbn1 Cannabinoid receptor 1 (CB1) (CB-R) = Cxcr3 C-X-C chemokine receptor type 3 = Dfy Duffy antigen/chemokine receptor = Edg1 Sphingosine 1-phosphate receptor (Lysophospholipid receptor B1) = Ednra Endothelin receptor type A (ET-AR) ↑ (5.40) F2r Proteinase activated receptor 1 precursor (PAR-1) ↑ (2.13) F2rl2 Proteinase activated receptor 3 precursor (PAR-3) = Gpr73 Prokineticin receptor 1 (PK-R1) = Mc2r Melanocortin-2 receptor (MC2-R) ↑ (4.23) Npy1r Neuropeptide Y receptor type 1 (NPY1-R) = Ptger1 Prostaglandin E receptor 1 (subtype EP1) = Ptger4 Prostaglandin E receptor 4 (subtype EP4) ↓ (2.01) Ptgir Prostaglandin I receptor (IP) = Rho Rhodopsin = Tbxa2r Thromboxane A2 receptor =
CLASS B GPCRs GENE GENE RSK2 -/- SYMBOL INFORMATION EXPRESSION
Calclr Calcitonin gene-related peptide type 1 (CGRP) ↑ (2.28) Cd97 CD97 antigen ↑ (2.14) Pthr1 Parathyroid hormone receptor 1 ↓ (4.18)
CLASS C GPCRs GENE GENE RSK2 -/- SYMBOL INFORMATION EXPRESSION
Gabbr1 Gamma-aminobutyric acid (GABA-B) receptor, 1 =
Other GPCRs GENE GENE RSK2 -/- SYMBOL INFORMATION EXPRESSION
Fzd1 Frizzled homolog 1 (FZ-1) = Fzd2 Frizzled homolog 1 (FZ-2) = Fzd7 Frizzled homolog 7 (FZ-7) = Smo Smoothened homolog precursor (SMO) =
215 Adenosine A2B Receptor (Adora2B)
Adenosine is a purine nucleoside that is ubiquitously released by sodium-
dependent transporters, is formed extracellularly by the breakdown of released ATP, and
is one of the primary mediators of metabolic distress. Adenosine mediates its action
through the adenosine receptors, Class A GPCRs, of which four subtypes have been
identified: A1, A2A, A2B, and A3. The A1 and A3 adenosine receptors are coupled to Gαi and inhibit adenylate cyclase, whereas the A2A and A2B receptors both couple to Gαs and stimulate adenylate cyclase. In addition, the A2B receptor has also been shown to couple
to Gαq. Our microarray data show a 2.73-fold increase in A2B gene expression in the absence of RSK2 (Table 6-1).
The A2A receptor displays high affinity for adenosine, whereas the A2B receptor displays low affinity for adenosine (Feoktistov and Biaggioni, 1997). Unfortunately, it has proven difficult to fully characterize the A2B receptor due to a lack of A2B selective agonists. A2B receptors are believed to be involved in the adenosine-induced vasodilation
(Webb et al., 1992), as well as playing a neuroexcitatory role in the brain (Phillis et al.,
1993). A2B receptors have also been shown to induce long-term potentiation in the CA1
region of the rat hippocampus in a manner consistent with a postsynaptic site of action
(Kessey et al., 1997). In addition, the A2B receptor has been argued to be a presynaptic
receptor that enhances the neurally mediated release of acetylcholine resulting in the
induction of contraction in bronchial smooth muscle (Walday and Aas, 1991).
216 Endothelin Receptor Type A (Ednra)
The endothelin (ET) family is comprised of three peptides, ET-1, ET-2, and ET-3
(Yanagisawa et al., 1988). The ET peptides are 21 AA, contain two intramolecular
disulfide bridges, and are generated by a two step processing pathway involving
conversion of prepro-ETs to pro-ETs via prohormone- and furin-like convertases and the
subsequent hydrolyzation of a specific Trp-Val(or Ile) bond by endothelin-converting enzymes (ECEs) to generate the mature active forms of the ETs (Delarue et al., 2004).
The ETs exert their action through the endothelin receptors, Class A GPCRs, of which two subtypes, ETA and ETB, have been cloned and which are both coupled to Gαq (Arai et al., 1990; Sakurai et al., 1990) The endothelin receptors differ in their affinities for the different ET members, with the ETA receptor displaying a higher affinity for ET-1 and
ET-2 than ET-3, and ETB showing no ET ligand preference. Our microarray studies
demonstrated a 5.40-fold increase in ETA receptor (Ednra) mRNA expression in the
absence of RSK2 (Table 6-1).
ET stimulation has a number of cardiovascular effects including coronary
vasoconstriction, the secretion of atrial natiuretic peptide and nitric oxide, and the
facilitation of cardiac hypertrophy (Hu et al., 1988; Ishikawa et al., 1988; Moravec et al.,
1989). In addition, ET receptor antagonists have been shown to improve cardiovascular
dysfunction and to prolong the survival of experimental animals with congestive heart
failure (Sakai et al., 1996). Interestingly, individuals with Coffin-Lowry Syndrome have
a much higher incidence of cardiovascular disease, cardiac dysfunction, and
217 cardiomyopathy, as discussed in Chapter 1. In addition to these cardiovascular effects,
the ETs have been reported to modulate the activities of a number of endocrine glands
including the anterior pituitary, thyroid, parathyroid, gonads, and the adrenal gland
(Masaki, 1993). As the mRNA for the ETA receptor is increased dramatically in the absence of RSK2 and ET receptor antagonists improve cardiovascular dysfunction, investigation into the potential role of RSK2 in the expression of the ETA receptor may provide valuable insight into a number of the cardiovascular complications associated with Coffin-Lowry Syndrome. Another point of interest is that several studies have shown that insulin and insulin-like growth factor-1 (IGF-1) treatment increases the number of ETA receptors in vascular smooth muscle cells (Kwok et al., 2005). Our microarray data show that IGF-1 expression is increased an average of 4.46-fold in the absence of RSK2 (there are two different gene probe sets for IGF-1, see Supplemental
Microarray Data). Therefore, the increase in expression in the ETA receptor that is
observed in the absence of RSK2 may be an indirect result of the increase in IGF-1
expression in the absence of RSK2, but this matter will require further study.
Protease-Activated Receptor 1 (F2r)
The protease-activated receptors are Class A GPCRs that have a unique method of
activation. Four PAR subtypes have been cloned, designated PAR-1, PAR-2, PAR-3, and
PAR-4, and are coupled via Gαq, Gα12/13, and Gαi to a variety of signaling pathways. Of these PARs, PAR-1, PAR-3, and PAR-4 are activated by the serine protease thrombin
(Ishihara et al., 1997; Vu et al., 1991; Xu et al., 1998), whereas PAR-2 is activated by
218 multiple trypsin-like serine proteases including: trypsin, mast cell tryptase, neutrophil
proteinase 3, tissue factor/factor VIIa/factor Xa, and membrane-tethered serine protease-
1, but is not cleaved by thrombin (Coughlin, 2000). Thrombin is the main effector protease of the coagulation cascade, a series of zymogen conversions that take place when coagulation factors in the plasma contact tissue factor expressed either in the extravascular compartment or recruited to a site of injury. In this cascade, tissue factor acts as a co-factor for the activation of zymogen factor X by factor VIIa. Factor Xa along with factor Va then converts prothrombin to thrombin (Coughlin, 2000).
Thrombin’s method of activation of the PARs is unique among GPCRs. Thrombin binds to the N-terminal domain of PAR-1, PAR-3, and PAR-4 and cleaves it to generate a new receptor N-terminus, which then acts as a tethered ligand that binds to the heptahelical bundle of the receptor, activating it (Coughlin, 2000). Thrombin binds the N- terminal domains of PAR-1, PAR-3, and PAR-4 through thrombin-interacting sequences that fall both N-terminal and C-terminal to the thrombin cleavage site. Interestingly,
PAR-4 lacks one of these thrombin-interacting sequences, and as such has a much lower affinity for thrombin (Xu et al., 1998). For PAR-1, this cleavage occurs at Arg41 resulting in the unmasking of a new N-terminus whose first 6 amino acids are SFLLRN.
The addition of this peptide, also known as Thrombin Receptor Activating Peptide
(TRAP), results in the direct activation of PAR-1 in the absence of thrombin (Vu et al.,
1991). As the cleavage and activation of PAR-1 by thrombin is irreversible, signaling is terminated by rapid phosphorylation of the receptor resulting in G-protein uncoupling,
219 followed by internalization of the receptor and sorting to the lysosomal compartment for degradation (Coughlin, 1999).
