Regulation of the Dopamine D1-D2 Receptor Heterooligomer

Home , Cholecystokinin receptor, GPCR oligomer, Smoothened

Vaneeta Verma

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Pharmacology and Toxicology University of Toronto

Regulation of the Dopamine D1-D2 Receptor Heterooligomer

Vaneeta Verma

Doctor of Philosophy

Department of Pharmacology and Toxicology

University of Toronto

2011

ABSTRACT

Dopamine receptors are members of the G protein-coupled receptor superfamily and play important roles in neuronal transmission. A D1-D2 receptor heterooligomer generating a G- protein linked PLC-dependent intracellular calcium signal was previously identified. The discovery of this dopamine mediated calcium signal implicated a direct link between dopamine receptors and calcium generation, but its regulation remained to be elucidated. By measuring calcium signaling with Fluo-4 fluorescence or cameleon FRET, rapid desensitization of the calcium signal in heterologous cells and striatal neurons was demonstrated by pre-treatment with SKF 83959, which selectively activates D1-D2 receptor heteromers, or SKF 83822 which only activates D1 receptor homooligomers. Although SKF 83822 was unable to activate D1-D2 receptor heteromers, it still permitted desensitization of the calcium signal. This suggested that occupancy of the D1 receptor binding pocket by SKF 83822 resulted in conformational changes sufficient for desensitization without activation of the heteromer. BRET and co- immunoprecipitation studies indicated an agonist induced interaction between the D1-D2 receptor heteromer and GRK2. Increased expression of GRK2 led to a decrease in the calcium signal and decreased expression of GRK2 led to an increased calcium signal. Expression of the catalytically inactive and RGS mutated GRK2 constructs each led to a partial recovery of the GRK2-attenuated calcium signal. These results indicated that desensitization of the D1-D2 receptor heteromer mediated calcium signal can occur by agonist occupancy even without activation and is regulated by two distinct functions of GRK2. Immunocytochemistry and calcium assays demonstrated that recycling of internalized D1 and D2 receptors and ii resensitization of the desensitized calcium signal occurred after dopamine pre-treatment but not SKF 83959, suggesting that the trafficking and resensitization response associated with the D1- D2 receptor heteromer is differentially regulated by specific ligands. Overall, these results suggest that D1-D2 receptor heterooligomers are uniquely regulated from their constituent receptors which are not coupled to Gq.

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ACKNOWLEDGEMENTS

First and foremost, I would like to extend my sincerest gratitude to my supervisor, Dr. Susan George. The outstanding mentorship I received from her over the years has taught me valuable lessons that I will carry forward throughout my career. Her expertise and guidance have been instrumental to my successes that I have achieved during my doctoral studies and have helped me attain skills that are key for becoming a strong, independent scientist. Dr. George has been an inspiration to me and working under her supervision has been an honor and a privilege. I would also like to thank my co-supervisor, Dr. Brian O’Dowd. I appreciate his profound insight, mentorship and guidance that have been immensely beneficial to this work. Thank you to the funding agencies that helped support this research. These include the Natural Sciences and Engineering Research Council of Canada, Peterborough K.M Hunter Graduate Scholarship, Canadian Institutes of Health Research and the National Institutes of Health. I would also like to thank my committee members, Dr. Denis Grant and Dr. Peter McPherson, as well as my examination committee, Dr. Ali Salahpour, Dr. Denis Grant, Dr. Scott Heximer, Dr. Jose Nobrega, and Dr. Stephen Ferguson for their valuable advice, insightful comments and providing different perspectives on this work. A special thanks to all of the members of the lab for creating a friendly and motivating environment to work in. Specifically, Theresa Fan, Tuan Nguyen, and Tony ji for providing me with exceptional technical assistance. Their skills, time and energy are highly appreciated and greatly contributed to this work. I would also like to send a special thanks to Dr. Melissa Perreault, Dr. Ahmed Hasbi, Dr. Christopher So, Dr. Asim Rashid, and Dr. Michael Kong. They have been my mentors and friends, always giving me sound advice and never hesitating to provide either an ear to listen or a hand to help. I am very grateful for their efforts which were pivotal to the success of this work. I would like to thank my family for their constant patience and encouragement and always believing in me. Last but not least, I would like to thank my friends for always being there for me in good times and in bad, helping make my time at graduate school fun and enjoyable and providing great memories that will last a life time.

TABLE OF CONTENTS

ABSTRACT ii

AKNOWLEDGEMENTS iv

TABLE OF CONTENTS v

LIST OF PUBLICATIONS viii

LIST OF FIGURES ix

SUMMARY OF ABBREVIATIONS xii

1 INTRODUCTION 1

1.1 Overview of G protein-Coupled Receptors 1 1.1.1 G Proteins 2 1.2 Control of Receptor Signaling 4 1.2.1 Agonist-dependent GPCR Desensitization 4 1.2.2 Agonist-dependent GPCR Internalization 7 1.2.3 GPCR Recycling and Degradation 9 1.3 Introduction to GPCR Oligomerization 10 1.3.1 Evidence for GPCR Oligomerization 11 1.3.2 GPCR Stoichiometry 12 1.3.3 Structural Features of GPCR Oligomers 14 1.4 Regulation of GPCR Oligomers 16 1.4.1 Agonist-dependent Desensitization of GPCR Oligomers 16 1.4.2 Agonist-dependent Internalization of GPCR Oligomers 17 1.4.3 Post Endocytic Sorting of GPCR Oligomers 19 1.4.4 Stability of GPCR Oligomers 20 1.5 Dopamine 21 1.5.1 Dopamine Pathways and Functions 22 1.6 Dopamine Receptors 23 1.7 Dopamine D1 Receptors 24 1.7.1 Cellular Signaling of D1 Receptors 24 1.7.2 Desensitization of D1 Receptors 27 1.7.3 Internalization of D1 Receptors 29 1.7.4 Resensitization and Recycling of D1 Receptors 32 1.7.5 D1 Receptor Homooligomers and Heterooligomers 33 1.8 Dopamine D2 Receptors 35 1.8.1 Cellular Signaling of D2 Receptors 35 1.8.2 Desensitization of D2 Receptors 35 1.8.3 Internalization of D2 Receptors 37

1.8.4 Resensitization and Recycling of D2 Receptors 39 1.8.5 D2 Receptor Homooligomers and Heterooligomers 39 1.9 The D1-D2 Receptor Heterooligomer 41 1.9.1 D1-D2 Receptor Heterooligomers in vitro and in vivo 42 1.9.2 Activation of the D1-D2 Receptor Heterooligomer in Striatum 43 1.9.3 Functional Consequences of D1-D2 Receptor Heterooligomer 44 Mediated Signaling 1.9.4 Pharmacology of the D1-D2 Receptor Heterooligomer 46 1.9.5 D1-D2 Receptor Heterooligomer Desensitization 49 1.9.6 D1-D2 Receptor Heterooligomer Internalization 50 1.10 Research Rationale and Objectives 50

2 MATERIALS AND METHODS 53

2.1 Cell Culture 53 2.2 Transient Transfections in HEK293T Cells 53 2.3 Measurement of the Calcium Signal in HEK293T Cells 54 2.4 Membrane Preparation and Radioligand Saturation Binding Assay 55 2.5 Intact Cell Radioligand Binding Assay 56 2.6 Immunocytochemistry of HEK293T Cells 56 2.7 Neuronal Cultures 57 2.8 Immunocytochemistry of Cultured Neurons 58 2.9 Confocal Microscopy FRET 58 2.10 Measurement of the Calcium Signal in Primary Striatal Neurons 59 2.11 Immunoprecipitation 61 2.12 BRET Assay 62 2.13 SDS-Polyacrylamide Gel Electrophoresis 62 2.14 Statistical Analysis 63

3 RESULTS 64

3.1 Activation of the D1-D2 Receptor Heteromer Mediated Calcium Signal 64 in HEK293T Cells 3.2 Desensitization of the D1-D2 Receptor Heteromer Mediated Calcium Signal 64 in HEK293T Cells 3.2.1 Desensitization Through Selective Occupancy 71 of the D1 Receptor 3.3 Activation of the D1-D2 Receptor Heteromer Mediated Calcium Signal 74 in Primary Striatal Neurons 3.4 Desensitization of the D1-D2 Receptor Heteromer Mediated Calcium Signal 77 in Primary Striatal Neurons 3.5 Role of GRK2 in Regulating the D1-D2 Receptor Heteromer Mediated 80 Calcium Signal 3.5.1 Evaluation of GRK2 Functional Domains in Regulating 80 the D1-D2 Receptor Heteromer Mediated Calcium Signal

3.5.2 Knockdown of GRK2 in HEK293T Cells and Striatal Neurons 85 3.5.3 D1-D2 Receptor Heteromer Interaction with GRK2 85 3.6 Resensitization of the D1-D2 Receptor Heteromer Mediated Calcium Signal 92 in HEK293T Cells 3.7 Internalization and Recycling of D1 and D2 Receptors Following Treatment 92 with Dopamine or SKF 83959

4 DISCUSSION 98

4.1 Desensitization of the D1-D2 Receptor Heteromer Mediated Calcium Signal 98 4.2 Internalization of the D1-D2 Receptor Heteromer and Resensitization of the 105 Associated Calcium Signal 4.3 Related Studies 109 4.4 Novel Findings and General Conclusions 110 4.5 Significance and Future Studies 115 4.6 Concluding Remarks 124

5 REFERENCES 125

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LIST OF PUBLICATIONS

1. Kong MM*, Verma V*, O’Dowd BF, George SR (2011). The role of palmitoylation in directing dopamine D1 receptor internalization through selective endocytic routes. Biochem Biophys Res Commun. 405:445-9 * These authors contributed equally to this work

2. Verma V, Hasbi A, O’Dowd BF, George SR (2010). Dopamine D1-D2 receptor heteromer- mediated calcium release is desensitized by D1 receptor occupancy with or without signal activation: dual functional regulation by G protein-coupled receptor kinase 2. J Biol Chem. 285:35092-103.

3. Perreault M*, Verma V*, O’Dowd BF, George SR (2009). Regulation of dopamine receptor trafficking and responsiveness. (Review) The Dopamine Receptors, Second Edition. * These authors contributed equally to this work

4. So CH, Verma V, Alijaniaram M, Cheng R, Rashid AJ, O'Dowd BF, George SR (2009). Calcium signaling by dopamine D5 receptor and D5-D2 receptor heterooligomers occurs by a mechanism distinct from that for dopamine D1-D2 receptor heterooligomers. Mol Pharmacol 75: 843-54.

5. So CH, Verma V, O’Dowd BF, George SR (2007). Desensitization of the dopamine D1 and D2 receptor hetero-oligomer mediated calcium signal by agonist occupancy of either receptor. Mol Pharmacol 72:450-62.

6. Rashid AJ, O’Dowd BF, Verma V, George SR (2007). Neuronal Gq/11 coupled dopamine receptors: an uncharted role for dopamine (Review). Trends in Pharmacology 28:551-5.

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LIST OF FIGURES

Figure 1-1. Schematic diagram of the classical regulation of GPCR signaling. 6

Figure 1-2. General structure of a GRK protein. 8

Figure 1-3. Schematic diagram of the dopamine D1 receptor. 25

Figure 1-4. Schematic diagram of the dopamine D2 receptor. 26

Figure 1-5. Differential agonist activation of the D1-D2 receptor 48 heterooligomer and D1 receptor homooligomer

Figure 3-1. Specificity of dopamine receptor agonists activating the 65 D1-D2 receptor heteromer calcium signal in cells stably expressing the D1 and D2 receptors.

Figure 3-2. The D1-D2 receptor heteromer mediated calcium signal is 67 desensitized by prior treatment with dopamine agonists for 5 min.

Figure 3-3. The D1-D2 receptor heteromer mediated calcium signal is 68 desensitized by prior treatment with dopamine agonists for 10 min.

Figure 3-4. The D1-D2 receptor heteromer mediated calcium signal is 69 desensitized by prior treatment with dopamine agonists for 30 min.

Figure 3-5. The ATP induced calcium signal is not reduced by prior treatment 70 with agonist, SKF 83959

Figure 3-6. Effect of the adenylyl cyclase inhibitor, SQ 22536, on the SKF 83822 72 induced desensitization of the D1-D2 receptor heteromer mediated calcium signal.

Figure 3-7. Effect of dopamine receptor antagonists on the desensitization 73 of the D1-D2 receptor heteromer mediated calcium signal in D1-D2 receptor stably expressing cells.

Figure 3-8. Effect of D2 receptor agonist, quinpirole, on the desensitization 75 of the D1-D2 receptor heteromer mediated calcium signal in HEK 293T cells stably expressing D1 and D2 receptors.

Figure 3-9. Specificity of dopamine receptor agonists activating the 76 D1-D2 receptor heteromer calcium signal in primary striatal neurons.

Figure 3-10. The D1-D2 receptor heteromer mediated calcium signal is desensitized 78 in striatal neurons by prior treatment with dopamine agonists for 30 min.

Figure 3-11. Effect of dopamine receptor antagonists on the desensitization 79 of the D1-D2 receptor heteromer mediated calcium signal in striatal neurons.

Figure 3-12. Increased expression of GRK2 led to a concentration dependent 81 decrease of the D1-D2 receptor heteromer activated calcium signal.

Figure 3-13. Effect of catalytic domain mutated or RGS domain mutated GRK2 83 on the D1-D2 receptor heteromer mediated calcium signal.

Figure 3-14. Effect of GRK2, catalytic domain mutated or RGS domain mutated 84 GRK2 on the D1-D2 receptor heteromer mediated calcium signal after agonist pretreatment with dopamine agonists for 30 min.

Figure 3-15. Decreased expression of GRK2 by siRNA led to significant 86 recovery of the D1-D2 receptor heteromer mediated calcium signal after pretreatment with either SKF 83959 or SKF 83822 in the HEK 293T D1-D2 receptor heteromer stable cell line.

