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THE REGULATION of G PROTEIN-COUPLED RECEPTOR (GPCR) SIGNAL TRANSDUCTION by P90 RIBOSOMAL S6 KINASE 2 (RSK2)

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THE REGULATION of G PROTEIN-COUPLED RECEPTOR (GPCR) SIGNAL TRANSDUCTION by P90 RIBOSOMAL S6 KINASE 2 (RSK2) 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
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