In our experimental data outlined in Chapter 5, we discovered that PAR-1 signaling, as measured by phosphoinositide hydrolysis and by calcium mobilization, is increased dramatically in the absence of RSK2. As demonstrated in Chapter 5, the reintroduction of RSK2 into the RSK2 -/- background resulted in a partial reversion of phenotype for the PAR-1 receptor. For each of the other receptors tested, namely the 5-
HT2A receptor, the bradykinin-B receptor, and the P2Y-purinergic receptor, reintroduction of RSK2 into the RSK2 -/- fibroblasts resulted in at least a 50% reduction of the RSK2 -/- maximal phosphoinositide hydrolysis back toward RSK2 +/+ fibroblast maximal signaling. However, reintroduction of RSK2 into the RSK2 -/- fibroblasts only resulted in approximately a 20% reduction of PAR-1 signaling back toward RSK2 +/+ fibroblast maximal signaling. Our microarray data show a 2.13-fold increase in PAR-1 (F2r) expression in the absence of RSK2 (Table 6-1) and this increase in receptor expression may account for the smaller phenotype reversion seen for the PAR-1 receptor when
RSK2 was reintroduced into the RSK2 -/- background.
The activation of the PARs plays a role in a number of physiologic processes including both vasodilation and vasoconstriction, calcium-dependent chloride secretion in the intestinal epithelial cells, inflammatory signaling cascades, and bone cell resorption
(Flynn and Buret, 2004). Most importantly, PAR signaling largely accounts for platelet activation by thrombin and the downstream signaling by the PARs play a role in both
220 hemostasis and thrombosis (Coughlin, 2000). In addition, the PAR-1 receptor may also
contribute to blood vessel development as 50% of PAR-1 knockout mice die between
embryonic days 9.5 and 10.5 due to defects in vascular integrity (Connolly et al., 1996).
Due to the role of the PAR-1 receptor in both cardiac function as well as bone resorption, further investigation into the role of RSK2 in PAR-1 receptor signaling and regulation may provide additional insights into a variety of the pathological conditions found in
Coffin-Lowry Syndrome.
Melanocortin-2 Receptor (Mc2r)
The melanocortin-2 receptor was cloned in 1992 and later determined to be the receptor for adrenocorticotropin (ACTH) (Clark and Weber, 1998; Mountjoy et al.,
1992). The characterization of MC2R has proven extremely difficult in heterologous systems. An adrenocortical Y1 cell line derivative Y6 is the primary model system used to study MC2R (Schimmer et al., 1995). The reason for this difficulty is that in Y6 cells,
the MC2R is cell surface expressed, but in all other lines tested, MC2R is found in a
perinuclear location that co-localizes with the endoplasmic reticulum (ER) and the trans-
golgi (Clark et al., 2003). It is largely believed that the lack of cell-surface expression in
all other tested cell lines is due to the absence of an ER or golgi network resident
chaperone protein such as the RAMPs (McLatchie et al., 1998), which are required for
CGRP receptor membrane transport (as will be described later in this chapter) or DRiP78
type molecules, which associate with the D1-dopamine receptor (Bermak et al., 2001).
MC2R is coupled to Gαq, and desensitized rapidly following stimulation in a PKA
221 dependent manner involving Ser208 in the third intracellular loop (Baig et al., 2001).
MC2R internalizes via clathrin-coated pits and not via caveolae, in a PKA and arrestin
insensitive but in a GRK2 sensitive manner (Baig et al., 2002). We have found that
MC2R gene expression is up-regulated 4.23-fold in the absence of RSK2 (Table 6-1).
Prostaglandin E Receptor 4, Subtype EP4 (Ptger4)
Prostanoids are a class of compounds including prostaglandin E2 (PGE2), prostaglandin F2α (PGF2α), prostaglandin D2 (PGD2), prostaglandin I2 (PGI2), and
thromboxane A2 (TxA2), which are produced by the initial actions of cyclooxygenases on
arachidonic acid (Regan, 2003). There are two cyclooxygenases, COX-1 and COX-2,
which convert arachidonic acid to PGH2, the initial metabolite in prostanoid synthesis
that is converted to all of the other prostanoids through the action of specific synthases
(Smith et al., 2000). The prostanoids bind to prostanoid receptors, members of the Class
A GPCR superfamily. In particular PGE2 binds to and activates four different prostaglandin E receptors, which are designated as subtypes EP1, EP2, EP3, and EP4. Our
microarray demonstrated a 2.01-fold decrease in expression of the gene for the
prostaglandin EP4 receptor in the absence of RSK2 (Table 6-1).
Of the prostaglandin E receptors, the EP4 and EP2 prostaglandin E receptors
show the most similarity, couple to Gαs, and induce smooth muscle relaxation (Jabbour
and Sales, 2004). However, given their similarity, there are major differences in the
desensitization and internalization of EP2 and EP4 receptors, whereby EP4 receptors
222 show a rapid desensitization and internalization while EP2 receptors do not.
Desensitization and internalization of the EP4 receptor requires elements in the C-
terminal tail of the receptor, which contains potential PKA and G protein-coupled
receptor kinase consensus phosphorylation motifs (Desai et al., 2000). The EP4 receptor
is known to activate the ERK MAPK cascade via a phosphoinositide-3 kinase (PI3K)
dependent mechanism, which results in the expression of the early growth response factor-1 (EGR-1), another dysregulated gene in the RSK2 -/- fibroblasts that will be discussed in a later section of this chapter. Another point of interest is that EP2 and EP4 agonists have been shown to stimulate bone growth, and that EP2 and EP4 knockout mice have bone growth deficiencies, showing a potential role of the EP2 and EP4 receptors in osteoclast mediated bone formation (Yoshida et al., 2002). As discussed in
Chapter 1, RSK2 plays a role in osteoclast function and Coffin-Lowry Syndrome patients show a variety of skeletal deformations, including slower bone growth, so there
may be a functional link with the EP4 receptor worth exploring (Yang et al., 2004).
Calcitonin Gene-Related Peptide Type 1 Receptor (Calclr)
Calcitonin gene-related peptide α (CGRPα) is an alternatively spliced gene
product of the calcitonin gene and is a 37 AA peptide with a characteristic seven amino
acid ring linked by a disulfide bridge between positions 2 and 7, and an amidated N-
terminus (Rosenfeld et al., 1983). A second homologue, CGRPβ, was characterized,
which has a high sequence homology with CGRPα, although it is not derived from the
calcitonin gene (Amara et al., 1985). The CGRP peptide family also includes the peptides
223 amylin and adrenomedullin (ADM) (Kitamura et al., 1993). All of these CGRP family members are widely distributed in peripheral tissues and in the CNS and have somewhat overlapping effects, such as extremely potent vasodilation, but they also show distinct biological actions as well. Due to the overlapping vasodilatory effects combined with the unique actions of each of the CGRP family peptides, it was hypothesized that all of the
CGRP compounds activate similar types of receptors but that distinct CGRP, amylin, and
ADM receptors likely exist (Juaneda et al., 2000).
A great deal of complication arose with the attempts to discover the receptors for the CGRP family peptides. The Calcitonin gene-related peptide type 1 receptor (CRLR,
Calclr), was cloned from rat pulmonary blood vessels in 1993 and was shown to have the highest expression in the lung, a tissue known to be enriched in CGRP and ADM receptor binding sites (Han et al., 1997; Njuki et al., 1993). However, full identification of the
CGRP receptor would have to wait for a few years until the discovery of a novel family of transmembrane proteins known as receptor activity modifying proteins (RAMPs)
(McLatchie et al., 1998). RAMPs facilitate the intracellular translocation of CRLR and its insertion into the plasma membrane. Additionally, the various RAMPs (RAMP1,
RAMP2, and RAMP3) dramatically alter the pharmacological profile of CRLR in addition to a second human calcitonin receptor, hCTR2. In addition to RAMP alteration of the pharmacological properties of the calcitonin receptors, another chaperone known as receptor component protein (RCP) may be necessary for CGRP receptor activity.
Therefore, current data indicates that the CGRP1 receptor is a complex of CRLR with
RAMP1 and RCP, the amylin receptor is a complex of hCTR2 with RAMP1 or RAMP3,
224 and that the ADM receptor is a complex of CRLR with RAMP2 or RAMP3. Regardless,
these receptors are Class B GPCRs that are coupled via Gαs to adenylate cyclase activation (Juaneda et al., 2000). We have found that Calclr gene expression (CRLR) is increased 2.28-fold in the absence of RSK2 (Table 6-1).
CD97 Antigen (CD97)
Epidermal growth factor-seven transmembrane (EGF-TM7) receptors are a family of Class B GPCRs with a unique structural arrangement. These GPCRs have a number of tandemly arranged EGF domains in their extracellular N-terminal region and a conserved cleavage motif known as the GPCR-proteolytic site (GPS). In addition to these two features, EGF-TM7 family GPCRs have a great number of alternatively spliced transcripts that result in various isoforms that have differing numbers and arrangements of the EGF domains (Kwakkenbos et al., 2004). CD97 is a member of this EGF-TM7 family, which is found to be broadly expressed, found on most hematopoitic cells including activated lymphocytes, monocytes, macrophages, dendritic cells, and granulocytes (Eichler et al., 1994; Hamann et al., 2000; Jaspars et al., 2001). CD97 is the only EGF-TM7 family receptor that has been detected on active lymphocytes to date
(Jaspars et al., 2001). The endogenous ligand of CD97 is the CD55/decay accelerating factor (DAF), which binds in the EGF domain region of CD97 (Hamann et al., 1996).
DAF protects host cells from complement-mediated damage by accelerating the decay of
C3/C5 convertases in the classical complement pathway (Lublin and Atkinson, 1989).