Figure 3-16. Decreased expression of GRK2 by siRNA led to significant 87 recovery of the D1-D2 receptor heteromer mediated calcium signal after pretreatment with either SKF 83959 or SKF 83822 in striatal neurons.

Figure 3-17. Immunocytochemistry of striatal neurons in culture 88 showing endogenously expressed GRK2 localization before and after exposure to either 100nM dopamine, SKF 83959 or SKF 83822 for 5 min.

Figure 3-18. Co-immunoprecipitation of HA-D1 receptor and GRK2 90 with Flag-D2 receptor from P2 membranes expressing Flag-D2 receptor, HA-D1 receptor and GRK2 after the HEK 239T cells were treated with vehicle, 1 μM dopamine, SKF 83959, or SKF 83822 for 5 min.

Figure 3-19. BRET detection of Rluc-D1 and GFP-GRK2 interaction after 91 either 1 or 10 min treatment with either vehicle, 1 μM dopamine, SKF 83959, or SKF 83822.

Figure 3-20. Resensitization of the D1-D2 receptor heteromer mediated 93 calcium signal in HEK 293T cells stably expressing D1 and D2 receptors.

Figure 3-21. Trafficking of the D1 and D2 receptors after treatment with 94 dopamine in HEK 293T cells stably expressing D1 and D2 receptors. x

Figure 3-22. Trafficking of the D1 and D2 receptors after treatment with 95 SKF 83959 in HEK 293T cells stably expressing D1 and D2 receptors..

Figure 3-23. Agonist induced internalization of the D1 receptor in HEK 293T 97 cells expressing the D1 receptor alone or co-expressing both the D1 and D2 receptors.

SUMMARY OF ABBREVIATIONS

AFM Atomic Force Microscopy

AFU Absolute Fluorescence Units

AMPH Amphetamine

ATP Adenosine Triphosphate

BDNF Brain Derived Neurotrophic Factor

BRET Bioluminescence Resonance Energy Transfer

CaMKII Ca2+ / Calmodulin Dependent Kinase II cAMP Cyclic Adenosine Monophosphate cDNA Complementary Deoxyribonucleic Acid

CFP Cyan Fluorescent Protein

CHO Chinese Hamster Ovary

D2L Dopamine D2 Long

D2S Dopamine D2 Short

ECL Enhanced Chemiluminescence

EGTA Ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid

FRET Fluorescence Resonance Energy Transfer

FRAP Fluorescence Recovery After Photobleaching

GABA γ-aminobutyric acid

GASP G Protein Coupled Receptor Associated Sorting Protein

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GDP Guanosine Diphosphate

GFP Green Fluorescent Protein

GPCR G Protein-Coupled Receptor

GRK G Protein Receptor Kinase

GTP Guanosine Triphosphate

HA Hemagglutinin

HBSS HEPES-buffered Saline

HEK Human Embryonic Kidney

HEPES 4-(2-hydroxyethyl)-1-Piperazineethanesulfonic Acid

H89 N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline

IC3 Intracellular Loop

IP3 Inositol 1,4,5 Trisphosphate

L-Dopa L-dihydroxyphenlalanine

MAPK Mitogen-activated Protein Kinase

PBS Phosphate Buffered Saline

PH Pleckstrin Homology

PKA Protein Kinase A

PKC Protein Kinase C

PMCAs Plasma Membrane Calcium-ATPases

PLC Phospholipase C

PSD-95 Post Synaptic Density-95 xiii

PTX Pertussis Toxin

RGS Regulator of G Protein Signaling

Rluc Renilla luciferase

SDS Sodium Dodecyl Sulfate

SKF 81297 6-chloro-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol

SKF 83822 ([R/S]-6-chloro-7, 8-dihydroxy-3-allyl-1-[3-methyl-phenyl]-2,3,4,5-tetrahydro- 1H-3-benzazepine)

SKF 83959 6-chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3- benzazepine

SEM Standard Error of the Mean siRNA Small Interfering Ribonucleic Acid

SQ 22536 9-(Tetrahydro-2-furanyl)-9H-purin-6-amine

TM Transmembrane Domain

YFP Yellow Fluorescent Protein

VTA Ventral Tegmental Area

ZIP Zeta interacting protein

xiv 1

1 INTRODUCTION

1.1 Overview of G protein-Coupled Receptors

Heptahelical G protein-coupled receptors (GPCRs) form a large superfamily of cell- surface receptors that respond to a diverse array of sensory and chemical stimuli, such as light, odors, hormones and neurotransmitters. There are over 800 GPCRs in the human genome making them the largest superfamily of plasma membrane proteins involved in signal transduction (Lagerstrom and Schioth, 2008).

All GPCRs have a structurally similar core of seven transmembrane (TM) α helices, three intracellular loops, three extracellular loops, an amino terminal extracellular domain and a carboxyl terminal intracellular domain. Crystallography of the prototypical family 1 GPCR, rhodopsin, in 2000 represented a milestone in the GPCR field and revealed that the TMs are arranged in a ring-like fashion, forming a tightly packed helical bundle (Palczewski et al., 2000).

In the last ten years several other GPCRs have been crystallized, including the human β2 adrenergic (Cherezov et al., 2007; Rasmussen et al., 2007), turkey β1 adrenergic (Warne et al.,

2008), human A2A adenosine (Jaakola et al., 2008), human CXCR4 chemokine (Wu et al., 2010) and the human D3 dopamine receptor (Chien et al., 2010).

The GPCR superfamily is classified into six main families or classes determined by protein sequence similarity (Davies et al., 2007). These include family 1 or class A rhodopsin like, family 2 or class B secretin like, family 3 or class C metabotropic glutamate, family 4 or class D pheromone, family 5 or class E cAMP, and family 6 or class F frizzled/smoothened receptors. Family 1 is the largest and most studied of the classes, accounting for ~80% of the entire GPCR superfamily. Family 2 is much smaller with approximately 53 receptors, including

2 receptors for the gastrointestinal peptide hormone family, corticotrophin-releasing hormone,

calcitonin and parathyroid hormone. Family 3 has only approximately 19 receptors including γ-

aminobutyric acid (GABA) receptors, metabotropic glutamate receptors, calcium sensor

receptors and a few taste receptors (Foord et al., 2005). Families 4, 5 and 6 are additional minor

classes that are considerably smaller (Davies et al., 2007).

1.1.1 G Proteins

Agonist stimulated GPCRs undergo conformational rearrangements that allow their

activation of G proteins which leads to modulation of different intracellular effectors. The

classical model of signal transduction cascades mediated by GPCRs involves the binding of an

extracellular ligand to the receptor binding pocket. This leads to a conformational change of the

receptor and allows activation of heterotrimeric G proteins. Heterotrimeric G proteins consist of

one Gα, Gβ and Gγ subunit. There are 27 Gα subunits, 5 Gβ subunits, and 14 Gγ subunits in

total. Receptor activation catalyzes the exchange of guanosine diphosphate (GDP) for guanosine

triphosphate (GTP) on the guanine nucleotide binding/GTPase domain of inactive Gα subunits.

This results in the dissociation of the Gα-GTP and Gβγ subunits, which can then activate or

inhibit various intracellular effectors or enzymes, such as adenylyl cyclases, guanylyl cyclases,

phospholipases and ion channels (Wess, 1998).

Four main Gα families have been described that are based on the primary sequence similarity of the Gα unit. These include Gαs, Gαi, Gαq and Gα12. The Gαs family contains both

Gαs and Gαolf proteins. The enzyme adenylyl cyclase is activated by Gαs protein subtypes,

resulting in an increased production of cAMP by the enzyme. Gαolf proteins have specific

3 expression in the olfactory epithelia and striatum. The Gi family includes Gαi, Gαo, Gαt and

Gαz proteins. Adenylyl cyclase production is inhibited by the activation of Gαi/o protein subtypes. Except for Gαz, members of the Gαi family are pertussis toxin (PTX) sensitive. Gαi proteins are inactivated by PTX through ADP-ribosylation, which prevents Gαi proteins from coupling to the GPCR (Milligan and Kostenis, 2006). The Gαq family consists of Gαq, Gα11,

Gα14, Gα15, and Gα16. All Gαq proteins are activators of phospholipase C-β (PLC) resulting in production of the second messengers, inositol (1,4,5)-trisphosphate (IP3) and diacylglycerol.

The Gα12 family consists of Gα12 and Gα13 proteins and regulate a group of Rho guanine nucleotide exchange factors (RhoGEFs). While full characterization of Gα12/13 still remains incomplete, accumulating evidence indicates that Gα12/13-mediated signaling pathways are involved in a variety of physiological processes, including embryonic development, cell growth, cell polarity and migration, angiogenesis, platelet activation, the immune response, apoptosis, and neuronal responses (Suzuki et al., 2009).

There are 5 Gβ and 14 Gγ subunits in total. The Gβ and γ subunits form a functional unit that can only be dissociated by denaturing conditions. Distinct combinations of Gβγ subunits result in specific signaling activity and there are currently a number of proteins that either

interact with or are regulated by Gβγ subunits. Many of the processes dependent on Gβγ

downstream signaling are sensitive to PTX, which selectively modifies the Gαi proteins

(Smrcka, 2008). For example, Gβγ subunits released from Gαi heterotrimers can activate PLCβ.

Although most Gβγ-dependent signaling appears to arise from Gαi proteins, there are a few

examples of PTX-insensitive processes reported to be mediated by Gβγ subunits, as suggested

for the M3 muscarinic Gq coupled receptor (Stehno-Bittel et al., 1995).

4 1.2 Control of GPCR Signaling

GPCR signaling is also regulated at the receptor level by distinct processes that include

desensitization, internalization and resensitization. Desensitization is an adaptive process used

by cells to wane membrane signaling and internalization refers to the removal of GPCRs from

the cell surface. The receptors can then recycle back to the plasma membrane allowing for

resensitization of the signal or be retained in intracellular compartments or be targeted for

degradation. These processes are central to the continued maintenance or resultant termination

of the receptor mediated signal (reviewed by Ferguson, 2001).

Several other mechanisms are also used to regulate GPCR signaling. Blocking the

function of presynaptic neurotransmitter transporters that serve to remove endogenous ligands

from the extracellular space surrounding a GPCR increases receptor activation. Additionally,

extracellular ligands are degraded by enzymes, such as acetylcholinesterases to control

transmitter levels (Appleyard, 1994). Furthermore, termination of GPCR signaling can occur at

the level of the G protein by interacting with Regulator of G Proteins Signaling (RGS) proteins,

which increase the rate of hydrolysis of GTP that is bound to Gα subunits and therefore

decrease signaling (reviewed by Hollinger and Hepler, 2002 and Ross and Thomas, 2001).

1.2.1 Agonist-dependent GPCR Desensitization

Activation of a GPCR by its agonist can initiate a process known as desensitization, an adaptive process used by cells to wane membrane signaling and avoid potentially harmful effects that can result from excessive cell stimulation. There are two types of desensitization, homologous and heterologous. Homologous desensitization is an agonist dependent process, with only the activated receptors desensitized. Heterologous desensitization refers to the activation of

5 one receptor leading to a decreased response of an unrelated, unactivated receptor. In either type, there is functional uncoupling of the receptor from heterotrimeric G proteins in response to receptor phosphorylation at serine and threonine residues on intracellular domains and the carboxyl terminal domain (reviewed by Ferguson, 2001). A schematic of the classical regulation of GPCR signaling is presented in Figure 1-1. GPCR phosphorylation is mediated by both second messenger dependent kinases, such as cAMP-dependent protein kinase (PKA) and calcium activated protein kinase C (PKC), and second messenger independent kinases, such as G protein- coupled receptor kinases (GRKs), casein kinases I and II and tyrosine kinases (Bohm et al.,

1997). Second messenger activated protein kinase phosphorylation of GPCRs is generally involved in heterologous desensitization and phosphorylation by GRKs is involved in homologous desensitization (Bunemann et al., 1999).

There are seven known GRK subtypes based on sequence and functional similarity

(Sterne-Marr and Benovic, 1995). These kinases phosphorylate agonist occupied GPCRs, thus mediating homologous desensitization. GRK2, GRK3, GRK5, and GRK6 account for the regulation of most GPCRs throughout the body (Gainetdinov et al., 2004). All GRKs contain an amino terminal RGS-like domain, a central protein kinase domain, and a variable carboxyl terminal pleckstrin homology (PH) domain (Figure 1-2). Some GRKS also induce receptor desensitization independent of receptor phosphorylation. For example, the RGS domains of both

GRK2 and GRK3 have been demonstrated to interact with Gq proteins, and therefore sequestering them and making them unavailable for further signaling (Carman et al., 1999).

Additionally, the carboxyl terminal domain of GRK2 binds to Gβγ subunits to sequester these proteins (Tobin, 2002). GRKs can also serve as adaptor proteins that facilitate receptor internalization by interacting with endocytic machinery components such as clathrin (Mangmool et al., 2006).

Figure 1-1: Schematic diagram of the classical regulation of GPCR signaling. (1) Desensitization of GPCRs results from the binding of arrestins to agonist occupied receptors following phosphorylation of the receptor by GRKs. Arrestin binding leads to receptor and heterotrimeric G protein uncoupling resulting in termination of signaling by effectors. Arrestins also act as adaptor proteins, binding to components of the clathrin endocytic machinery including clathrin and adaptin, AP-2. (2) GPCR sequestration occurs via dynamin and clathrin coated pits. Once internalized, the receptor exhibits either a Class A or Class B pattern of arrestin interaction and trafficking. Class A GPCRs, rapidly dissociate from arrestin upon internalization. These receptors are trafficked to an acidified endosomal compartment, where the ligand is dissociated and the receptor dephosphorylated by a GPCR-specific protein phosphatase, PP2A, and are subsequently recycled to the plasma membrane (3). Class B GPCRs, form stable receptor-arrestin complexes. These receptors accumulate in endocytic vesicles and are either targeted for degradation or slowly recycled to the membrane (Reproduced with permission from (Luttrell and Lefkowitz, 2002)).

7 1.2.2 Agonist-dependent GPCR Internalization

Upon agonist stimulation, GPCRs can also be removed from the cell surface into specific intracellular compartments through the process of internalization. This process decreases the number of GPCRs from the cell surface and therefore limits their interaction with agonists in the extracellular space. The prototypical and most recognized agonist induced internalization pathway of GPCRs is through clathrin coated pits. This pathway involves the binding of a group of intracellular proteins known as arrestins to a GRK phosphorylated receptor. This interaction results in uncoupling of the receptor from its G protein and facilitates GPCR mobilization in clathrin-coated pits by functioning as a proximal endocytic adaptor molecule to recruit clathrin and adaptin protein, AP2 (Gainetdinov et al., 2004) (Figure 1-1). The GTPase dynamin then pinches off the clathrin coated vesicle (Damke et al., 1994). Arrestin association is driven by recognition of GRK phosphorylation sites on the receptor and the active conformation of the receptor (Gainetdinov et al., 2004). The arrestins include two visual arrestins (rod and cone) which are associated with rhodopsin in the retina and arrestin2 and arrestin3 which are ubiquitously expressed.

Several studies have shown that there are differences in the translocation kinetics of arrestin2 and arrestin3 to GPCRs, resulting in two classes (A and B) based on their internalization properties (Oakley et al., 1999; Oakley et al., 2001; Oakley et al., 2000). For Class A receptors, such as the β2 adrenergic receptor and the dopamine D1 receptor, arrestin3 translocates to the receptor more readily than arrestin2. The arrestin dissociates from the receptor at or near the plasma membrane and does not co-internalize with the receptor, allowing for dephosphorylation and rapid recycling of the receptor back to the cell surface. In contrast, Class B receptors such as

AT1a angiotensin receptor and V2 vasopressin receptor, do not display a preference for either arrestin isoform.

Figure 1-2: General structure of a GRK protein. GRKs consist of an amino terminal RGS region (~185 amino acids), a central protein kinase catalytic domain (~270 amino acids), and a variable carboxyl-terminal domain (~105-230 amino acids). Some of the specific interactions elucidated include GPCRs and Gq with the GRK2 RGS domain and Gβγ with the GRK2 carboxyl terminal PH domain. (Adapted with permission from (Pao and Benovic, 2002)).

9 These receptors form a stable complex with arrestin and traffic together into endocytic compartments resulting in inefficient dephosphorylation and much slower recycling of the receptor (Figure 1-1). Specific clusters of serine and thereonine residues in the carboxyl tail have been demonstrated to be the molecular determinant that defines these internalization properties as demonstrated by studies using chimeric GPCRs in which this region is exchanged between Class

A and B receptors (Oakley et al., 2001).

1.2.3 GPCR Recycling and Degradation

Once GPCRs are internalized, they may either enter the recycling pathway or degradation pathway. In the recycling pathway the internalized receptors are dephosphorylated by a GPCR- specific phosphatase and recycled back to the cell surface to prevent prolonged desensitization and allow for the resensitization of the signal. In contrast, the receptors can also be targeted to lysosomes where they are proteolytically degraded, resulting in receptor downregulation. These two opposing processes are regulated by discrete cellular mechanisms that include the recognition of specific sorting motifs within the carboxyl tail, as shown for the protease-activated receptor 1

(PAR1) (Trejo and Coughlin, 1999). Selective interactions of the cytoplasmic tail of GPCRs with sorting proteins, such as sorting nexin 1 (SNX-1), ezrin-radixin-moesin-binding phosphoprotein

(EBP50) and G protein-coupled receptor association protein (GASP) have also demonstrated importance in post endocytic sorting of GPCRs (Cao et al., 1999; Wang et al., 2002; Whistler et al., 2002). Lysosomal targeting of GPCRs can also occur by the posttranslational attachment of ubiquitin, as demonstrated for the yeast α factor mating receptor (Terrell et al., 1998) and the human CXCR4 chemokine receptor (Marchese and Benovic, 2001).

10 1.3 Introduction to GPCR Oligomerization

Although GPCRs were traditionally believed to exist and function as single monomeric entities, it is now widely accepted that GPCRs exist as higher-order molecular forms such as dimers and larger oligomers (George et al., 2002). Oligomerization was shown to occur not only between the same receptors, forming homooligomers of which a homodimer is the smallest unit, but also between different receptors, forming heterooligomers. Heterooligomers can not only form within GPCR families, as was shown for the serotonin (Xie et al., 1999) and opioid families

(Fan et al., 2005; George et al., 2000), but also between unrelated members of different families as observed between the somatostatin and dopamine receptors (Rocheville et al., 2000) and the adenosine and dopamine receptor (Gines et al., 2000), to name a few.

The existence of receptor-receptor interactions adds a level of complexity to the regulation of GPCR functions. Homooligomerization of GPCRs can increase the cooperativity between GPCR binding sites. For example, ligand binding of one receptor may affect the affinity of a second receptor within the oligomer. This was suggested by studies on the leukotriene B4 receptor, where agonist binding to one receptor induced specific changes in the conformation of the ligand binding pocket of the other receptor within the biochemically isolated dimer (Mesnier and Baneres, 2004). Additionally, activation of several receptors within an oligomer by a single ligand may lead to amplification of the signal (George et al., 2002).

Heterooligomerization between GPCRs can result in novel features such as altered binding profiles (Ferre et al., 2007; George et al., 2000) unique trafficking pathways (AbdAlla et al., 2000) and unprecedented effects upon receptor signaling such as second messenger activation

(Gines et al., 2000) or a switch in the signaling pathway (Rozenfeld and Devi, 2007). GPCR heterooligomerization may also be relevant to disease states as has been shown for the

11 Angiotensin AT1 and Bradykinin B2 receptors. In this case, altered heterooligomerization between these two receptors was postulated to be involved in the pathogenesis of angiotensin II hypersensitivity in preeclampsia (AbdAlla et al., 2001). Additionally, heterodimerization between the B2-adrenoreceptor and prostaglandin receptors resulted in a reduced bronchodilator response to B2-adrenoreceptor agonists and therefore was suggested have an impact on asthma (McGraw et al., 2006). A dysregulation of metabotropic glutatmate 2 and serotonin 5HT2A receptor heteromer expression in cortical tissue was suggested to result in abnormal signaling that could predispose schizophrenic patients to psychosis (Gonzalez-Maeso et al., 2008). Furthermore, an increased expression of dopamine D1-D3 receptor heteromers was hypothesized to be involved in

L-dopa induced dyskinesia in patients with Parkinson’s disease (Fiorentini et al., 2008;

Marcellino et al., 2008b).

1.3.1 Evidence for GPCR Oligomerization

Although the concept of oligomerization has only become widely accepted as a vital characteristic of GPCRs in the last 10 years, there were earlier observations from radiation inactivation, chemical crosslinking, and radioligand binding studies that GPCRs might be organized as oligomers (Bouvier, 2001). The discovery that the GABABR1 and GABABR2 receptors formed functional heterooligomers that allowed for proper GABABR1 transport and

GABABR2 function in 1998 (Jones et al., 1998; Kaupmann et al., 1998; White et al., 1998) was instrumental in making the concept of GPCR oligomerization widely accepted.

Today there are a number of techniques that are used to provide evidence for GPCR oligomerization, including Bioluminescence Resonance Energy Transfer (BRET), Fluorescence

Resonance Energy Transfer (FRET), co-immunoprecipitation and functional complementation.

12 One important finding that demonstrated very convincing evidence for the existence of GPCR oligomers was through the use of atomic force microscopy (AFM). AFM is a technique used to study surface morphology and the properties of molecules, often at atomic resolution. Through

AFM, rhodopsin, the prototypic GPCR of many family 1 GPCRs, was shown to exist on mouse retinal disc membranes as dimers arranged in rows as oligomers (Fotiadis et al., 2003), suggesting that other family 1 receptors may have the ability to also orient themselves in a similar arrangement. Another key technique that has proven useful in supporting the concept of GPCR oligomerization is x-ray crystallography, which results in high resolution structural images.

Recently, the chemokine CXCR4 receptor was crystallized revealing a structure in favor of oligomer formation as a similar dimer interface was observed in multiple crystal forms (Wu et al.,

2010). Additionally, the crystal structure demonstrated that binding by a ligand induced a conformational change within the homoooligomeric receptor complex, providing structural evidence in support of cooperative binding observed by some ligands.

1.3.2 GPCR Stoichiometry

Although it is known that GPCRs can form receptor-receptor interactions, the actual proportion of receptors that make up the functional unit of a GPCR complex is still unclear. Most methodologies do not distinguish if the GPCR exists in a dimer or higher-order oligomeric complex and there is still controversy about the issue of whether a receptor is functional in a monomeric form. Some earlier immunoprecipitation studies performed in both heterologous cells and native brain tissue revealed the presence of monomers, dimers and oligomers, but these results can also be influenced by the experimental conditions employed, such as denaturing of receptors due to solubilization with detergents. Monomeric forms have been shown to be present

13 for some GPCRs including rhodopsin receptor (Jastrzebska et al., 2004), neurokinin-1 receptor

(Meyer et al., 2006), N-formyl peptide receptor (Gripentrog et al., 2003), muscarinic M2 (Park and Wells, 2003), and somatostatin receptors SSTR1 and SSTR5 (Patel et al., 2002). However, as mentioned previously, the experimental conditions employed in these studies can result in disruption of interactions mediating oligomerization, challenging the physiological relevance.

More recent studies using energy transfer and fluorescence complementation studies actually postulate assemblies of GPCRs into larger oligomeric complexes. This has been proposed for dopamine D2 receptors (Fotiadis et al., 2006; Guo et al., 2008), GABAB receptors (Maurel et al.,

2008), adenosine A2 receptors (Vidi et al., 2008), B2-adrenoreceptors (Dorsch et al., 2009; Fung et al., 2009) and serotonin 5HT1A receptors (Ganguly et al., 2011).

Furthermore, despite the widely accepted notion that GPCRs exist as dimers or oligomers, some recent studies of monomeric GPCRs reconstituted in high density lipoprotein- like particles demonstrate that the monomeric form is sufficient for functional interactions with G proteins or arrestins. This efficient activation of G proteins was shown for the β2 adrenergic receptor (Whorton et al., 2007) and rhodopsin receptor (Whorton et al., 2008), with monomeric rhodopsin also being capable of binding to arrestin (Tsukamoto et al., 2010). Yet, although these studies indicate a monomer can function in a reconstituted particle, the physiological relevance for the existence of functional GPCRs as monomers in their native environment is still questionable. Indeed, rhodopsin has been observed to exist as higher order oligomers in vivo using AFM (Fotiadis et al., 2003). Furthermore, it is known that for other non GPCR systems, monomers are not functional and oligomerization is required. This has been demonstrated for tyrosine kinases (Lemmon et al., 1994), cytokine receptors (Constantinescu et al., 2001), nuclear steroid receptors (Whitfield et al., 1999), ion channels (Papazian, 1999) and N-methyl-D-aspartic

14 acid (NMDA) receptors (Wenthold et al., 2003). Thus, this requirement for oligomerization may hold true for GPCRs as well.

1.3.3 Structural Features of GPCR Oligomers

GPCR oligomerization occurs through direct interactions between receptors that are mediated by both covalent and non covalent bonds, including disulphide, hydrophobic and electrostatic interactions (Kumar and Nussinov, 2002). These interactions have been suggested to be mediated by several structural regions of a GPCR, including extracellular, intracellular and

TMs.

The importance of the amino terminal domain in GPCR homooligomerization varies depending on the family. The involvement of this domain has been revealed for the family 3 metabotropic glutamate 1 receptors based on X-ray crystallographical data of its extracellular binding region, (Kunishima et al., 2000). Cysteine residues localized on the amino terminal domain formed a disulphide linked homodimer both in its resting and ligand occupied state.

Disulphide bonds in the amino terminal domain of the calcium sensing receptor were also shown to be critical for intermolecular receptor interactions (Ray et al., 1999). A role for the amino terminus in receptor interactions has also been implicated for family 1 GPCRs, such as the bradykinin B2 receptor, as shown by using mutant bradykinin B2 receptors lacking the amino terminal domain or peptides corresponding to the amino terminus to block interactions (AbdAlla et al., 1999).

The carboxyl terminal domain has also been shown to mediate interactions in GPCR homooligomers and heterooligomers. The carboxyl terminal domain was suggested to be

15 important in the homooligomerization of δ-opioid receptors (Trapaidze et al., 1996), and cannabinoid CB1 receptors (Wager-Miller et al., 2002). For GPCR heterooligomers, the most detailed evidence for the carboxyl terminal domain comes from studies on the family 3

GABABR1-GABABR2 receptor heterooligomer. These studies demonstrated that heteromer formation required an assembly of coil-coiled alpha helices contained in the carboxyl terminal domain (Margeta-Mitrovic et al., 2000). Carboxyl terminal interactions were also shown to mediate interactions between the family 1 adenosine A2A-dopamine D2 receptor heterooligomers, where the carboxyl terminal domain of the A2A receptor interacted with the third intracellular loop of the D2 receptor (Canals et al., 2004).

Interactions involving different TM’s have also been suggested for GPCR homooligomers and heterooliogmers. There are several examples that demonstrate the significance of TM’s in mediating interactions between GPCR homooligomers. The importance of contact between TM 4 and 5 between rhodopsin monomers to form dimers was demonstrated as well as contacts between TM 1 and 2 of one rhodopsin dimer connecting to TM 5 and 6 of another rhodopsin dimer leading to the formation of dimeric rows (Liang et al., 2003).