Additionally, CD97 has been shown to be important for leukocyte migration and for host
225 defense (Leemans et al., 2004). Our microarray studies have revealed a 2.14-fold increase
in expression of CD97 in the absence of RSK2 (Table 6-1).
Parathyroid Hormone Receptor 1 (Pthr1)
Parathyroid hormone (PTH) regulates both calcium and phosphate metabolism in
bone and in the kidney via the activation of adenylate cyclase through Gαs and phospholipase Cβ through Gαq/11 (Bringhurst et al., 1993; Iida-Klein et al., 1997). PTH
acts in the kidney by activating 1-alpha-hydroxylase, the enzyme responsible for
hydroxylating 25-hydroxyvitamin D. In addition, PTH inhibits phosphate resorption in
the kidney through the blockade of sodium-dependent phosphate co-transport (Schipani
and Provot, 2003). In the bone, PTH results in the activation of osteoclasts leading to
increased osteoclast number and increased bone resorption (Solimando, 2001). In
addition to PTH, parathyroid hormone-related peptide (PTHrP) is another related peptide
whose aberrant expression causes hypercalcemia of malignancy syndrome (HCM).
Hypercaclemia is the direct result of increased bone resorption, resulting in increased
serum calcium levels and is caused by PTHrP-stimulated adenylate cyclase activity.
Both PTH and PTHrP bind to the same receptor, parathyroid hormone receptor 1
(Pthr1) (Juppner et al., 1991). Pthr1 is a member of the class B GPCRs, which are
characterized by a long extracellular N-terminal domain that contains six absolutely
conserved cysteine residues (Schipani and Provot, 2003). This 585 AA GPCR is
characterized by a long N-terminal, a long C-terminal, and a long 1st extracellular loop.
226 We have found that the expression of the Pthr1 gene probe is decreased 4.18-fold in the
absence of RSK2 (Table 6-1). As previously discussed, it has been well established that
PTH/PTHrP and the Pthr1 receptor have a role in endochondral bone formation and in
osteoclast function. Intriguingly, as discussed in Chapter 1, RSK2 also plays a role in
osteoclast function, primarily through the activation of the transcription factor ATF4
(Yang et al., 2004). It would prove interesting to investigate any possible links between
RSK2 and the function of the Pthr1 receptor, as this may provide greater insight into the skeletal defects of Coffin-Lowry Syndrome.
6.6 The Role of RSK2 in Biogenic Amine Synthesis Pathways
An important observation of the RSK2 -/- and RSK2 +/+ fibroblast microarray data is that RSK2 -/- fibroblasts show a 3.4-fold increase in expression of DDC in the absence of RSK2. DDC, whose gene product is the Aromatic L-Amino Acid
Decarboxylase (AADC), is an enzyme involved in catecholamine and serotonin biosynthesis (Figure 6-3). AADC is responsible for the decarboxylation step in both catecholamine and serotonin (5-HT) synthesis, resulting in the formation of dopamine from L-DOPA and 5-hydroxytryptamine (5-HT) from 5-hydroxytryptophan (5-HTP)
(Brodie et al., 1962). In the human brain, AADC levels are lower than what are found in other species. Due to this, it has been hypothesized that AADC may become the rate- limiting step in monoamine synthesis due to expression and that alterations in AADC expression may be a mechanism for the control of catecholamine and serotonin biosynthesis (Brodie et al., 1962). As described in Chapter 4, the absence of RSK2
227 results in an increase in signaling of the 5-HT2A receptor to PI hydrolysis, calcium mobilization, and to the MAPK cascade. Taken together, our microarray analysis data suggest a role of RSK2 in the regulation of DDC expression, whereby increased expression of AADC in the human brain could potentially augment the RSK2 -/- phenotype by providing higher levels of endogenous serotonin.
228
Figure 6-3 - RSK2 +/+ and -/- fibroblasts display a few differences in biogenic amine synthesis enzyme expression. Figure 6-3 shows the microarray data of biogenic amine synthesis overlaid with gene-expression color criterion and fold-changes from the programs GenMAPP and MAPPFinder. Gray colored genes are those expressed in RSK2
-/- and RSK2 +/+ fibroblasts that show no change in gene expression. White colored genes are not expressed in either RSK2 -/- or RSK2 +/+ fibroblasts. Green colored genes are those genes in the RSK2 -/- fibroblasts that show greater than a 2-fold increase in expression over the RSK2 +/+ fibroblasts. Red colored genes are those genes in the
RSK2 -/- fibroblasts that show greater than a 2-fold decrease in expression compared the
RSK2 +/+ fibroblasts.
229 Figure 6-3
230 6.7 The Role of RSK2 in Cell Cycle Control Pathways
There are a number of genes involved in cell-cycle control pathways that are differentially expressed in the absence of RSK2. These genes fall into several families, the cyclins, the cyclin dependent kinase inhibitors (CKIs), DNA damage checkpoint control proteins, minichromosome maintenance (MCM) proteins, and the E3 ubiquitin ligase MDM2 (Figure 6-4). In the following section, each of these genes will be discussed in the context of their function, whether or not they have any known relation to
RSK2 or RSK2 signaling pathways, and potential signaling changes that may result from the alterations in gene expression of the proteins in the absence of RSK2.
231
Figure 6-4 - RSK2 +/+ and -/- fibroblasts display a number of differences in cell cycle control gene expression profiles. Figure 6-4 shows the microarray data of cell cycle control pathways overlaid with gene-expression color criterion and fold-changes from the programs GenMAPP and MAPPFinder. Gray colored genes are those expressed in RSK2 -/- and RSK2 +/+ fibroblasts that show no change in gene expression. White colored genes are not expressed in either RSK2 -/- or RSK2 +/+ fibroblasts. Green colored genes are those genes in the RSK2 -/- fibroblasts that show greater than a 2-fold increase in expression over the RSK2 +/+ fibroblasts. Red colored genes are those genes in the RSK2 -/- fibroblasts that show greater than a 2-fold decrease in expression compared the RSK2 +/+ fibroblasts.
232 Figure 6-4
233 Cyclins and Cyclin-Dependent Kinase Inhibitors
The eukaryotic cell cycle is divided into four stages: G1, S, G2 and M. G1 and G2 are the so called gap stages between the stages of DNA replication (S) and mitosis (M).
The transitions between the various stages of the cell cycle are controlled primarily through the sequential activation and deactivation of the cyclin-dependent kinases
(Cdks), whose activity is controlled through the periodic synthesis and degradation of the family of proteins known as cyclins. Cyclins were first identified in marine invertebrates as proteins whose accumulation and degradation oscillated during the cell cycle
(Rosenthal et al., 1980), and at least 16 mammalian cyclins have been identified (Johnson and Walker, 1999). Different cyclins are associated with select classes of Cdks and act at varying points in the cell cycle.
The product of the gene CCNE2 is Cyclin E2 (Figure 6-4), which our microarray data showed has a 2.3-fold decrease in expression in the absence of RSK2. Cyclin E2 is the cyclase known to associate with the cyclin-dependent kinase Cdk2 in a functional catalytically active kinase complex that phosphorylates H1 and Rb but not p53 (Gudas et al., 1999). This Cyclin E2-Cdk2 complex functions at the G1-to-S phase transition
(Johnson and Walker, 1999). The activity of Cyclin E2 peaks as cells approach S-phase and kinase activity corresponds with Cdk2 phosphorylation and activation by CDK activating kinase (CAK) and a decrease in p27Kip1 levels (Gudas et al., 1999). A second
cyclin, Cyclin B2 (gene symbol CCNB2), showed a 2.1-fold decrease in expression in the absence of RSK2 (Figure 6-4). Cyclin B2 interacts primarily with the cyclin-dependent
234 kinase Cdk1 and is involved in the G2-to-M phase transition. Mitotic B-type cyclins activate the p34cdc2 protein kinase to form maturation promoting factor, which is required for cells to undergo mitosis (Brandeis et al., 1998). Cyclin B2 knockout mice are viable, but are smaller in body size (Brandeis et al., 1998). As there is a decrease in expression of both the G1-to-S phase and G2-to-M phase cyclins in the absence of RSK2, one would expect RSK2 -/- fibroblasts to display a slower cell doubling time than the RSK2 +/+ fibroblasts. Although the doubling time has not been precisely measured, I have observed that RSK2 -/- fibroblasts double approximately every 26 hours, whereas RSK2 +/+ fibroblasts double approximately every 12 hours.
In addition to alterations in the expression patterns of the E and B class cyclins, the expression of a number of cyclin-dependent kinase inhibitors (CKIs) is altered in the absence of RSK2. The genes altered fall into two different classes of CKIs, the Cip/Kip class (Cdk-interacting protein/kinase inhibitory protein) and the INK4 class (inhibitor of
Cdk4). The altered genes include CDKN1A, CDKN1C, CDKN2A, and CDKN2B. The gene product of CDKN1A is the CKI known as p21Cip1/WAF1, which is the founding member of the Cip/Kip family, known to inhibit multiple Cdks including both Cdk4/6 and Cdk2 by binding to cyclin-Cdk complexes (el-Deiry et al., 1993). p21Cip1 is known to be the primary mediator of p53-dependent cell cycle arrest (Lohrum and Vousden, 2000), which is of interest as p53 is a known substrate for RSK2 phosphorylation (as previously described in Chapter 1). The CDKN1A gene showed a 7.4-fold increase in expression the absence of RSK2 (Figure 6-4). CDKN1C is a second member of the Cip/Kip class of
CKIs whose gene product is p57Kip2 (el-Deiry et al., 1993). Intriguingly, p57Kip2 has been
235 established as the only CKI that is required for mouse embryogenesis through gene
disruption studies (Zhang et al., 1997). The CDKN1C gene showed a 6.6-fold decrease in
expression in the absence of RSK2 (Figure 6-4).
CDKN2A and CDKN2B fall into the second class of CKIs, INK4, known for
their ability to inhibit cdk4, specifically inhibiting the cyclin-D dependent kinases (Ruas and Peters, 1998). CDKN2A is the founding member of this class whose gene product is p16INK4a (Serrano et al., 1993). The CDKN2A gene showed a 2.1-fold decrease in
expression in the absence of RSK2 (Figure 6-4). CDKN2B is a second member of the
INK4 CKIs whose gene product is p15INK4b (Hannon and Beach, 1994). The CDKN2B
gene showed a 2.6-fold decrease in expression in the absence of RSK2 (Figure 6-4). The
CKIs described above act primarily at the G1-to-S phase transition. As all of these CKIs,
with the exception of CDKN1A, show a decrease in expression, one would predict that
RSK2 knock-out would increase the rate of the G1-to-S phase transition due to a decrease
in CKI inhibition. This increase in G1-to-S phase rate would result in the quickening of
the cell cycle, due to a shortening of the G1 phase. These predictions also fall in line
with the experimental observation of a shortened cell doubling time in RSK2 knock-out
cells as previously described.
CHK1
The DNA damage checkpoint gene CHEK1, which encodes the protein Chk1,
was originally identified in fission yeast as a kinase required for the DNA damage
236 checkpoint (Walworth et al., 1993). Chk1 has been determined to be phosphorylated in a
Rad3 dependent manner in response to activation of the DNA damage response
checkpoint (Walworth and Bernards, 1996). Chk1 is known to phosphorylate and interact
with CDC25 (Furnari et al., 1997), which inhibits CDC25 phosphatase activity (Furnari
et al., 1999), and decreases nuclear localization of CDC25 (Lopez-Girona et al., 1999).
The CHEK1 gene expression is down-regulated 2.1-fold in the absence of RSK2 (Figure
6-4).
MCM Family Proteins
MCM2 and MCM5 are “minichromosome maintenance” proteins. This protein
family was identified in the 1980s for Saccharomyces cerevisiae mutants that have defects in maintaining a simple minichromosome (Maine et al., 1984). The MCM proteins are members of the AAA ATPase family, which form large ATP dependent complexes that are often heterohexamers. The MCM proteins, specifically MCM2-7, are essential for replication initiation and elongation in eukaryotic cells (Bell and Dutta,
2002; Labib and Diffley, 2001). It has been suggested that Mcm2, Mcm3, and Mcm5 negatively regulate the active Mcm4,6,7 complex (Ishimi et al., 1998; Lee and Hurwitz,
2001; You et al., 1999). Additionally, interferon signaling has been shown to drive the association of MCM5 and STAT1 (signal transducer and activator of transcription 1),
which may interfere with MCM5’s role in replication through sequestration (DaFonseca
et al., 2001). It is interesting to note that phosphorylation of STAT1 on Ser727 is
absolutely essential for STAT activation and that phosphorylation of STAT1 on Ser727 is
237 absent in RSK2 -/- cells (Zykova et al., 2005). Our microarray data show that the gene expression of MCM2 is decreased 2.1-fold and that the gene expression of MCM5 is decreased 2.5-fold in the absence of RSK2 (Figure 6-4).
MDM2
Murine double minute 2 (MDM2) is a known binding partner of the tumor suppressor p53 and inhibits p53-mediated transactivation (Momand et al., 1992). It has been demonstrated that the over-expression of MDM2 is a mechanism by which a cell can inactivate p53 in the process of transformation. Our microarray data show a 7.1-fold up-regulation of MDM2 gene expression in the absence of RSK2 (Figure 6-4).
Biochemically, MDM2 functions as an E3 ubiquitin ligase responsible for the ubiquitination and subsequent degradation of p53 (Haupt et al., 1997; Honda et al., 1997;
Kubbutat et al., 1997). Intriguingly, MDM2 is known to interact with p300/CBP, a known binding partner of RSK2. The MDM2 interaction with p300/CBP results in a cooperative polyubiquitination and degradation of p53 (Grossman et al., 1998; Kawai et al., 2001). Of interest is the fact that the phosphorylation of p53 on Ser15 inhibits the binding of MDM2 (Shieh et al., 1997) and that p53 is known to be phosphorylated on
Ser15 by RSK2 (Cho et al., 2005).
238 6.8 The Role of RSK2 in Insulin Signaling Pathways
As discussed in Chapter 1, RSK2 is a known target in insulin signaling pathways
and is directly activated via PDK1 and ERK1/2 phosphorylation following insulin
stimulation. As previously discussed, the primary role or RSK2 in insulin signaling
pathways is to act as a transcriptional regulator. As would be expected, there are a
number of alterations in insulin signaling pathway gene expression in the absence of
RSK2. These genes that show alterations in expression include the Serum- and
glucocorticoid-regulated kinase (SGK), phosphatidylinositol 3 kinase regulatory subunit
polypeptide 4 (PI3KR4), the growth factor receptor-bound adapter protein Grb14, the guanine nucleotide exchange factor SOS2, c-Cbl-associated protein (CAP), EHD2, immediate early growth response gene 1 (EGR1), and Trib3 (Figure 6-5). None of these
gene products fall into a particular family within the insulin signaling pathway. As such,
in the following section, each of these genes will be discussed separately. The known
functions of these proteins and how they function within the insulin signaling pathways
will be discussed in addition to any potential relation to RSK2 or RSK2 signaling
pathways.
239
Figure 6-5 - RSK2 +/+ and -/- fibroblasts display a number of differences in insulin
signaling pathway gene expression profiles. Figure 6-5 shows the microarray data of insulin signaling pathways overlaid with gene-expression color criterion and fold- changes from the programs GenMAPP and MAPPFinder. Gray colored genes are those expressed in RSK2 -/- and RSK2 +/+ fibroblasts that show no change in gene expression.
White colored genes are not expressed in either RSK2 -/- or RSK2 +/+ fibroblasts. Green colored genes are those genes in the RSK2 -/- fibroblasts that show greater than a 2-fold increase in expression over the RSK2 +/+ fibroblasts. Red colored genes are those genes in the RSK2 -/- fibroblasts that show greater than a 2-fold decrease in expression compared the RSK2 +/+ fibroblasts.
240 Figure 6-5
241 Serum- and Glucocorticoid-Regulated Kinase (SGK)
Serum- and glucocorticoid-regulated kinase (SGK) is involved in transducing cell
survival signals and proliferative responses (Brunet et al., 2001; Buse et al., 1999;
Mikosz et al., 2001; Webster et al., 1993; Xu et al., 2001). We found that the SGK gene
showed a 2.6-fold decrease in expression in the absence of RSK2 (Figure 6-5). SGK is
primarily known to be involved in the control of epithelial sodium channel (ENaC)
activity and sodium homeostasis (Alvarez de la Rosa et al., 1999; Chen et al., 1999;
Kamynina and Staub, 2002; Naray-Fejes-Toth et al., 1999; Shigaev et al., 2000). Like
RSK2, SGK is a member of the AGC family of serine/threonine protein kinases, and is
phosphorylated by PDK1, resulting in it’s activation (Le Good et al., 1998). The SKG
promoter is a target of p53 (Maiyar et al., 1997), which is a known substrate of RSK2
(Cho et al., 2005).
Phosphatidylinositol 3 Kinase Regulatory Subunit, Polypeptide 4 (PIK3R4)
The product of the gene PIK3R4 is the phosphatidylinositol 3 kinase (PI3K),
regulatory subunit, polypeptide 4, which showed a 2.1-fold decrease in expression in the
absence of RSK2 (Figure 6-5). This regulatory subunit is primarily known as p150. PI3K is involved in the synthesis of phospotidylinositol-3-phosphate [PtdIns(3)P], where it is associated with endomembranes such as the trans-Golgi network by its interaction with membrane-bound proteins, one of which is the adapter protein p150 (Panaretou et al.,
242 1997). These adaptors contribute to the stimulation of PtdIns(3)P production. Little is known about this regulatory adapter protein.
Grb14
The growth factor receptor-bound (Grb) family of proteins are adapter proteins that lack any sort of enzymatic activity. Grb14, a member of this family, showed a 4.6- fold decrease in expression in the absence of RSK2 in our microarray studies (Figure 6-
5). Functionally, Grb14 has been shown to bind to activated insulin receptors and to act as a scaffold for the recruitment of PDK1, one of the kinases responsible for RSK2 activation, to activated insulin receptors (King and Newton, 2004). Grb14 has been argued to have a role in the regulation of insulin sensitivity, in the development of insulin resistance, and primarily appears to play an inhibitory role on insulin signaling (Holt and
Siddle, 2005).