Endogenous cysteine residues located in TM 3 and 4 have also been shown to play a role in homodimerization of the serotonin 5HT4 receptor (Berthouze et al., 2005). Additinally, TM 4 was shown to play a role in the homodimerization of serotonin 5HT2C receptors (Mancia et al., 2008) and α1β adrenoreceptors (Carrillo et al., 2004; Lopez-Gimenez et al., 2007). Furthermore, for the dopamine D2 receptor, TM 4 interactions were found to form part of the homo dimerization interface (Guo et al., 2003; Lee et al., 2003), as well TM 6 and 7 (Ng et al., 1996). For GPCR heterooligomers, TM 4 was reported to mediate interactions between serotonin 5HT2A- metabotropic glutamate 2 receptor heterooligomers (Gonzalez-Maeso et al., 2008) and

16 corticotropin releasing hormone- arginine vasotocin VT2 receptor heterooligomers (Mikhailova et al., 2008).

In 2000, two different models were proposed to describe how TM interactions can mediate GPCR oligomerization, which were the domain swapping and contact model. In the domain swapping model, TM’s of different GPCRs are swapped so that there is a reciprocal exchange of receptor domains. In contrast, the contact dimer model suggests GPCRs interact laterally to form dimers (Gouldson et al., 2000). Although the domain swapping model has shown validity for some GPCRs, such as the muscarinic M3-adrenergic α2 heteromer (Maggio et al.,

1993), more recent studies provide evidence that is more in favor of the contact dimer model, such as the demonstration of rhodopsin monomers organized into two dimensional arrays of dimers (Fotiadis et al., 2003; Liang et al., 2003).

1.4 Regulation of GPCR Oligomers

1.4.1 Agonist-dependent Desensitization of GPCR Oligomers

GPCR homooligomerization has been demonstrated to be important in recruiting scaffolding proteins, such as arrestins, for desensitization. For example, the muscarinic M3 receptor homooligomer was shown to recruit arrestin2 only when there was co-expression of wild type M3 receptors (Novi et al., 2005). When the constituent receptors within the M3 receptor homooligomer included the wild type M3 receptor and a mutated M3 receptor, there was no arrestin2 recruitment upon agonist activation. Homooligomerization also allowed for cross phosphorylation of a nonfunctional chemokine CCR5 receptor that homooligomerized with a

17 functional CCR5 receptor (Huttenrauch et al., 2005), suggesting homooligomerization can function to amplify desensitization.

GPCR heterooligomerization has also been reported to result in altered desensitization properties. For example, formation of the somatostatin SSTR2A and SSTR3 heterooligomer resulted in slower agonist induced desensitization compared to what was observed for their respective homooligomers (Pfeiffer et al., 2001). Similar to homooligomerization, GPCR heterooligomerization also allowed for cross phosphorylation of the CCR5 receptor by a GRK dependent mechanism upon agonist activation of the C5a receptor within a chemokine C5a-CCR5 receptor heteromeric complex (Huttenrauch et al., 2005). Formation of μ-opioid-somatostatin

SST2A receptor heterooligomers, adenosine A2A-dopamine D2 receptor heterooligomers, and μ- opioid- chemokine CCR5 receptor heterooligomers, also each led to a cross-desensitization of receptor function (Chen et al., 2004; Hillion et al., 2002; Pfeiffer et al., 2002).

1.4.2 Agonist-dependent Internalization of GPCR Oligomers

GPCR homooligomers have been shown to internalize as a homooligomeric complex. For example, it was shown that agonist occupancy of only one monomer within the β2-adrenoceptor homodimer was sufficient to cause co-internalization of each constituent receptor, indicating the activated β2-adrenoceptor can internalize as a dimeric complex (Sartania et al., 2007).

Similar to homooligomerization, many GPCR heterooligomers have been shown to internalize as a complex after activation of either member within the complex. This has been demonstrated for the adenosine A2A-dopamine D2, somatostatin SSTTR2a-μ-opioid, and

α1Β adrenergic -α1D adrenergic receptor heterooligomers (Hague et al., 2004; Hillion et al.,

18 2002; Pfeiffer et al., 2002). It is suggested that activation of either member within the heteromeric complex can recruit the necessary endocytic machinery, for receptor internalization. GPCR heterooligomerization can also affect the endocytic properties of the receptors. Indeed, GPCR heterooligomerization has been demonstrated to both inhibit and induce receptor internalization, depending on the constituent receptors that form the receptor heterooligomer. For example, although the β1 adrenergic receptor does not significantly internalize and the β2 adrenergic receptor significantly internalizes upon agonist activation, heterooligomer formation of the two of these receptors led to inhibition of agonist induced endocytosis of the β2 adrenergic receptor

(Lavoie et al., 2002). Likewise, heterooligomer formation of the κ-opioid receptor with either the

δ opioid-receptor or β2 adrenergic receptor resulted in inhibition of internalization of these receptors (Jordan and Devi, 1999; Jordan et al., 2001). In contrast, it has also been shown that receptors that could not internalize as homooligomers were able to internalize as a result of heterooligomer formation. This was demonstrated for the somatostatin SSTR1 receptor, which did not internalize as a homooligomer, but did endocytose when in a heterooligomer formation with the SSTR5 receptor (Rocheville et al., 2000).

In addition to altering the extent of internalization, heterooligomerization has also been shown to change the endocytic pathway used by receptor homooligomers after agonist activation.

This was demonstrated for the angiotensin II AT1-bradykinin 2 receptor heterooligomer that internalized in a dynamin dependent manner even though each constituent homooligomer endocytosed independently of dynamin (AbdAlla et al., 2000).

GPCR heterooligomerization can also alter the complement of scaffolding proteins, such as arrestins, that are recruited during endocytosis and may explain some of the changes in endocytic properties that are observed upon heteromer formation. For example, although the thyrotropin releasing hormone TRHR2 recruited arrestin3 when expressed as a homooligomer, it

19 recruited arrestin2 when it formed a heterooligomer with the TRHR1 receptor and thus changed the rate of receptor internalization (Hanyaloglu et al., 2002).

1.4.3 Post Endocytic Sorting of GPCR Oligomers

Homooligomerization of GPCRs has been shown to affect membrane trafficking of the receptors after agonist induced endocytosis. This was demonstrated for the endocytosed β2 adrenergic receptor that was sorted to a degradative fate as opposed to recycling upon homooligomerization with a recycling defective mutant version of the β2 adrenergic receptor that was directed to lysosomes (Cao et al., 2005).

Heterooligomerization of GPCRs has also been demonstrated to change the endocytic fate of a receptor. Activation of the chemokine CXCR4-chemokine CXCR5 heterooligomer by a

CXCR5 ligand not only resulted in co-internalization of the receptors, but was also suggested to change the trafficking of the co-internalized CXCR4 receptor from its degradative pathway to the

CXCR5’s recycling pathway (Contento et al., 2008).

As mentioned earlier, GPCRs can be designated as Class A or Class B GPCRs, depending on the pattern of arrestin recruitment and post endocytic sorting. However, this designation of Class A or B has been shown to change for GPCRs upon heterooligomerization.

For example, this has been demonstrated for the vasopressin V1a-vasopressin V2a receptor heterooligomer (Terrillon et al., 2004). When expressed as homooligomers, the V1a receptor behaves as a class A receptor with arrestin dissociation from the receptor and rapid recycling, and the V2a receptor is designated as class B, without arrestin dissociation from the receptor and retainment in endosomes. Yet, upon V1a-V2a receptor heterooligomer formation, co-activation of

20 both receptors resulted in recruitment of the complex with arrestin into endocytic compartments that did not enable V1a recycling. In contrast, activation of the V1a receptor alone recruited the heteromeric complex into endosomes without arrestin and resulted in rapid recycling of the heterooligomer (Terrillon et al., 2004).

1.4.4 Stability of GPCR Oligomers

Even though there is much evidence in support of GPCRs assembling into dimers, tetramers, or even higher order oligomers, the stability of these complexes is still not known.

Earlier studies which indicate that dimer assembly may occur during biosynthesis and travel to the cell surface in a dimeric form, suggest a stable and static interaction between the constituent receptors of the GPCR oligomer (reviewed by Lohse, 2010). However, more recent reports actually suggest that GPCR oligomers may behave in a more transient and dynamic state, where the associated receptors also dissociate into monomers in seconds or less. This was suggested for both the B1-adrenergic receptor (Dorsch et al., 2009) and D2 dopamine receptors (Fonseca and

Lambert, 2009), as shown by fluorescence recovery after photo bleaching (FRAP). FRAP experiments use receptors that are tagged with fluorophores and an area of the cell membrane is bleached; this is followed by monitoring the subsequent return of fluorescence into the bleached area. In the reported studies, a fraction of receptors on the cell surface was immobilized by crosslinking with an antibody, and the mobility of the non-crosslinked receptors was monitored by using FRAP. Immobilization of receptors that are part of stable dimers or oligomers also immobilize associated receptors that are not directly crosslinked. Based on these reports, it has been proposed that GPCRs may exist in a transient monomer-dimer equilibrium state that may shift as GPCRs move through various cellular compartments that they encounter from synthesis

21 to degradation (reviewed by Lambert, 2010). Overall, the dynamics of GPCR assemblies of monomers into dimers or higher order oligomers is still not definitely known and is major topic of current research.

1.5 Dopamine

The catecholamine neurotransmitter dopamine has fundamental roles in regulating a wide variety of functions such as locomotion, cognition, reward and emotion. Dopamine also regulates prolactin release from the pituitary gland, modulates sensory perception in the retina and olfactory bulb, as well as controls body temperature and food intake (Callier et al., 2003). At the peripheral level, it controls renal function, gastrointestinal motility, and blood pressure (Missale et al., 1998). Dysregulation of this system in brain has been implicated in a number of pathological conditions such as schizophrenia, drug addiction, attention deficit hyperactivity disorder and Parkinson’s disease (Pivonello et al., 2007).

Dopaminergic neurons synthesize and release dopamine through two reactions. First, the

aromatic amino acid, tyrosine, is converted into L-3,4 dihydroxyphenylalanine (L-Dopa) by the

enzyme, tyrosine hydroxylase (rate limiting step) and then L-Dopa is decarboxylated by aromatic

L-amino acid decarboxylase to produce dopamine (Vallone et al., 2000). As a typical

neurotransmitter, dopamine is released into the synapse after stimulation by an action potential

via a calcium dependent mechanism. In the synaptic cleft, dopamine transmits the signal by

binding to dopamine receptors on pre and post synaptic sites. The dopamine signal is terminated

by the process of reuptake by a presynaptic Na+/Cl- dependent transporter that removes

dopamine from the extracellular space soon after its release, thus regulating its concentration.

After reuptake, dopamine can then be re-packaged for synaptic release. Alternatively it can be

22 inactivated by its metabolic enzymes monoamine oxidase and catechol-O-methyltransferase

(Elsworth and Roth, 1997).

1.5.1 Dopamine Pathways and Functions

There are four main dopamine-mediated pathways in the brain that include the nigrostriatal, mesocortical, mesolimbic and tuberoinfundibular neurons. The nigrostriatal pathway originates in the dopamine-synthesizing neurons of the substantia nigra compacta and extends to the striatum (caudate putamen). This pathway is primarily responsible for the control of movement. Pathological conditions such as Parkinson’s disease demonstrate the importance of dopamine in the control of movements. This disease is characterized by a degeneration of dopaminergic neurons in the substantia niagra resulting in a reduction of circulating dopamine in the striatum leading to motor impairment. In Parkinson’s disease all components of the nigrostiatal dopamine pathway degenerate and therefore the dopamine loss is accompanied by a significant reduction in other neurochemical markers of presynaptic dopamine neurons, including the dopamine metabolite homovanillic acid as well as the synthesizing enzymes L- tyrosine hydroxlase and L-dopa decarboxylase. The degree of striatal dopamine loss is correlated with the severity of the motor symptoms of Parkinson’s disease (Lang and Lozano, 1998).

The mesolimbic pathway innervates the ventral striatum (nucleus accumbens) from the ventral tegmental area (VTA) and is involved in modulating motivated behaviour. This system has been implicated in reward mechanisms and psychomotor effects generated by drugs of abuse, such as cocaine and amphetamine. Administration of psychostimulants and drugs of abuse elicit an increase in dopamine release in the mesolimbic areas, whereas withdrawal of these drugs results in a reduction in dopaminergic transmission. In intracranial self-stimulation experiments performed in rats, the rewarding properties of stimulation results in dopamine release in the

23 prefrontal cortex and nucleus accumbens (Di Chiara and Imperato, 1988; Jackson and Westlind-

Danielsson, 1994).

The mesocortical pathway also arises from the VTA, but extends to various regions of the

frontal cortex, where it is involved with aspects of learning and memory. Dopamine transmission

in this system has been demonstrated to play a role in transient changes of impulse activity in

motivational and attention processes that are essential to learning and cognitive behaviour

(Schultz et al., 1993).

Lastly, the tuberoinfundibular pathway arises from the arcuate nucleus of the

hypothalamus and terminates in the median eminence of the hypothalamus. This pathway is

responsible for transporting dopamine to the anterior pituitary gland (Vallone et al., 2000). This

dopaminergic system results in inhibition of prolactin release. Prolactin is produced in the lactotrophs of the anterior pituitary gland and plays a significant role in lactation. Loss of

dopaminergic control or drugs with anti-dopaminergic activity can result in over production of prolactin leading to suppression of secretion hormones such as LH and FSH by inhibiting GnRH

release (Doppler, 1994).