SOS2
Ras is a membrane-associated GTPase involved in signal transduction that is activated by receptor tyrosine kinases (RTKs) via Ras-specific guanine-nucleotide exchange factors (GEFs) (Lowy and Willumsen, 1993). Son of Sevenless (SOS) 1 and 2 are GEFs constitutively bound to the SH3 domain of the adapter protein Grb2 and couple
RTKs to Ras activation (Esteban et al., 2000). In our studies, we found a 3.0-fold up- regulation of SOS2 expression in the absence of RSK2 (Figure 6-5). SOS1 participates in
243 both short and long-term signaling whereas SOS2-dependent signals are only short term
(Qian et al., 2000). SOS1 is absolutely essential for development, whereas SOS2 is completely dispensable (Esteban et al., 2000). Due to SOS2’s non-essential function, it has proven difficult to determine differing functions of SOS2 from SOS1.
c-Cbl-Associated Protein (CAP)
There are two primary signal transduction pathways that are required for insulin- stimulated glucose transport, one of which is an insulin receptor substrate/PI3-kinase pathway (Khan and Pessin, 2002) and the second of which is PI3-kinase independent and involves the c-Cbl-associated protein (CAP) and the tyrosine phosphorylation of c-Cbl
(Saltiel and Pessin, 2002). Our microarray data showed a 3.0-fold up-regulation of CAP in the absence of RSK2 (Figure 6-5). CAP is known to associate with the adapter protein
APS and this CAP-APS complex associates with the insulin receptor following insulin stimulation. This CAP-APS association with the insulin receptor promotes the association of CAP with c-Cbl (Ahn et al., 2004). Following insulin receptor phosphorylation of c-
Cbl, the CAP/c-Cbl complex migrates to lipid rafts where CAP interacts with flotillin through it’s sorbin homology (SoHo) domain (Kimura et al., 2001). The CAP/c-Cbl lipid raft association allows the C3G/Crk complex to be recruited to lipid rafts where C3G activates the small G-protein TC10 (Chiang et al., 2001). TC10 activation is required for
GLUT4 translocation from intracellular vesicles to the plasma membrane for the control of glucose homeostasis (Saltiel and Pessin, 2002).
244 EHD2
GLUT4 is the major glucose transporter (James et al., 1989), is mostly stored in
intracellular sites in the basal state, and insulin stimulates glucose uptake by the
recruitment of GLUT4 from these storage sites to the plasma membrane (Cushman and
Wardzala, 1980). GLUT4 constantly recycles between the plasma membrane and these
storage sites through endocytosis, exocytosis, and vesicular trafficking (Czech and
Corvera, 1999). EHD2 is a member of the Eps15 homology-domain containing proteins
and has been shown to form a complex with GLUT4 (Park et al., 2004). In our studies,
we found a 3.6-fold decrease in expression of EHD2 in the absence of RSK2 (Figure 6-
5). The association of EHD2 with GLUT4 has been shown to be required for the endosomal transport of GLUT4 by coupling GLUT4 transport via clathrin-mediated endocytosis to the actin cytoskeleton (Guilherme et al., 2004).
EGR1 and Trib3
The immediate early growth response gene 1 (EGR1) is a transcription factor often found over-expressed in prostate cancers (Eid et al., 1998; Thigpen et al., 1996).
We found a 2.3-fold up-regulation of the EGR1 gene in the absence of RSK2 (Figure 6-
5). EGR1 is thought to promote cell growth and desensitization to cell death (Virolle et al., 2003). Interestingly, EGR1 is known to interact with p300/CBP, known binding partners of RSK2, and to modulate their activity (Yu et al., 2004). Trib3 was identified in a screen searching for proteins that modulate AKT activity (Du et al., 2003). Trib3 is a
245 homolog of the Drosophila protein tribbles, a protein that inhibits mitosis early in
development by promoting the ubiquitination and degradation of the Drosophila CDC25
homolog (Seher and Leptin, 2000). RSK2 -/- fibroblasts showed a 2.0-fold decrease in
expression of Trib3 (Figure 6-5). The over-expression of Trib3 blocks insulin and IGF1
stimulated AKT phosphorylation and activity, and block in AKT phosphorylation has
been shown to not be due to alterations in the ubiquitination or degradation of AKT (Du
et al., 2003).
6.9 Discussion
A microarray analysis of RSK2 -/- and RSK2 +/+ fibroblasts has shown no global
change in gene expression resulting from RSK2 knockout. RSK2 mRNA was
significantly reduced in the RSK2 -/- fibroblasts compared to the RSK2 +/+ fibroblasts,
consistent with the knock-out of RSK2 in the RSK2 -/- fibroblasts. These data are further
supported by the lack of RSK2 expression by Western blot as demonstrated in Chapter
4. The genes probes for RSK1 and RSK3 also show a slight decrease in mRNA
expression, which was confirmed at the protein level for RSK1 via Western blot as
shown in Chapter 4. It is an intriguing possibility that the expression of the RSKs may
be coordinately regulated, though this remains to be seen.
As previously stated, 5-HT2A receptors were shown to be expressed at equivalent levels in both RSK2 -/- and RSK +/+ fibroblasts through both RT-PCR studies and saturation binding studies. Importantly, these microarray data demonstrate that other 5-
246 HT2 class receptors are not expressed. Therefore, alterations in 5-HT2A signaling in RSK2 knock-out fibroblasts are not due to signaling via 5-HT2B or 5-HT2C receptors. The
signaling phenotype of 5-HT2A receptors is also not due to alterations in the expression patterns of members of the GPCR signaling cascades or in the expression patterns of
MAPK cascade members (Chapter 4, Figures 4-3 and 4-4). As such, the signaling phenotypes observed for the 5-HT2A receptor are not due to gene expression differences.
It should be noted, however, that microarray data only demonstrate mRNA expression
and not protein expression. Therefore, there may be alterations in protein expression for
these pathways, but this will require further study.
As discussed in Chapter 1, RSK2 plays a role in osteoclast function, primarily
through the activation of the transcription factor ATF4 (Yang et al., 2004). Coffin-Lowry
Syndrome patients show a variety of skeletal deformations, including slower bone
growth; therefore, any alterations in gene expression that may contribute to this
phenotype are worth exploring. As such, the analysis of differences in the expression
levels of the GPCRs has provided a number of interesting points of study, including a
decrease in expression of both prostaglandin E4 receptors and the parathyroid hormone
receptor. As previously described, EP2 and EP4 agonists have been shown to stimulate
bone growth, and that EP2 and EP4 knockout mice have bone growth deficiencies,
showing a potential role of the EP2 and EP4 receptors in osteoclast mediated bone
formation (Yoshida et al., 2002). Additionally, it has been well established that
PTH/PTHrP and the Pthr1 receptor have a role in endochondral bone formation and in osteoclast function. It would prove interesting to investigate any possible links between
247 RSK2 and the function of the prostaglandin EP4 and the parathyroid hormone receptor,
as this may provide greater insight into the skeletal defects of Coffin-Lowry Syndrome.
Our microarray analysis has given us a great number of insights into the role of
RSK2 in maintenance of cell cycle control. For these pathways, the most interesting
alterations in gene expression are the down-regulation of the ‘minichromosome
maintenance’ protein MCM5 and the up-regulation of MDM2. Both MCM5 and MDM2
interact with and are regulated by known binding partners or substrates of RSK2. For
MCM5, as previously stated, interferon signaling has been shown to drive the association
of MCM5, which may interfere with MCM5’s role in replication through sequestration
(DaFonseca et al., 2001). Since STAT1 is a substrate for RSK2, the phosphorylation of
STAT1 on Ser727 is absent in RSK2 -/- cells, and this phosphorylation of Ser727 is
essential for STAT activation (Zykova et al., 2005), it will prove interesting to further examine the role of RSK2 in both interferon signaling pathways and in DNA replication.
A similar case exists for MDM2, with RSK2 acting as a binding partner or a kinase toward several known MDM2 interacting proteins including p300/CBP and p53. As
RSK2 is known to phosphorylate p53 on Ser15 (Cho et al., 2005) and the interaction of
MDM2 with p53 is inhibited by the phosphorylation of Ser15 (Shieh et al., 1997) , one would expect that p53 polyubiquitination and degradation would be inhibited in RSK2 knock-out cells. The RSK2 knock-out fibroblasts will prove to be a valuable tool for the dissection of the role of RSK2 in cell cycle progression.
248 Serotonin receptors are known to have a widespread distribution (e.g. platelets,
gastrointestinal smooth muscle, uterine smooth muscle, kidneys, the cardiovascular
system and the brain) and the distribution of RSK2 is ubiquitous. Given this, it is likely
that the interaction of RSK2 with the 5-HT2A receptor represents a functionally
significant protein-protein interaction. Our studies suggest that RSK2 associates with 5-
HT2A receptors, regulating signal transduction, and that this regulation is likely though the phosphorylation of the 5-HT2A receptor. As shown in Chapter 4, the phosphorylation of the 5-HT2A receptor in vitro by active RSK2 in combination with the lack of rescue of
the PI hydrolysis phenotype by the introduction of ‘kinase-dead’ RSK2 mutants into the
RSK2 knock-out fibroblasts points to the logical conclusion that the PI hydrolysis
phenotype is due to 5-HT2A receptor phosphorylation. It will require further study to
determine the site(s) of phosphorylation of RSK2 on the 5-HT2A receptor. As shown in
Chapter 4, ‘kinase-dead’ mutants of RSK2 remain associated with the 5-HT2A receptor.
Whether or not the direct interaction of these two proteins has additional functional consequences on 5-HT2A receptor signaling remains to be seen. To address the above possibilities extensive in vitro studies need to be performed. These studies will give us crucial insights into modulation of 5-HT2A receptor function and regulation.
As described in Chapter 1, the 5-HT2A receptor is the site of action of most
(Nichols, 2004; Roth et al., 2002), but not all (Roth et al., 2002; Sheffler and Roth, 2003)
hallucinogens. Importantly, many 5-HT2A receptor antagonists are effective antipsychotic drugs (Meltzer et al., 2004; Roth et al., 2004). As previously described, 5-HT2A receptors
249 play a number of prominent roles in both the central nervous system and in the periphery,
including memory and cognition (Umbricht et al., 2003; Williams et al., 2002), the
modulation of mood and perception, platelet aggregation, and smooth muscle contraction
(Roth et al., 1998b). Dysregulation of 5-HT2A receptor function has been implicated in various mood and psychological disorders, including depression and psychosis. Since 5-
HT2A receptor function is significantly augmented in RSK2 knock-down fibroblasts; one can predict that RSK2 null mutations will cause psychosis due to hyperactive 5-HT2A receptors. Additionally, it has been hypothesized that AADC may become the rate-
limiting step in monoamine synthesis due to expression in the human brain, thereby
limiting the levels of endogenous serotonin. As previously stated, our microarray data
show an increase in expression of AADC in RSK2 knock-out cells. It is worth exploring
the potential role of RSK2 in the regulation of AADC expression, as increased expression
of AADC in the human brain could potentially augment the RSK2 -/- phenotype by
providing higher levels of endogenous serotonin.
Taken together, our microarray analysis study shows that in RSK2 knock out
fibroblasts there is a significant down regulation in the mRNA levels of RSK2, which is
shown to be absent at the protein level by Western blot. Our studies provide evidence for
role of RSK2 in regulating the functional activity of 5-HT2A serotonin receptors in addition to a number of other GPCRs. Our current studies indicate that RSK2 is likely exerting its effect on 5-HT2A receptor signaling through the direct phosphorylation of the
5-HT2A receptor. Whether the effect RSK2 exerts on other GPCR signaling is due to
250 receptor phosphorylation remains to be seen. It will prove interesting to determine the
site(s) of phosphorylation of the 5-HT2A receptor by RSK2. Knowledge of the site(s) of
phosphorylation will allow for the investigation of a number of aspects of 5-HT2A receptor regulation including trafficking, internalization, down-regulation, and desensitization. Answers to these questions will clarify RSK2’s role in serotonergic signaling and provide a further understanding of the clinical features of Coffin-Lowry
Syndrome.
251 CHAPTER 7: Implications and Future Directions
G protein-coupled receptors (GPCRs) are the largest family of cell surface molecules involved in signal transduction, comprising 1-2% of the human genome
(Kroeze et al., 2003b). Importantly, at least one third (Robas et al., 2003) and perhaps as many as half (Flower, 1999) of currently marketed drugs target GPCRs, although only
10% of GPCRs are proven drug targets (Vassilatis et al., 2003). Due to the prevalence of
GPCRs as a drug target, decades of research in both the academic and the private sector have been performed to further characterize GPCR ligand recognition sites and to identify novel GPCR ligands. To this end, drug discovery for GPCRs has primarily relied on serendipity, molecular library screens, comprehensive mutagenesis, and molecular modeling studies. Unfortunately, it has proven difficult to crystallize GPCRs, and to date, only one GPCR, bovine rhodopsin, has had its crystal structure solved (Palczewski et al.,
2000). Even though only one GPCR has had its crystal structure solved, the structure of bovine rhodopsin has proven useful as a template for the molecular modeling of a variety of other GPCRs. Since GPCRs are a common target of drugs, a great deal of research has been performed over the last several decades to advance our understanding of GPCR structure, function, and regulation. Many of these studies have made use of the β- adrenergic receptor as a model for GPCR function and regulation (Lefkowitz et al.,
1998).
Signaling diversity by GPCRs is generated in a variety of ways. As stated in
Chapter 1, GPCRs respond to a large variety of extracellular stimuli including light,
252 neurotransmitters, odorants, biogenic amines, lipids, proteins, amino acids, hormones,
nucleotides, and chemokines (Kroeze et al., 2003b). The paradigm of GPCR signaling
involves the transmission of these extracellular stimuli into an intracellular signal via the
activation and dissociation of heterotrimeric G proteins (Cabrera-Vera et al., 2003). The
heterotrimeric G proteins add to the diversity of GPCR signaling as there are at least 18
different human Gα proteins (Hermans, 2003; Wong, 2003), which form complexes with
Gß subunits and Gγ subunits, of which there are at least 5 types and 11 types, respectively
(Hermans, 2003). However, the vast repertoire of signaling from the varied GPCRs can not be accounted for by classical models of G-protein coupling and activation of second- messenger-generating enzymes (Pierce et al., 2002).
Therefore, there has been an increasing focus on understanding the regulation of
GPCRs by the complex interactions of various intracellular domains of the GPCRs with numerous intracellular proteins (Bockaert et al., 2003; Hall and Lefkowitz, 2002). As stated in Chapter 1, GPCRs have been shown to interact with a number of scaffolding proteins, chaperones, and other accessory proteins (Brady and Limbird, 2002; Hall and
Lefkowitz, 2002). The interaction of GPCRs with these proteins allows the generation of a richer signaling diversity through the alteration of ligand recognition, the
compartmentalization of signaling complexes, and through the regulation of GPCR
trafficking (Brady and Limbird, 2002). Further understanding of the regulation GPCR
signaling via interactions with these scaffolding, accessory, and other proteins will
advance our understanding of the regulation of GPCR signaling and may present novel
targets for drug development and for disease treatment.
253
Although many studies have used ß-adrenergic receptors as prototypical GPCRs,
it has become increasingly clear that much more can be learned by systematic study of
other GPCRs. In our laboratory, we primarily study 5-HT2A receptors, GPCRs that play
crucial roles in the modulation of perception, cognition, and emotion (Jakab and
Goldman-Rakic, 1998; Kroeze and Roth, 1998b; Roth, 1994; Roth et al., 1999a). Our
studies of the 5-HT2A receptor have shown, as described in Chapter 1, that 5-HT2A receptor internalization and desensitization can occur by arrestin-independent pathways in HEK-293 cells (Bhatnagar et al., 2001; Gray et al., 2003a), pointing to cell type specific effects of 5-HT2A receptor regulation which diverge from the ß-adrenergic receptor paradigm. In addition, within the last decade, a number of 5-HT2A receptor interacting proteins (See Chapter 1) have been identified. These 5-HT2A receptor interacting proteins have been demonstrated to have a role in the trafficking, function, and regulation of 5-HT2A receptors (Bhatnagar et al., 2004; Gray et al., 2003a; Robertson
et al., 2003; Turner and Raymond, 2005; Xia et al., 2003a). Since the majority of these
proteins interact with the intracellular loops and the C-terminal tail of the 5-HT2A receptor and the i3 loop is known to be important for G-protein coupling (Hyde and Roth,
1997; Oksenberg et al., 1995), we sought to identify other proteins that could bind to the i3 loop of the 5-HT2A receptor and to investigate the role of these proteins in 5-HT2A receptor signaling.
254 7.1 RSK2 Associates with the 5-HT2A Receptor and Attenuates Signaling
In my studies I identified RSK2 as a potential 5-HT2A receptor-interacting protein
via a yeast two-hybrid screen and confirmed that full-length RSK2 associates with 5-
HT2A receptors in vivo and in vitro through co-immunoprecipitation studies (Chapter 3).
Further immunocytochemical and immunohistochemical studies indicated that 5-HT2A receptors and RSK2 show overlapping-subcellular distributions in HEK-293 cells, in rat brain prefrontal cortex, and in the rat brain globus pallidus (Chapter 3). Together, these studies verified the yeast two-hybrid “hit” and provided evidence that 5-HT2A receptors and RSK2 are expressed in overlapping distributions in vivo, where their interaction may have functional consequences on 5-HT2A receptor regulation. In order to determine the
functional consequences of this protein-protein interaction we wanted to make use of a
system where 5-HT2A receptors and RSK2 are endogenously expressed. We chose this route for our study as there are a number of potential confounds in undertaking ‘protein- protein’ interaction studies in an over-expression system. In over-expression systems, the functional consequences of receptor–G-protein activation may vary from cell to cell, depending on (1) the receptor and its level of expression; (2) the repertoire of effector
molecules expressed within a given cell; and (3) saturation of the system by the
expression of non-physiological levels of the proteins under study. Such complexities
make any results obtained in transfected cell lines difficult to relate to signaling changes
that may occur in vivo. To this end, we were fortuitous to obtain fibroblasts from RSK2
+/+ and RSK2 -/- mice (Bruning et al., 2000), which we determined to endogenously
express similar levels of 5-HT2A receptors via quantitative RT-PCR and radioligand
255 binding studies (Chapter 4). These RSK2 -/- and RSK2 +/+ fibroblasts therefore
provided a model system to study the effects of RSK2 on 5-HT2A receptor signaling.