1.6 Dopamine Receptors

The dopamine receptors are members of the GPCR super family and mediate the effects of dopamine. There are five dopamine receptors, D1, D2, D3, D4 and D5 which are divided into two distinct subclasses: the D1-like receptors, of which the D1 and D5 are members, and the D2- like receptors, of which the D2, D3 and D4 receptors, are members. The D1 and D5 receptors share a high similarity in overall sequence homology (80% with the TMs and 50% overall) and the D2, D3 and D4 receptors share a high sequence homology with each other (46% for D2 and

24 D3; 53% for D2 and D4 (O'Dowd, 1993)). Two alternatively spliced transcripts are generated from the D2 receptor gene and code for the D2L (long) and D2S isoforms (short). The D2L isoform differs from the D2S by the insertion of 29 amino acids in the putative third intracellular loop of the receptor (Neve et al., 2004). The D1 and D2-like receptors differ in their structure and their ability to link to second messenger systems. Schematic diagrams of D1 and D2 receptor are presented in figures 1-3 and 1-4.

1.7 Dopamine D1 Receptors

1.7.1 Cellular Signaling of D1 Receptors

The major signaling pathway of the D1 receptor is stimulation of adenylyl cyclase via activation of Gs and Golf proteins while the D2 receptor primarily signals through Gi or Go proteins to inhibit adenylyl cyclase. The overall amino acid homology for human D1 and D2 receptors is 29% but within the TMs, the two receptors are 44% identical (O'Dowd, 1993). The

D1-like receptors have a relatively short third intracellular loop and a long carboxyl terminal domain while D2-like receptors have a relatively large third intracellular loop and short carboxyl terminal domain. The cAMP pathway is the most well known signaling pathway associated with the D1 receptor. D1 receptor adenylyl cyclase activation resulting in cAMP accumulation was observed in cells (Le Crom et al., 2004) and adenylyl cyclase V was determined to be the isoform of the adenylyl cyclases that was preferably activated by D1 receptors in brain (Lee et al., 2002).

To activate adenylyl cyclase, the D1 receptor couples to Gs (Pedersen et al., 1994) and Golf proteins (Zhuang et al., 2000). The D1 receptor was also observed to couple to Gq/11 proteins in the striatum, hippocampus and amygdala (Jin et al., 2001). Furthermore, the D1 receptors have also been shown to activate ion channels, PLC and various kinases in addition to adenylyl cyclases (Gerfen et al., 2002; Maurice et al., 2001; Jin et al., 2001).

Amino Terminal Domain

Extracellular

ICL1 Intracellular

ICL2

ICL3

Carboxyl Terminal Domain

Figure 1-3: Schematic diagram of the dopamine D1 receptor. The human dopamine D1 receptor consists of 446 amino acids and has a relatively short third intracellular loop (IC3) and long carboxyl tail.

Amino Terminal Domain

Extracellular

ICL1

Carboxyl Terminal ICL2 Domain

ICL3

Figure 1-4: Schematic diagram of the dopamine D2 receptor. The human D2 receptor has a relatively large ICL3 and short carboxyl terminal domain. The red residues on the ICL3 illustrate the 29 amino acid difference between the long and short isoforms of the D2 receptor. The short isoform is a 414 amino acid protein and the long isoform is a 443 amino acid protein.

27 1.7.2 Desensitization of D1 Receptors

Desensitization of D1 receptors has been extensively studied over the past several years

and indicates that dopamine-induced attenuation of signaling by these receptors occurs within

minutes of exposure (Gardner et al., 2001; Jackson et al., 2002; Lamey et al., 2002; Mason et al.,

2002; Ng et al., 1994; Ng et al., 1995; Ng et al., 1997). As with the majority of GPCRs, the

predominant form of D1 receptor desensitization has been identified as being mediated through

GRKs. Truncated mutant constructs of the rat D1 receptor have shown that multiple residues

located downstream of Gly379 in the distal carboxyl terminus regulated dopamine-mediated

phosphorylation and desensitization of the D1 receptor, which was suggested to reflect the

removal of potential GRK2 and/or GRK3 phosphorylation sites (Jackson et al., 2002). Carboxyl terminal sequences located upstream of Gly379 (between Cys351 and Gly379) were shown to be important for phosphorylation but not for desensitization (Jackson et al., 2002). Site directed mutagenesis studies of the human D1 receptor, on the other hand, have provided evidence to suggest that GRK2 acts as a critical regulator of rapid agonist-induced receptor desensitization through phosphorylation of a single motif containing the residues Thr360 and Glu359 in the proximal segment of the carboxyl terminus (Lamey et al., 2002). Both of these studies have used differential methodologies and different species of the D1 receptor which may play a role in the discrepant results observed. Site directed mutagenesis studies may be a more reliable method for identifying the importance of specific residues since there is no change in the intact structure of the receptor. Carboxyl terminal truncations, however, can alter the structure of the receptor which permits access to previously sterically hindered receptor domains, such as the third intracellular loop.

The third cytoplasmic loop has also been implicated in desensitization of the D1 receptor.

It was previously demonstrated that the mutation of specific residues in the third intracellular

28 loop did not affect desensitization of the D1 receptor (Lamey et al., 2002). However, a

subsequent report has demonstrated that these same residues were involved in D1 receptor phosphorylation and desensitization (Kim et al., 2004). A possible reason for this discrepancy may be the use of differential cell lines, where one study used Chinese Hamster Ovary (CHO) cells and the other Human Embryonic Kidney (HEK) 293 cells. It has been shown that the rate of agonist-induced desensitization of the D1 receptor in CHO cells occurs more slowly than in other cell types (Ventura and Sibley, 2000). Thus, it has been postulated that D1 receptor phosphorylation may be GRK isoform-dependent and these isoforms may be lacking in the CHO cell line (Kim et al., 2004).

Given the evidence demonstrating the importance of the carboxyl terminus and third intracellular loop, it has been proposed that D1 receptor phosphorylation takes place in both the carboxyl terminus and third intracellular loop in a sequential manner, where primary phosphorylation of the carboxyl terminus is permissive for secondary third intracellular loop phosphorylation, which then allows for the desensitization response (Kim et al., 2004).

In contrast to D1 receptor phosphorylation by GRK2 and GRK3, GRK4 has been shown to regulate the constitutive phosphorylation and desensitization of the D1 receptor without exposure to agonist (Rankin et al., 2006) suggesting that specific GRK isoforms may serve discrete functions in the regulation of dopamine receptor activity.

PKA may also play a role in homologous desensitization of the D1 receptor. Although it has been demonstrated that the mutation of a potential D1 receptor PKA phosphorylation site reduced the rate of agonist-induced desensitization (Jiang and Sibley, 1999), and moreover, that

D1 receptor desensitization was blunted in cells deficient in PKA (Ventura and Sibley, 2000), it has also been shown that the inhibition of PKA action, either by substitution mutations (Lamey

29 et al., 2002; Mason et al., 2002) or pharmacologically (Mason et al., 2002), appeared to have no effect on D1 receptor-mediated increases in cAMP. These studies were carried out in different cell lines and therefore suggest that the involvement of PKA for homologous desensitization of the D1 receptor may be cell type specific (Gardner et al., 2001).

1.7.3 Internalization of D1 Receptors

The acute administration of dopamine agonists has been demonstrated to induce a robust internalization response both in cultured cells and neurons (Martin-Negrier et al., 2000; Martin-

Negrier et al., 2006) as well as in vivo (Dumartin et al., 1998). While in the absence of agonist the D1 receptor remained predominantly on the cell surface, the addition of dopamine induced rapid internalization of approximately 70% of the receptors, with a half life of less than 5 min

(Vickery and von Zastrow, 1999). However, although endocytosis of the D1 receptor has been consistently documented in both heterologous expression systems and neuronal cultures, the underlying mechanisms have shown to be more variable. Although earlier studies have identified a role for PKA-mediated internalization in cells endogenously expressing the D1 receptor (Bates et al., 1993), mutagenesis of the PKA sites of the rat dopamine D1 receptor (Jiang and Sibley,

1999), the human D1 receptor (Lamey et al., 2002) and the non-human primate D1 receptor

(Mason et al., 2002) did not affect agonist-induced internalization.

Consistent with the role of GRKs in D1 receptor desensitization, this group of kinases also appears to play an important role in D1 receptor internalization, although the underlying mechanisms have yet to be fully elucidated. Receptor mutagenesis has revealed that specific residues in the distal portion of the carboxyl terminus (Thr446, Thr439, and Ser431) are involved in GRK2-mediated internalization of the D1 receptor (Lamey et al., 2002). However, D1

30 receptor mutants with carboxyl terminal truncations implied sequences located between Cys351

and Gly379 that are pivotal to receptor internalization, but not desensitization (Jackson et al.,

2002). Although there appear to be discrepancies regarding the relative importance of specific

residues in D1 receptor internalization, the carboxyl terminus seems to be essential in this stage

of the endocytic trafficking pathway. Yet, it has also been postulated that GRK-mediated D1

receptor phosphorylation on the third intracellular loop may be of relevance in promoting

receptor interactions with arrestins (Kim et al., 2004). Specifically, it has been suggested that the

phosphorylation of residues within the carboxyl terminus and third intracellular loop dissociates

the two domains allowing for arrestin to bind to the activated third loop (Kim et al., 2004).

Activation of the heterologously expressed D1 receptor leads to translocation of both arrestins2

and 3 to the cell membrane, with arrestin3 being the more predominant translocated subtype.

Following arrestin membrane localization, the D1 receptor is internalized and arrestin

subsequently dissociates from the receptor at or near the membrane (Kim et al., 2004; Oakley et

al., 2000; Zhang et al., 1999). Similarly, co-localization between endogenous D1 receptors and arrestins in rat neostriatal neuronal cultures demonstrated that the D1 receptor preferentially interacts with arrestin3 (Macey et al., 2005).

In addition to arrestins, studies assessing the internalization pathway of D1 receptor membrane trafficking have demonstrated the involvement of numerous other proteins, including the scaffolding proteins post synaptic density-95 (PSD-95), clathrin, caveolin-1, and the GTPase dynamin (Kong et al., 2007; Vickery and von Zastrow, 1999; Zhang et al., 2007). In cultured cells, the co-expression of PSD-95 with the D1 receptor resulted in a robust internalization of the receptor in the absence of agonist. Additionally, the abolishment of PSD-95 in mice accentuated

D1 receptor-mediated behavioral responses, suggesting that PSD-95 may also serve an inhibitory role in the regulation of D1 receptor signaling in vivo (Zhang et al., 2007). Evidence suggests

31 that the facilitation of D1 receptor internalization by PSD-95 is mediated through interactions

with the carboxyl terminus of the D1 receptor, and furthermore is dependent upon the presence

of dynamin (Zhang et al., 2007). As dynamin has been previously shown to be involved in

dopamine-induced clathrin-mediated endocytosis of the D1 receptor (Vickery and von Zastrow,

1999) these findings implicate the clathrin-mediated endocytic pathway in the constitutive internalization of the D1 receptor.

In addition to clathrin-mediated internalization, it has been shown in cultured cells that

the D1 receptor can be localized to low density caveolin-enriched membrane domains and can

associate with caveolin-1 in rat brain through a specific binding motif found in TM 7 (Kong et

al., 2007). Agonist stimulation of the D1 receptor caused translocation of the D1 receptor into

caveolin-1-enriched membrane fractions, which was determined to be the result of D1 receptor

endocytosis through caveolae. However, unlike the relatively rapid clathrin-dependent

mechanism of internalization in which approximately 70% of activated receptors were

internalized within 5 min (Vickery and von Zastrow, 1999), caveolin-dependent D1 receptor

endocytosis appeared to be kinetically slower, reaching approximately 55% internalization

within 45 min of agonist stimulation (Kong et al., 2007). Palmitoylation of the D1 receptor was

hypothesized to play a role in directing the receptor to the slower caveolae-dependent

internalization pathway as opposed to the accelerated clathrin-dependent endocytosis pathway

since a de-palmitoylated D1 receptor exhibited a significantly greater rate of internalization than

wild-type D1 receptor (Kong et al., 2011). These findings suggest that both clathrin- and

caveolin-mediated processes may play functionally distinct roles in regulating D1 receptor

responsiveness in vivo.

32 1.7.4 Resensitization and Recycling of D1 Receptors

Investigations into the trafficking fate of the D1 receptor after agonist induced

internalization have generally reported that the D1 receptor recycles back to the plasma

membrane (Bartlett et al., 2005; Dumartin et al., 1998; Jackson et al., 2002; Lamey et al., 2002;

Martin-Negrier et al., 2006; Vargas and Von Zastrow, 2004; Vickery and von Zastrow, 1999), as opposed to being targeted to lysosomes for receptor degradation. With the use of immunohistochemistry or fluorescence microscopy, the D1 receptor expressed in cultured cells or neurons was demonstrated to recycle back to the plasma membrane after removal of agonist within approximately 20-30 min (Martin-Negrier et al., 2006; Vargas and Von Zastrow, 2004;

Vickery and von Zastrow, 1999). In accordance with these studies, dopamine-stimulated D1 receptor phosphorylation has been shown to be rapidly reversed within 30 min. It was suggested that internalization was not mandatory for D1 receptor dephosphorylation since pretreatment of the cells with hypertonic sucrose or concanavalin A did not alter D1 receptor dephosphorylation after agonist removal (Gardner et al., 2001). Resensitization of the D1 receptor-mediated cAMP response occurred within 90 min (Thompson and Whistler, 2011). The efficient recycling of the

D1 receptor was reported to require a specific sequence within the proximal portion of the carboxyl terminus of the receptor (Vargas and Von Zastrow, 2004). This sequence spans amino acid residues 360-382 of the human D1 receptor and is distinct from those previously identified as being required for efficient recycling of other GPCRs (Cao et al., 1999; Cong et al., 2001;

Gage et al., 2001; Kishi et al., 2001; Tanowitz and von Zastrow, 2003). The importance of this sequence as a sorting signal was further established by demonstrating that the motif could induce the recycling of the δ-opioid receptor, a receptor that traffics preferentially to lysosomes after agonist induced internalization (Vargas and Von Zastrow, 2004).

33 Attempts have been made to elucidate the accessory proteins that may contribute to the

regulation of D1 receptor post-endocytic sorting. GASP has been shown to interact with the D1 receptor, and to a greater degree, the D2 receptor (Bartlett et al., 2005; Thompson et al., 2007).

However, while GASP was demonstrated to promote receptor degradation for the D2 receptor, a role in D1 receptor sorting was not observed (Bartlett et al., 2005). One study involving a number of mutant GPCRs including the D1 receptor demonstrated that the presence of a GASP interaction in and of itself is not sufficient to induce receptor degradation but rather it is the robustness of the GASP-receptor interaction that regulates the targeting to lysosomes. Although deletion of the recycling motif in the D1 receptor prevented recycling, it also was not targeted for degradation, suggesting preventing recycling does not necessarily promote D1 receptor degradation unless affinity for sorting proteins such as GASP that mediate degradation is altered as well (Thompson et al., 2007).

1.7.5 D1 Receptor Homooligomers and Heterooligomers

Similar to most GPCRs, D1 receptors have been demonstrated to exist as homooligomers, forming complexes with identical receptors. An appropriate D1 receptor homooligomeric conformation must exist for proper signaling and cell surface trafficking (George et al., 1998;

Kong et al., 2006). D1 receptors can also form heterooligomers with other dopamine receptor subtypes. The D1 receptor heterooligomerized with the D5 receptor but the physiological significance of this interaction was not determined (O'Dowd et al., 2005). D1 receptors formed heterooligomers with the D3 receptor with activation resulting in increased affinity of dopamine for the D1 receptor and potency of dopamine in stimulating adenylyl cyclase through the D1 receptor (Fiorentini et al., 2008). Activation of the D1-D3 receptor heteromer by a D1 agonist

34 also abolished agonist induced D1 receptor internalization, but paired stimulation of the D1-D3 receptor complex by both a D1 and D3 receptor agonist enabled co-internalization of the D1-D3 receptor complex, even though D3 receptors are not reported to significantly internalize when expressed alone (Kim et al., 2001). Additionally, stimulation of the D1-D3 heteromer by a D3 agonist potentiated D1 receptor mediated behavioral effects (Marcellino et al., 2008b).

Furthermore, D1 receptors can form heterooligomers with D2 receptors in cells and in vivo resulting in a Gq/11 protein-linked PLC mediated intracellular calcium signal that was not activated by either constituent receptor alone (Hasbi et al., 2009; Lee et al., 2004; Rashid et al.,

2007). The D1-D2 receptor heterooligomer will be discussed in more detail below.

Formation of D1 receptor heteromeric complexes with other GPCRs have also been reported, including the adenosine A1 receptor (Gines et al., 2000), ghrelin receptor (Jiang et al.,

2006), μ-opioid receptor (Juhasz et al., 2008), and histamine H3 receptor (Ferrada et al., 2009).

Formation of a D1-A1 receptor heterooligomer resulted in a decreased D1 receptor affinity for dopamine (Gines et al., 2000) and formation of a D1-ghrelin receptor heteromer resulted in a switch of G protein selectivity for the ghrelin receptor from Gq/11 to Gi/o proteins with activation leading to increased D1 receptor mediated cAMP signaling (Jiang et al., 2006). Activation of D1-

H3 receptor heteromers by H3 receptor agonists led to activation of the mitogen-activated protein kinase (MAPK) signaling cascade, an effect that was not seen in cells expressing H3 receptors alone (Ferrada et al., 2009) and formation of D1- μ-opioid receptor heterooligomers resulted in a significantly enhanced surface expression of the μ-opioid receptor (Juhasz et al., 2008).

35 1.8 Dopamine D2 Receptors

1.8.1 Cellular Signaling of D2 Receptors

Similar to the D1 receptor, the cAMP pathway is the most studied pathway associated with the D2 receptor. However, in contrast to the D1 receptor, the D2 receptor results in inhibition of adenylyl cyclase and a decrease in cAMP concentrations. The D2 receptor was found to preferably inhibit adenylyl cyclase V in brain (Lee et al., 2002). To inhibit adenylyl cyclase, the D2 receptor couples to Gαi and Gαo proteins as well as Gαz proteins (Albert et al.,

1990; Obadiah et al., 1999). D2 receptors are also capable of modulating other second messenger systems, including inositol phospholipid metabolism, arachidonic acid release, potassium currents and calcium currents (Banihashemi and Albert, 2002; Memo et al., 1992; Nilsson et al., 1998;

Rasolonjanahary et al., 2002). Additionally, more recent studies indicate that the D2 receptor can function through the protein kinase B (Akt) – glycogen synthase kinase 3 (GSK-3) signaling cascade through an arrestin 3 dependent mechanism (Beaulieu et al., 2007; Beaulieu et al., 2005;

Mannoury la Cour et al., 2011). Furthermore, the D2 receptor can affect dopamine mediated cAMP signaling by forming a complex with the prostate apoptosis response (Par-4) protein (Park et al., 2005).

1.8.2 Desensitization of D2 Receptors

Early studies examining the functional desensitization of D2 receptors have generated variable results but, in general, indicate that D2 receptors desensitize much more slowly than D1

receptors and require prolonged agonist treatment (Ng et al., 1997; Zhang et al., 1994). Similar to

the D1 receptor, the mechanisms underlying D2 receptor desensitization appear to involve

36 GRKs. Only by over expression of GRK2, GRK5 (Ito et al., 1999), or GRK3 (Kim et al., 2001) was there increased phosphorylation of the human D2 receptor and receptor internalization, indicating the sensitivity of the D2 receptor as a substrate for GRK phosphorylation is lower than the D1 receptor. A recent study demonstrated that although the D2 receptor is phosphorylated by

GRK2 and GRK3, D2 receptor GRK phosphorylation was not required for agonist induced receptor desensitization or internalization, but rather was shown to regulate post-endocytic trafficking of the receptor (Namkung et al., 2009a). Additionally, GRK2 was shown to constitutively attenuate D2 receptor signaling through a mechanism that required GRK2 kinase activity and Gβγ binding, but did not involve receptor phosphorylation (Namkung et al., 2009b).

Furthermore, GRK6 may also play a role in regulating desensitization of the D2 receptor. Gene deletion of GRK6 was shown to lead to enhanced coupling of D2 receptors to their respective G proteins in vivo, an effect that was associated with increased susceptibility to the locomotor activating effects of psychostimulants, suggesting that desensitization was inhibited due to

GRK6 knockout (Gainetdinov et al., 2003).

In addition to GRK mediated desensitization, the second messenger kinase, PKC, has been suggested to be involved in D2 receptor desensitization. PKC phosphorylation was shown to attenuate the ability of both the D2 receptor to inhibit cAMP accumulation (Cho et al., 2007;

Namkung and Sibley, 2004) and additionally, to induce specific effects on each D2 receptor isoform (D2L and D2S) with regards to receptor stimulated calcium mobilization (Morris et al.,

2007). It has been reported that although PKC is able to effectively desensitize D2S-induced increases in intracellular calcium, the D2L isoform is insensitive to PKC-induced desensitization of calcium signaling due to the presence of a pseudosubstrate domain. A pseudosubstrate domain is a site that resembles a substrate domain except that the serine phosphorylation site is replaced by alanine or other residues (Morris et al., 2007). This regulation of substrate sensitivity to PKC

37 appeared to be the result of intramolecular competition between different substrate domains on

the D2L receptor for PKC recognition and a pseudosubstrate domain, which is not found in the

D2S receptor. Given the importance of the D2 receptor in numerous physiological processes, the presence of pseudosubstrate domains may potentially have significant implications for the regulation of the receptor by PKC.

1.8.3 Internalization of D2 Receptors

The endocytosis of the D2 receptor is a highly complex process that has been shown to be both isoform and cell specific, as well as to exhibit both dynamin-dependent and independent mechanisms (Iwata et al., 1999; Kabbani et al., 2004; Kim et al., 2001; Vickery and von

Zastrow, 1999).

Internalization of the D2 receptor requires increased levels of GRKs and appears to be a relatively slow process taking approximately 2 hours to plateau (Ito et al., 1999; Itokawa et al.,

1996; Iwata et al., 1999). Whereas little or no internalization was observed in the absence of exogenous GRKs or in the presence of the dominant negative GRK2, co expression of GRK2,

GRK3 or GRK5 (Ito et al., 1999; Kim et al., 2001), caused significant D2 receptor internalization. PKC activation also led to 50% of the D2 receptor being internalized when PKC was over expressed (Namkung and Sibley, 2004). Mutagenesis studies suggest that both of the

PKC phosphorylation domains identified within the third intracellular loop were involved in regulating its internalization from the cell surface (Namkung and Sibley, 2004).

Similar to the D1 receptor, internalization of the D2 receptor involves translocation of arrestin2 and arrestin3 to the cell membrane (Kim et al., 2001; Macey et al., 2004) which

38 function to promote receptor internalization (Kim et al., 2004; Namkung et al., 2009a). The endogenous dopamine D2 receptor in neurons, however, has been shown to preferentially interact with arrestin2 (Macey et al., 2004). The D2 receptor isoforms also showed differential regulatory mechanisms for internalization. For example, although both isoforms displayed a similar level of phosphorylation and arrestin translocation, the actual internalization of the two isoforms were differentially regulated by GRKs and arrestins, where the internalization of the

D2S receptor was preferentially enhanced by GRK2 or GRK3, but the D2L receptor was preferentially enhanced by arrestin3 (Cho et al., 2006). As discussed previously, given that the

D2L and D2S receptor isoforms are structurally similar, with the exception of a 29 amino acid deletion in the third intracellular loop of the D2S receptor, it is plausible that this region may play a role in isoform specific trafficking.

In contrast to the D1 receptor, D2 receptor internalization appears to be mediated by specific dynamin isoforms, suggesting specificity between dynamin isoforms and dopamine receptor subtypes. It has been reported that the internalization of the D2S receptor is dynamin dependent, implicating the clathrin-coated endocytic pathway in the sequestration of this receptor

(Iwata et al., 1999; Kim et al., 2001; Kim et al., 2004). There are conflicting reports, however, as to the importance of dynamin-mediated mechanisms in the internalization of the D2L receptor.

While it has been suggested that the D2L receptor internalizes in a dynamin independent manner

(Kim et al., 2004; Vickery and von Zastrow, 1999), these studies assessed only the role of the dynamin-1 isoform, whereas the dynamin-2 isoform has been more recently implicated. In cultured cells and primary striatal neurons dynamin-2 was shown to localize to sites of D2 receptor internalization and associate with the D2 receptor in the rat brain (Kabbani et al., 2004).

Furthermore, when an anti-D2 receptor antibody and high-resolution immunoelectron microscopy was used to study internalization patterns of the D2 receptor in the primate prefrontal

39 cortex, the D2 receptor was demonstrated to undergo clathrin endocytosis via clathrin coated pits

and clathrin coated vesicles (Paspalas et al., 2006).

1.8.4 Resensitization and Recycling of D2 Receptors

Studies examining the trafficking of internalized D2 receptors have generally reported

targetting of the D2 receptors to lysomes for degradation in cells and neurons as opposed to

recycling (Bartlett et al., 2005). As discussed previously, unlike the D1 receptor, the sorting fate

of the D2 receptor appears to be mediated by GASP in non-neuronal cells. Moreover, it was

shown that dopaminergic neurons endogenously expressing GASP did not exhibit a functional

recovery of neuronal responses following D2 receptor agonist administration, whereas disrupting

the GASP- D2 receptor interaction facilitated the recovery of functional D2 receptor responses

(Bartlett et al., 2005). In addition to GASP, the PKC interacting protein, ZIP, has been shown to

associate with the D2 receptor in both cultured cells and endogenous brain tissue. Over

expression of ZIP reduced D2 receptor cell surface expression via enhanced trafficking of the

receptors to lysosomes, suggesting that the ZIP protein functions as a negative modulator of D2

receptor expression (Kim et al., 2008).

1.8.5 D2 Receptor Homooligomers and Heterooligomers

Similar to D1 receptors, D2 receptors exist as homooligomers forming complexes with identical receptors (Guo et al., 2003; Lee et al., 2000; Lee et al., 2003; Wurch et al., 2001) and require appropriate homooligomeric formation for proper function (Lee et al., 2000). D2 receptors also form heterooligomers with other dopamine receptor subtypes, including the D3,

40 D1, and D5 receptors. Formation of D2-D3 receptor complexes increased the selectivity of

antiparkinsonian drugs to these receptors (Maggio et al., 2003) as well as resulted in adenylyl cyclase inhibition by binding of D3 agonists that do not inhibit adenylyl cyclase through D3 receptor activation alone (Scarselli et al., 2001). As mentioned previously, D2 receptors can also form heterooligomers with D1 receptors in cells and in vivo resulting in a Gq/11 protein-linked

PLC mediated intracellular calcium signal that was not activated by either constituent receptor

alone (Hasbi et al., 2009; Lee et al., 2004; Rashid et al., 2007). The D1-D2 receptor

heterooligomer will be discussed in more detail below. Furthermore, D2 receptors were shown to

form complexes with D5 receptors resulting in a Gq/11 protein linked PLC mediated calcium signal from both extracellular and intracellular calcium stores (So et al., 2009), unlike the calcium release triggered by D1-D2 receptor heterooligomers which was only generated from intracellular stores. Additionally, unlike what is observed for D1 receptors, which activate significant intracellular calcium mobilization only within a complex with D2 receptors, a robust calcium signal from both intracellular and extracellular stores was triggered by D5 receptors expressed alone that was Gq/11 protein linked and PLC mediated. Heterooligomerization with the

D2 receptor led to an attenuation of this D5 receptor mediated signal.