Characterization of 5-HT2A receptor signaling in RSK2 -/- and RSK2 +/+
fibroblasts led to the discovery that 5-HT2A receptors display augmented signaling in the absence of RSK2. These alterations in 5-HT2A receptor signaling include an
augmentation of PI hydrolysis, augmented Ca2+ mobilization, and an augmentation in
both basal and 5-HT-stimulated p42/p44 ERK phosphorylation (Chapter 4). As
discussed in Chapter 4, there were a number of possible explanations for this phenotype that do not involve a direct effect of RSK2. First, the absence of RSK2 may have led to an alteration in the gene expression of genes involved in serotonergic signaling. Second,
5-HT2A receptor signaling could be enhanced by an increase in 5-HT2A receptor surface
expression in the absence of RSK2. Finally, an augmentation of 5-HT2A receptor- mediated PI hydrolysis could occur via the direct potentiation of G-protein activity in the absence of RSK2. To rule out these possibilities we conducted microarray studies, surface-biotinylation studies, and studies using a constitutively active form of Gαq,
Gαq(Q209L).
The microarray studies we performed demonstrated that no alteration occurs in either sertotonergic signaling genes, in the expression pattern of MAPK genes, or in phosphatase expression (See Chapter 4, Chapter 6, and appendix). Although microarray data only provide mRNA expression of genes, and not protein expression, these data suggest that alterations in 5-HT2A receptor signal transduction apparent in the
256 absence of RSK2 are not due to aberrant gene expression. Next, we performed surface-
biotinylation and PI hydrolysis studies on RSK2 -/- and RSK2 +/+ fibroblasts expressing
FLAG-5-HT2A receptors and found that although RSK2 -/- FLAG-5-HT2A stable lines demonstrated augmented PI hydrolysis compared to RSK2 +/+ FLAG-5-HT2A stable lines, there was no difference in the cell surface expression of FLAG-5-HT2A receptors.
Therefore, the augmentation of 5-HT2A signaling in RSK2 -/- fibroblasts can not be attributed to an increased cell surface expression of 5-HT2A receptors in the absence of
RSK2 (Chapter 4). Finally, we performed studies whereby we transfected either wild-
type Gαq or a constitutively active form of Gαq, Gαq(Q209L), into RSK2 -/- and RSK2 +/+
fibroblasts. These studies demonstrated that the basal signaling by Gαq(Q209L) is not
altered by RSK2 knock-out. Since Gαq(Q209L) signaling was not altered in the absence of RSK2, the augmentation of 5-HT2A receptor signaling does not occur due to direct
potentiation of G-protein activity, and therefore must occur at the level of the receptor.
Together, these studies imply a direct effect of RSK2 on 5-HT2A receptor signaling.
Having verified the direct effect of RSK2 on 5-HT2A receptor signaling, we next
sought to determine the mechanism by which RSK2 augments 5-HT2A receptor signaling.
As described in Chapter 1, RSK2 is a down-stream kinase of the MAPK cascade. It was
equally possible that RSK2 exerts its effects on 5-HT2A receptor signaling simply via a protein-protein interaction or by direct phosphorylation of the 5-HT2A receptor. To address this possibility, we conducted in vitro kinase assays whereby we attempted to phosphorylate immunopurified 5-HT2A receptors with active purified RSK2. As shown in
Chapter 4, we found that 5-HT2A receptors were phosphorylated by RSK2. Since 5-HT2A
257 receptors have more than 30 potential phosphorylation sites (Gray et al., 2003b) it was
not possible within these current studies to determine which of these represent the site(s)
of RSK2 phosphorylation. Therefore, to address the role of the RSK2 kinase activity in
another manner, we re-introduced both wild-type and kinase-dead RSK2 into the RSK2 -
/- fibroblasts and found that only the wild-type RSK2 could revert the 5-HT2A receptor PI hydrolysis phenotype (Chapter 4). The co-immunoprecipitation of kinase-dead RSK2 with the 5-HT2A receptor was found to be identical to wild-type RSK2, demonstrating that
the lack of a phenotype reversion by the kinase-dead RSK2 was not due to a lack of
protein-protein interaction with the 5-HT2A receptor (Chapter 4). Together these studies have demonstrated a novel role for RSK2 in the regulation of 5-HT2A receptor signaling.
7.2 Future Directions
Our current studies indicate that RSK2 is likely exerting its effect on 5-HT2A receptor signaling through the direct phosphorylation of the 5-HT2A receptor. A number of studies will be necessary to determine the site(s) of phosphorylation of the 5-HT2A receptor by RSK2. Initial characterization of the site(s) of phosphorylation can be accomplished through additional in vitro kinase assays. In these assays, each of the intracellular loops and the C-terminal tail of the 5-HT2A receptor can be expressed, purified, and subjected to in vitro kinase assays using active RSK2. Determination of the ability of active RSK2 to phosphorylate a sub-set of these 5-HT2A receptor intracellular domains will greatly reduce the number of potential phosphorylation site(s) that will need to be explored. Following general determination of the intracellular region of the 5-HT2A
258 receptor that is phosphorylated, site-directed mutagenesis studies in combination with
additional in vitro kinase assays will allow the determination of the phosphorylated residues. Alternatively, a proteomics approach could also be used to identify the phosphorylated 5-HT2A receptor residue. For this method, FLAG-5HT2A receptors would
be immunopurified and subjected to an in vitro kinase assay using cold ATP as substrate
with and without the presence of active RSK2. These immunoprecipitated 5-HT2A receptors could then be identified on silver-stained gels, specifically eluted, and the phosphorylated residue(s) identified by mass spectrometry.
A number of additional studies are warranted in the RSK2 -/- and RSK2 +/+ fibroblasts. The role of RSK2 in 5-HT2A receptor desensitization, internalization, and
resensitization has not been explored. Determination of the sites of 5-HT2A receptor phosphorylation will allow the production of stable lines of both wild-type and phosphorylation site(s) mutant 5-HT2A receptors in the RSK2 +/+ fibroblasts. These stable lines will be necessary to characterize the role of 5-HT2A receptor phosphorylation
due to the low expression of 5-HT2A receptors in the RSK2 -/- and RSK2 +/+ fibroblasts.
If the augmentation of 5-HT2A signaling in the absence of RSK2 is entirely due to receptor phosphorylation, these studies should recapitulate the RSK2 -/- phenotype in the
RSK2 +/+ fibroblasts for the 5-HT2A receptor. In addition, these stable lines will provide
epitope-tagged receptors, whose fate upon agonist and antagonist administration can be
more easily tracked via confocal microscopy studies. Finally, higher receptor expression
levels will provide a model system to better study the role of RSK2 in desensitization and
resensitization of 5-HT2A receptors. Answers to these questions will clarify RSK2’s role
259 in serotonergic signaling and provide a further understanding of the clinical features of
Coffin-Lowry Syndrome.
7.3 Implications of the Current Findings
We have identified RSK2 as a novel 5-HT2A receptor interacting protein and a modulator of 5-HT2A receptor signaling. RSK2 appears to act as a “tonic brake” on 5-
HT2A receptor signaling, attenuating PI hydrolysis, calcium mobilization, and p42/44
ERK phosphorylation. RSK2 likely accomplishes these effects through the
phosphorylation of the 5-HT2A receptor, although additional studies are required to
determine the site(s) of 5-HT2A receptor phosphorylation. The augmentation of 5-HT2A receptor signaling in the absence of RSK2 may be due to an increased constitutive activity of 5-HT2A receptors. This is supported our p42/44 ERK studies, wherein we found that the 5-HT2A receptor-selective antagonist, MDL100,907, attenuated the
increased basal p42/44 ERK phosphorylation found in the absence of RSK2.
These studies, together with our co-immunoprecipitation studies demonstrating
agonist-independent association of RSK2 with the 5-HT2A receptor and our studies
demonstrating that RSK2 phosphorylates the 5-HT2A receptor imply that RSK2 may constitutively phosphorylate the 5-HT2A receptor. A constitutive phosphorylation of 5-
HT2A receptors by RSK2 would be in accord with studies that have shown high levels of
basal phosphorylation of 5-HT2A receptors are found in HEK-293 cells when 5-HT2A receptors are over-expressed and that agonist exposure does not increase the level of 5-
260 HT2A receptor phosphorylation (Vouret-Craviari et al., 1995). If RSK2 indeed constitutively phosphorylates 5-HT2A receptors, these results make sense given the high
expression of RSK2 is in HEK-293 cells. Therefore, identification of the site(s) of 5-
HT2A receptor phosphorylation, as described above, will allow further characterization of
the role of RSK2 phosphorylation in 5-HT2A receptor regulation. Hypothetically, a constitutive phosphorylation of the 5-HT2A receptor may decrease the constitutive
activity of the 5-HT2A receptor by forcing a “pre-desensitized” state of the 5-HT2A receptor, which would display reduced signal transduction capacity. Hence one potential role of RSK2 could be to restrain 5-HT2A receptor signaling.