D2 receptors also form heterooligomers with other GPCRs. One of the most documented includes the dopamine D2-adenosine A2A receptor heterooligomer. A reduction in cAMP signaling by A2A receptor activation and a decrease in the affinity of D2 agonists for the D2 receptor were mediated by D2-A2A receptor heterooligomers (Kamiya et al., 2003; Kull et al.,

1999). Co-desensitization and co-internalization of both receptors within the heteromeric

receptor complex was observed after prolonged agonist treatment by either A2A receptor or D2

receptor agonists (Hillion et al., 2002). Dopamine D2-cannabinoid CB1 receptor heterooligomers

resulted in the CB1 receptor switching from Gi/o to Gs protein coupling (Jarrahian et al., 2004)

and stimulation of CB1 receptors decreased the affinity of D2 receptors for dopamine (Marcellino

et al., 2008a). D2-somatostatin SSTR5 receptor heterooligomer formation potentiated the effects

of dopamine and somatostatin agonists (Rocheville et al., 2000). Agonist-induced

heterodimerization of dopamine D2-somatostatin SSTR2 resulted in increased affinity for

dopamine and enhanced D2 receptor signaling as well as prolonged SSTR2 receptor

internalization (Baragli et al., 2007). Furthermore, dopamine D2-histamine H3 heteromer

formation was suggested to be responsible for an H3 receptor agonist inhibiting and antagonist

potentiating the locomotor activation induced by a D2 agonist (Ferrada et al., 2008).

1.9 The D1-D2 Receptor Heterooligomer

Although D1 and D2 receptors are biochemically and functionally distinct, some

physiological functions require the co-activation of both receptors (Capper-Loup et al., 2002;

Kita et al., 1999; Robertson and Robertson, 1987; Walters et al., 1987). At a mechanistic level this has been difficult to reconcile since co-activation of the D1 and D2 receptors can result in both opposing as well as synergistic physiological responses. The discovery, however, of a common functional output generated by the concurrent activation of D1 and D2 receptors within the same cells resulting in activation of a novel Gq/11-linked PLC-dependent calcium signal (Lee

et al., 2004) has provided a possible biochemical mechanism by which the D1 and D2 receptors work in concert to mediate these molecular and behavioral functions. The calcium signal was

inhibited by either a D1 or D2 receptor antagonist, indicating that both constituents of the

heterooligomer must be activated to elicit the signal. This calcium signal was independent of

extracellular calcium influx as well as other intracellular pathways that can trigger calcium

release, as shown by inhibitors of multiple components of these signaling cascades (Hasbi et al.,

42 2009; Lee et al., 2004). Additionally, the rate of the calcium signal propogation was temporally

similar to what has been detected for Gq-coupled P2Y1 purinergic receptors (Lee et al., 2004),

which result in a calcium signal that has a slower upstroke than the P2Y2 isoform of purinergic

receptors (Gallagher and Salter, 2003).

1.9.1 D1-D2 Receptor Heterooligomer in vitro and in vivo

Heterodimerization of D1 and D2 receptors was initially shown by

coimmunoprecipitation from rat and human brain (Lee et al., 2004). These findings were then

confirmed in HEK 293T cultured cells co-expressing both receptors by FRET (So et al., 2005),

co-trafficking studies (So et al., 2005), and visualization of D1-D2 receptor heteromer co- trafficking in live cells (O'Dowd et al., 2005).

More recently, quantitative FRET in situ has been utilized to verify the presence of D1-

D2 receptor heteromers both in neonatal cultured rat striatal neurons and adult striatum (Hasbi et al., 2009; Perreault et al., 2010). In neonatal cultured rat striatal neurons, immunocytochemistry revealed that D1 and D2 receptors were mainly expressed at the cell surface and on proximal neurites with a high degree of co localization. Localization of D2 receptors was also observed in the cytosol. Confocal FRET analysis of the natively expressed D1 and D2 receptors demonstrated a relative distance of 5-7 nm (50-70Å) in localized microdomains, thus indicating a

physical interaction between them. FRET efficiency ranged from 0.1-0.5, with a higher

efficiency in the soma and proximal dendrites and lower in distal processes (Hasbi et al., 2009).

In adult striatum, the highest degree of D1 and D2 receptor co localization was found in the cell

bodies of neurons in the nucleus accumbens and globus pallidus and the lowest incidence in the

caudate putamen. FRET analysis demonstrated that ~91% of the cell bodies in neurons co-

43 expressing D1 and D2 receptors in the nucleus accumbens exhibited heteromer formation whereas only ~24% of D1 and D2 receptor co expressing neurons in the caudate putamen formed the D1-D2 receptor complex. Furthermore, these D1-D2 receptor heteromers were also found on presynaptic terminals in the striatum (Perreault et al., 2010).

1.9.2 Activation of the D1-D2 Receptor Heterooligomer in Striatum

Activation of the D1-D2 receptor heteromer in adult rodent striatum induced activation of

35 Gq/11, as shown by direct [ S]GTPγS incorporation into Gq (Rashid et al., 2007). Gq activation did not occur in either D1 or D2 receptor knockout animals, emphasizing the importance for involvement of both receptors to induce Gq activation. Similar to the heterologous cells, treatment of rodents with either a D1 or D2 receptor antagonist blocked the effect of agonist activation, indicating that activation of both D1 and D2 receptor subtypes that were part of the heterooligomeric complex was necessary for generating a functional response.

Recently, activation of the D1-D2 receptor mediated calcium signal through the Gq/ PLC signaling pathway was also shown in neonatal cultured rat striatal neurons (Hasbi et al., 2009).

Similar to the heterologous cells, the calcium signal was inhibited by either a D1 or D2 receptor antagonist, was independent of extracellular calcium influx as well as other intracellular pathways that can trigger calcium release, as shown by inhibitors of multiple components of these signaling cascades. Preincubation with inhibitors of the Gq protein, IP3 receptors, or PLC resulted in significant inhibition of the D1-D2 receptor heteromer mediated calcium signal, confirming the involvement of the Gq/PLC pathway in these neurons. Furthermore, no significant difference in the calcium signal was observed in D5 knockout animals, but it was abolished in D1 knockout animals, confirming that the calcium mobilization was through the D1-

D2 receptor heterooligomer without involvement of the D5 receptor.

44 1.9.3 Functional consequences of D1-D2 Receptor Heterooligomer Mediated Signaling

Activation of calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) was the first

demonstration of a functional consequence of activation of the D1-D2 receptor heteromer

complex (Rashid et al., 2007). CaMKIIα plays a fundamental role in synaptic plasticity, and both

its translation and activity can be regulated by increases in intracellular calcium (Lisman et al.,

2002). Significant increases in phosphorylated CaMKIIα in neurons of nucleus accumbens shell

and caudate nucleus was demonstrated within 10 min of an intraperitoneally injection of D1 and

D2 agonists concurrently into mice and rats (Rashid et al., 2007). This effect did not occur in D1

or D2 receptor knockout animals and preadministration of a D1 or D2 antagonist also blocked

this effect, indicating the necessity for both D1 and D2 receptors.

D1-D2 receptor heteromer mediated induction of CaMKIIα and nuclear translocation

was also recently demonstrated to occur in cultured postnatal rat striatal neurons (Hasbi et al.,

2009). Within 2 min of agonist treatment, there were not only robust increases in phosphorylated

CaMKIIα levels, but also a significant increase of phosphorylated CaMKIIα in the nucleus. This

effect was blocked by D2 antagonist treatment and was absent in D1 knockout animals, but still

present in D5 knockout animals. Accordingly, levels of brain derived neurotrophic factor

(BDNF), a neurotrophin whose gene expression is modulated by isoforms of CaMKII (Takeuchi

et al., 2000), were also significantly increased within 1 hour of activation of the D1-D2 receptor heteromer in these neurons and the effect was blocked by D1 and D2 antagonist treatments, but still present in D5 knockout animals. Increased BDNF expression was also observed in the adult rat striatum, specifically in the nucleus accumbens, after activation of the D1-D2 receptor heteromer. Furthermore, activation of the D1-D2 receptor heteromer by dopamine agonists led to accelerated morphological maturation and differentiation of the cultured striatal neurons. These

45 results are consistent with the ability of dopamine and BDNF to promote neuronal maturation, differentiation, and survival (Iwakura et al., 2008). Thus, taken together, this data suggests that activation of the D1-D2 receptor heteromer signaling pathway is a key cascade involved in the development, maturation, and differentiation of striatal neurons, through mobilization of intracellular calcium through Gq, activation of PLC, followed by activation of CaMKIIα and

BDNF signaling.

Alterations in striatal D1-D2 receptor heteromeric function have also been demonstrated in response to amphetamine (AMPH) treatment (Perreault et al., 2010). It was shown that repeated AMPH treatment in rats significantly increased the proportion of the D1-D2 receptor heteromer in the agonist detected high affinity state in striatum and also increased the affinity of the dopamine agonist for the D1-D2 receptor complex by approximately 10-fold, as shown by radioligand binding assays. The striatal D1-D2 heteromer high affinity state was absent in D1 knockout animals, but still present in D5 knockout animals. AMPH treatment also resulted in increased sensitivity of striatal D1-D2 receptor heteromer G protein activation by dopamine, as shown by agonist induced GTPγS binding, indicating the D1-D2 receptor heteromer was functionally supersensitive in response to repeated increases in dopamine transmission.

Furthermore, changes in D1-D2 receptor heteromer function was also studied in human schizophrenia brain samples. Radioligand binding studies demonstrated that there was an approximate 10-fold increase in affinity of the D2 receptor within the D1-D2 receptor heteromer for agonist in schizophrenia brain globus pallidus, together with a concomitant increase in the levels of heteromeric D2 receptors in the high affinity state compared to that in normal individuals (Perreault et al., 2010). Moreover, there was enhanced D1 and D2 coupling in postmortem brain of subjects diagnosed with major depression and disruption of the complex resulted in anti-depressant like effects in rats (Pei et al., 2010).

46 Behavioral consequences of D1-D2 receptor heteromer signaling have also been

examined (Perreault et al., 2010). Selective activation of the D1-D2 receptor heteromer by SKF

83959 significantly increased the amount of time rats spent grooming, an effect that was greatly

attenuated by acute injection of the D2 receptor antagonist, raclopride. In contrast, SKF 83822, a drug that activates only D1 receptor homooligomers, attenuated grooming.

1.9.4 Pharmacology of the D1-D2 Receptor Heterooligomer

Initially, the pharmacology of the D1-D2 receptor heteromer was studied by investigating

any differences in ligand binding affinity for the D1-D2 receptor heteromer vs. D1 and D2

receptor homooligomers. Results from radioligand binding studies with dopamine and other

commonly used D1 and D2 dopamine agonists, such as SKF 81297 and quinpirole, demonstrated

no differences between the ligand binding pocket of the D1 or D2 receptors within the D1-D2

receptor heterooligomer compared to that within their respective homooligomers (Lee et al.,

2004; So et al., 2005). These unchanged binding characteristics were also similar for D1 and D2

receptor selective antagonists.

However, further studies on the pharmacology of the D1-D2 receptor heteromer revealed

pharmacological profiles with differential specificity for the D1-D2 receptor heteromer and their

respective homomers (Rashid et al., 2007). Radioligand binding assays demonstrated that the

commonly used D1 agonist, SKF 81297, displayed a high affinity for the D1 receptor in D1-D2

receptor heteromers and in D1 receptor homooligomers. This agonist demonstrated robust

activation of the D1 receptor homooligomers through the Gs-mediated adenylyl cyclase pathway

as well as activation of the D1-D2 receptor heteromer through the Gq-mediated PLC pathway.

Two other commonly used D1 agonists that were screened include SKF 83959 and SKF 83822,

both of which displayed a very high affinity for the D1 receptor in D1-D2 receptor

47 heterooligomers and in D1 receptor homooligomers, but displayed differential functional effects.

SKF 83959 was shown to selectively activate the D1-D2 receptor heteromer resulting in a robust

PLC dependent rise in intracellular calcium, without activating D1 receptor homooligomers

coupled to Gs mediated adenylyl cyclase activity. In contrast, SKF 83822 was shown to selectively activate D1 receptor homooligomer-Gs mediated adenylyl cyclase activity, without activating the D1-D2 receptor heteromer-Gq mediated calcium signal (Figure 1-5).

Furthermore, results from radioligand binding assays revealed that although SKF 81297 and SKF 83959 are full D1 agonists, they are also partial D2 agonists for the D2 receptor within the D1-D2 receptor complex (Rashid et al., 2007). This partial D2 receptor agonism was first suspected when these SKF compounds were shown to generate a calcium signal on their own with an approximately 70% lower peak height than that generated by dopamine in HEK 293T cells co-expressing D1 and D2 receptors. This signal could be blocked by D1 or D2 receptor antagonists and was not seen in HEK 293T cells expressing D1 receptors alone. Accordingly, when these SKF compounds were co-administered with the D2 agonist, quinpirole, the calcium signal was increased to the same extent as generated by dopamine. The calcium signal was not activated by the administration of quinpirole alone in HEK 293T cells co-expressing D1 and D2 receptors or in HEK 293T cells expressing the D2 receptor alone. Thus, these results suggested that these compounds could directly activate the D2 receptor within the D1-D2 receptor complex. This was confirmed with radioligand binding assays using PTX to uncouple Gi/o proteins from D2 receptor homomers leaving them with low affinity for agonists. The results indicated that SKF 81297 and SKF 83959 could bind to a high affinity PTX-resistant D2 site within the D1-D2 receptor heteromer in rat and mouse striatum as well as in HEK 293T cells co- expressing D1 and D2 receptors. This binding was absent in cells expressing only the D1 or D2 receptor and in D1 knockout animals (Rashid et al., 2007).

SKF 81297 SKF 81297 SKF 83959 SKF 83822

Figure 1-5: Differential agonist activation of the D1-D2 receptor heterooligomer and D1 receptor homooligomer. SKF 81297 activates both the D1-D2 receptor heteromer and D1 receptor homooligomer, while SKF 83959 selectively activates D1-D2 receptor heterooligomer- Gq mediated signaling and SKF 83822 only activates D1 receptor homooligomer-Gs mediated signaling.