As described in Chapter 1, the 5-HT2A receptor is the site of action of most
(Nichols, 2004; Roth et al., 2002), but not all (Roth et al., 2002; Sheffler and Roth, 2003)
hallucinogens and the site of action of many antipsychotic drugs (Meltzer et al., 2004;
Roth et al., 2004). It has been suggested that the inability of pyramidal neurons to
attenuate firing in the absence of stimuli, due to either hyperactive 5-HT2A receptors or
abnormally high levels of 5-HT2A receptors, mediates some of the psychotic symptoms in schizophrenia (Jakab and Goldman-Rakic, 1998). Importantly, as described in Chapter
1, substantial RSK2 expression is detected through-out the neocortex with the strongest detection in layers V and VI (Zeniou et al., 2002a), where 5-HT2A receptors are also highly expressed (Willins et al., 1997b). It has been hypothesized that RSK1 and RSK3 may not be able to compensate for the lack of RSK2 in these brain regions due to the absence of RSK1 and RSK3 mRNA transcripts in these brain regions (Prabhakaran et al.,
2000; Shimamura, 1995; Zeniou et al., 2002a). Therefore, since 5-HT2A receptor function
261 is significantly augmented in RSK2 knock-down fibroblasts; one can predict that RSK2
null mutations may cause psychosis due to hyperactive 5-HT2A receptors present in pyramidal neurons. This is an intriguing hypothesis given that female heterozygote carriers of Coffin-Lowry Syndrome have an incidence of psychotic behavior, schizophrenia, and depressive psychosis (Manouvrier-Hanu et al., 1999). However, a great deal of additional study will be necessary to fully appreciate the role of RSK2 in the regulation of 5-HT2A receptors.
7.4 The Alteration of GPCR Signaling by RSK2
In addition to our studies with the 5-HT2A receptor, we also chose to explore the
role of RSK2 in the signaling of a variety of other GPCRs. We made use of our
previously conducted microarray studies to identify a number of GPCRs expressed in
RSK2 -/- and RSK2 +/+ fibroblasts (Chapter 5). From these studies we demonstrated
that RSK2 knock-out augments adenosine 5’-triphosphate (ATP)-stimulated P2Y-
purinergic receptor, bradykinin-stimulated bradykinin-B receptor, and thrombin receptor
activating peptide (TRAP)-stimulated PAR-1 thrombin receptor signaling, as measured
by PI hydrolysis (Chapter 5). When we measured calcium signaling by these Gαq- coupled GPCRs, we found variable effects of RSK2 knock-out on signaling. In addition, we also found variable effects on agonist potency for both PI hydrolysis and calcium mobilization, implying that RSK2 does not play the same role for all GPCRs. In accord with this, three patterns emerged for PI hydrolysis and calcium mobilization, one characterized by 5-HT2A and P2Y receptors, a second class characterized by the PAR-1
262 thrombin receptor, and a final class characterized by the Bradykinin-B receptor, implying
that RSK2 does not regulate all GPCRs in the same manner. Interestingly, our microarray
data show a 2.13 fold increase in PAR-1 (F2r) expression in the absence of RSK2 (See
Chapter 6), and this increase in receptor expression may account for the alterations seen
in PAR-1 receptor signaling.
We also found that isoproterenol-stimulated β1-adrenergic receptor, a Gαs- coupled GPCR, signaling was augmented in the RSK2 knock-out fibroblasts, demonstrating that the effects of RSK2 on GPCRs signaling are not restricted to Gαq- coupled GPCRs (Chapter 5). Taken together these findings describe an entirely novel mode of GPCR regulation. Our studies on PAR-1-thrombinergic, P2Y-purinergic,
Bradykinin-B, and β1-adrenergic receptors only provide an initial characterization of the role of RSK2 in the signaling by these GPCRs. Therefore, in order to determine the role of RSK2 in PAR-1, P2Y, Bradykinin-B, and β1-adrenergic receptor signaling, it will be necessary to conduct additional studies with these GPCRs, similar to those conducted with the 5-HT2A receptor in order to rule-out non-specific effects of RSK2 knock-out.
7.5 Microarray Data Implications
As discussed in Chapter 1, individuals with Coffin-Lowry Syndrome have an
increased risk of cardiac abnormalities and cardiomyopathy, suggesting an indispensable
role for RSK2 in cardiovascular function (Hunter, 2002). Coffin-Lowry Syndrome
patients also show a variety of skeletal deformations, including slower bone growth.
263 Indeed, RSK2 has been recently been shown to play an indispensable role in osteoclast
function, primarily through the activation of the transcription factor ATF4 (Yang et al.,
2004). The microarray studies that we conducted in RSK2 -/- and RSK +/+ fibroblasts identified the dysregulation of a number of genes that may be involved in the pathogenesis of Coffin-Lowry Syndrome, specifically in cardiovascular and bone development and function These dysregulated genes include the endothelin receptor A
(ETA), the protease-activated receptor-1 (PAR-1). The Prostaglandin E receptor 4,
subtype EP4 (EP4), and the Parathyroid hormone receptor 1 (Pthr1) (Chapter 6).
As discussed in Chapter 6, endothelin (ET) stimulation has a number of
cardiovascular effects including coronary vasoconstriction, the secretion of atrial
natiuretic peptide and nitric oxide, and the facilitation of cardiac hypertrophy (Hu et al.,
1988; Ishikawa et al., 1988; Moravec et al., 1989). In addition, ET receptor antagonists
have been shown to improve cardiovascular dysfunction and to prolong the survival of
experimental animals with congestive heart failure (Sakai et al., 1996). We found an up-
regulation of ETA receptor mRNA in RSK2 -/- fibroblasts in our studies (Chapter 6).
Since ET receptor antagonists improve cardiovascular dysfunction, this implies that an increase in ET receptor expression would be detrimental to cardiac function.
In addition to an increase in ETA mRNA, we also found an increase in PAR-1 mRNA in RSK2 -/- fibroblasts. As described in Chapter 6, the activation of the PARs plays a role in a number of physiologic processes including both vasodilation and vasoconstriction and bone cell resorption (Flynn and Buret, 2004), are highly expressed
264 in the heart, and have many cardiovascular functions (Steinberg, 2005). In addition, PAR signaling largely accounts for platelet activation by thrombin and the downstream signaling by the PARs play a role in both hemostasis and thrombosis (Coughlin, 2000).
Intriguingly, the PAR-1 receptor may also contribute to blood vessel development as
50% of PAR-1 knockout mice die between embryonic days 9.5 and 10.5 due to defects in vascular integrity (Connolly et al., 1996). Due to the role of the PAR-1 receptor in both cardiac function as well as bone resorption and the role of ETA in cardiac function, further investigation into the role of RSK2 in regulating these receptors may provide additional insights into a variety of the cardiovascular conditions associated with Coffin-
Lowry Syndrome.
Our microarray studies have also indicated a down-regulation of both prostaglandin E4 receptors and the Pthr1 receptor. As previously described in Chapter 6, prostaglandin EP2 and EP4 agonists have been shown to stimulate bone growth and EP2 and EP4 knockout mice have bone growth deficiencies, showing a potential role of the
EP2 and EP4 receptors in osteoclast mediated bone formation (Yoshida et al., 2002). The decrease in prostaglandin EP4 receptor mRNA found in the absence or RSK2 would be expected to hinder bone-growth if this decrease is carried though to the protein level. It has also been well established that the Pthr1 receptor plays a role in endochondral bone formation and in osteoclast function. This is intriguing in light of the role RSK2 plays in osteoclast function, primarily through the activation of the transcription factor ATF4
(Yang et al., 2004). The investigation of any possible links between RSK2 and the function of the Pthr1 receptor or the prostaglandin EP4 receptor this may provide greater
265 insight into the skeletal defects of Coffin-Lowry Syndrome. Together these data
demonstrate that dysregulation of GPCR expression may also account for a number of the
physiological dysfunctions found in Coffin-Lowry Syndrome.
7.6 Final Words
In this thesis, I have highlighted the importance of RSK2 in the regulation of 5-
HT2A receptor signal transduction. Although my studies clearly demonstrate an interaction of 5-HT2A receptors with RSK2 and the in vitro phosphorylation of 5-HT2A receptors by RSK2, the exact mechanism by which RSK2 regulates 5-HT2A signaling remains elusive. Examination of the role of RSK2 in other Gαq- and Gαs-coupled GPCR signaling revealed that all GPCRs are not regulated in the same manner as the 5-HT2A receptor by RSK2. These differences in GPCR regulation by RSK2 provide yet another level of complexity to GPCR signal transduction, which we have only begun to understand. This novel pathway of GPCR regulation will provide new opportunities for the development of strategies to therapeutically manipulate GPCR function, providing additional avenues for drug development and for the understanding of diseases associated with dysfunction in GPCR signaling.
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