49 1.9.5 D1-D2 Receptor Heterooligomer Desensitization

Although little is known regarding the regulation of D1-D2 heterooligomer

responsiveness, it has been shown that desensitization of the agonist-induced calcium signal

occurs within minutes of agonist exposure and is initiated by agonist occupancy of either

receptor subtype, even though the signal is generated only by occupancy of both receptors (So et

al., 2007). Additionally, the attenuation of receptor internalization did not result in a concomitant

decrease in the magnitude of the desensitization, suggesting desensitization of the signal

occurred prior to recruitment of the complex into vesicles by endocytic machinery. Although

GRKs 5 or 6 or any of the second messenger kinases such as PKA, PKC, CaMKII, or casein kinases I and II did not play a role in the desensitization, GRKs 2 and 3 appeared to have a role in the extent of desensitization. Inhibition of GRK2-mediated phosphorylation, however, did not inhibit this desensitization (So et al., 2007), suggesting that in addition to phosphorylating receptors, GRKs may also mediate signal desensitization by phosphorylation independent mechanisms. It has been suggested that GRK2 and GRK3 may sequester Gq/11 proteins, which

interact with the RGS domain on these GRKs (Iwata et al., 2005). Thus, this may provide a

mechanism by which GRKs 2 and 3 contribute to desensitization of the calcium signal mediated

by the D1-D2 receptor heterooligomer (So et al., 2007). It is of note, however, that

heterooligomeric D1 and D2 receptors exhibit conformational changes that permitted cross

phosphorylation of the D2 receptor by selective D1 receptor activation (So et al., 2005), a finding

that implicates a discrete mechanism by which the D1 receptor within the D1- D2 complex may

regulate heterooligomer functioning.

50 1.9.6 D1-D2 Receptor Heterooligomer Internalization

Given the relatively recent discovery of the D1-D2 receptor heteromer, there is much as yet unknown regarding the regulation and trafficking of this complex. It has been determined

that selective agonist occupancy by either a D1 agonist or D2 agonist leads to D1-D2 receptor heteromer co-internalization (So et al., 2005). This interesting finding indicates that activation of only one receptor within the D1-D2 complex is sufficient for internalization, whereas co- activation of the D1 and D2 receptors is required for the PLC-mediated calcium signal. It was also shown that heteromerization resulted in altered steady-state cellular distribution of the D1

and D2 receptors within HEK 293T cells that was distinct from that of the D1 and D2 receptor

homomers (So et al., 2005). Together, these findings emphasize the unique trafficking responses of the heteromer compared to its constituent D1 and D2 receptors, a characteristic that may elucidate differences in physiological function.

1.10 Research Rationale and Objectives

Since it is well documented that activation of the D1-D2 receptor heteromeric complex results in calcium signaling in vivo, it is important then to determine how this signal is regulated.

For dopamine receptors, regulatory processes, such as desensitization, may be relevant to the outcome of hyper-dopaminergic states, such as schizophrenia, as well as to the development of therapeutic tolerance in the treatment of dopamine related diseases. When I started my PhD

studies, although much was known about how signals generated from D1 and D2 receptor

homooligomers are regulated, the regulation of the D1-D2 receptor heteromer was much less

defined. However, it was known that pre-treatment of the D1-D2 receptor heteromer with

dopamine resulted in rapid desensitization of the D1-D2 receptor mediated calcium signal that

51 occurred at the receptor level and was independent of intracellular calcium store depletion, suggesting that the calcium signal desensitization might occur at the level of the receptor complex. Additionally, several second messenger kinases were tested as potential mediators of

D1-D2 receptor heteromer calcium signal desensitization, but only GRK 2 or 3 were shown to play a role.

Furthermore, when I began my PhD, the pharmacology of the D1-D2 receptor heteromer had just been thoroughly investigated with selective dopaminergic agonists. It was specifically identified that the agonist, SKF 83959, selectively activated the D1-D2 receptor heteromer, while

SKF 83822 only activated the D1 homooligomer and SKF 81297 activated both the D1 and D1-

D2 receptor complexes.

Given this knowledge that the D1-D2 receptor heteromer could be selectively activated, I was able to use the dopaminergic agonists, all of which have equivalent ability to bind with high affinity to the D1 receptor, but exhibit differential abilities to activate the D1-D2 receptor heteromeric pathway as pharmacological tools to help elucidate the regulatory characteristics of the D1-D2 receptor heterooligomer and distinguish them from D1 and D2 receptor homooligomeric units. Investigating the regulatory characteristics of the D1-D2 receptor heterooligomer was the overall goal of the project.

Hypothesis: Given that co-activation of the dopamine D1-D2 receptor heterooligomer results in a rapid and robust Gq-mediated calcium signal, the regulation of this complex will include mechanisms that are distinct from that regulating its constituent receptors which are not coupled to Gq.

52 The specific hypotheses were:

1) The D1-D2 receptor complex will desensitize following agonist activation and may

include mechanisms involving mediators such as GRK2.

2) After agonist activation, the D1-D2 receptor heteromeric complex will internalize and

result in resensitization of the calcium signal.

3) Selective agonists will have specific roles in desensitization, internalization and

resensitization of the D1-D2 receptor heteromeric complex and its associated calcium signal.

The objectives of the project were to:

1) Determine desensitization properties of the D1-D2 receptor heterooligomer and identify

mechanisms involved.

2) Evaluate agonist induced internalization of the D1-D2 receptor heterooligomer and

resensitization of the calcium signal.

53 2 MATERIALS AND METHODS

2.1 Cell Culture - All cell culture and transfection reagents were obtained from Invitrogen

(Carlsbad, CA). HEK293T cells were maintained as monolayer cultures at 37 oC in advanced minimum essential medium supplemented with 6% fetal bovine serum, 300 μg/ml Zeocin, and antibiotic-antimycotic. Stable cell lines co-expressing the amino-terminal hemagglutinin (HA) epitope-tagged human D1 receptor and amino-terminal FLAG epitope-tagged human D2 receptor were created in HEK293T cells using the bicistronic pBudCE 4.1 vector (Invitrogen,

Burlington, ON, Canada). Briefly, the D1 receptor cDNA was inserted into the EF1 α multicloning site and the D2 receptor cDNA into the CMV site. For stable cell lines expressing either D1 or D2 receptors, HA-D1 receptor cDNA or FLAG-D2 receptor cDNA was introduced alone into the pBUDCE4.1 vector. Antibiotic-resistant clones (selected with 300μg/ml zeocin) of each transfection were isolated and tested for expression of corresponding receptors using saturation binding analysis. All experiments were performed with the long isoform of the D2 receptor.

2.2 Transient Transfections in HEK293T Cells- Cells were grown to 80% confluency before being transfected with Lipofectamine 2000 reagent (Invitrogen). Experiments were performed 48 hours post transfection. For studies involving GRKs, transient transfections of cDNA encoding

GRK2, K220R-GRK2, D110A-GRK2, or R106A/K220R-GRK2 in the mammalian expression vector pcDNA3 or enhanced green fluorescent protein-GRK2 (EGFP-GRK2) in the mammalian expression vector pEGFP-N were performed. GRK constructs were a kind gift from Dr. Jeffrey

Benovic (Thomas Jefferson University, Philadelphia, PA). For small interfering RNA (siRNA) silencing of gene expression, chemically synthesized double-stranded siRNA duplexes (with 3’

54 dTdT overhangs) were purchased from Qiagen Inc. (Mississauga, ON, Canada). GRK2 siRNA

(5’-AAGAAGUACGAGAAGCUGAG-3’) and a nonsilencing RNA duplex (5’-

UUCUCCGAACGUGUCACGU-3’) that was used as a control for the siRNA experiments were

transfected with a final concentration of 40 nM. The effect of siRNA transfection was assessed

by immunoblotting. Average percent protein knockdown in these experiments was 69% of basal

levels.

2.3 Measurement of the Calcium Signal in HEK293T Cells- Calcium mobilization assays

were carried out using a Flex station multiwell plate fluorometer (Molecular Devices, Sunnyvale,

CA, USA). Stably transfected HEK 293T cells were seeded in black microtiter plates at a density

of 1.5 x 105 cells/well grown for 24 h. The cells were then loaded with 2 μM Fluo-4

acetoxymethyl ester indicator dye (Invitrogen) in advanced minimum essential medium

supplemented with 2.5 mM probenecid (Sigma Aldrich, Oakville, ON, Canada) and 250 μM

Ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA) (Sigma) for 1 h. The

cells were then washed twice with Hanks’ balanced salt solution (HBSS) supplemented with 20

mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 250 μM EGTA. Baseline

fluorescence values were measured for 15 sec and changes in fluorescence corresponding to

alterations in intracellular calcium levels upon the addition of agonists thereafter were recorded.

Fluorescence values were collected at 3 sec intervals for 100 sec and the difference between

maximum and minimum fluorescence values for each agonist concentration was determined and

analysed using Prism software (GraphPad, San Diego, CA, USA). For desensitization studies,

cells were pre-treated with agonists in serum-free advanced minimum essential medium in a dose

and time response manner and washed off with HBSS supplemented with 250 μM EGTA before calcium measurement. For resensitization studies, the cells were incubated at 37 oC for up to 3

55 hrs after agonist wash off before calcium measurement. For inhibitor studies, cells were pre-

treated with 10 μM SQ 22536 1 h before agonist pre-treatment, or 10 μM SCH 23390 or raclopride 10 min before agonist pre-treatment. SKF 81297, SKF 83959, SKF 83822, dopamine, quinpirole, SCH 23390, raclopride, ATP, H-89 and SQ 22536 were purchased from Sigma.

2.4 Membrane Preparation and Radioligand Saturation Binding Assay- HEK 293T cells co- expressing D1 and D2 receptors were first treated with either vehicle or 100 nM SKF 83959 in advanced minimum essential medium supplemented with 2.5 mM probenecid for 30 min at 4oC.

The cells were then washed twice in cold HBSS with probenecid, collected, and centrifuged at

2000 RPM for 10 min at 4 oC to obtain a pellet. Cell lysates were prepared by disruption with a polytron homogenizer (Kinematica, Basel, Switzerland) in ice cold lysis buffer (5 mM Tris-HCl and 2 mM EDTA) containing protease inhibitors (5 μg/ml leupeptin, 10 μg/ml benzamidine, and

5 μg/ml soybean trypsin inhibitor). Lysates were centrifuged at 800 RPM for 10 min and the supernatant was collected. Membrane fractions were prepared by centrifuging the supernatant at

13000 RPM for 20 min. Membrane protein was determined by the Bradford assay according to the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA). Saturation binding assays were performed in 1ml antagonist binding buffer (50 mM Tris HCl, 5 mM EDTA, 1.5 mM CaCl2, 5 mM MgCl2, 5 mM KCl, 120 mM NaCl) with 35 μg of membrane homogenate and increasing concentrations (0.05 to 4 nM) of [3H]SCH 23390. Non specific binding was determined by 10

μM (+)-butaclamol. Incubation was performed for 2 h at room temperature. At the end of the

incubation, bound ligand was isolated by rapid filtration through a 48-well cell harvester

(Brandel, Montreal, QC, Canada) using Whatman GF/C filters (Whatman, Clifton, NJ, USA).

56 Data were analyzed by nonlinear regression analysis using GraphPad Prism for the determination

of dissociation constants (Kd) and the density of receptors (Bmax).

2.5 Intact Cell Radioligand Binding Assay- HEK 293T cells expressing D1 receptors, D2 receptors or both D1 and D2 receptors were treated with either vehicle, 100 nM SKF 83959 or 1

μM dopamine for 5 or 30 min in advanced minimum essential medium at 37ºC. The cells were then washed once in acidic buffer (1mM EDTA, 50mM TrisHCl, pH 5) and twice in warm PBS.

Dissociated cells were collected in cold PBS and centrifuged at 1000g for 10 min at 4oC. The resultant pellet was gently resuspended in antagonist binding buffer and protein estimation was determined by the Bradford assay according to the manufacturer’s instructions. 35 μg of protein was incubated with 1 nM [3H]SCH 23390 (Kd = ~ 0.2 nM) or 2 nM [3H]Raclopride (Kd = ~ 1

nM) to a final volume of 1ml. Non specific binding was determined by 10 μM (+)-butaclamol.

Incubation was performed for 3 h on ice to prevent receptor internalization. At the end of the

incubation, bound ligand was isolated by rapid filtration, as described above. To ensure that

[3H]SCH 23390 or [3H]Raclopride measured cell surface receptors, the assay was conducted at 4

ºC and specific binding to cell surface receptors was calculated by the displacement of

radioligand by 100 μM dopamine. Since dopamine does not cross the cell membrane it would

only displace radioligand from receptors on the cell surface. Non- specific binding accounted for

approximately 5% of total bound ligand.

2.6 Immunocytochemistry of HEK293T Cells- HEK 293T cells stably expressing HA-D1 and

Flag-D2 receptors were grown on glass coverslips in 6 well plates for 24 hr. The cells treated

with 1 μΜ dopamine or SKF 83959 for 30min in advanced minimum essential medium at 37oC

57 and then washed three times with PBS. After removal of agonist, the cells were incubated at

37oC for 0 hr, 1.5 hr or 3 hr before fixing with 4% paraformaldehyde for 30 min at 37oC. The

primary antibodies used were rat anti-HA (Roche, Laval, QC, Canada, 1:500) and mouse anti-

Flag (Sigma, 1:500). The secondary antibodies used were anti-rat conjugated to fluorophore

Alexa Fluor 488 and anti-mouse conjugated to flurophore Alexa Fluor 647 (Invitrogen 1:500).

Paraformaldehyde-fixed cells were incubated with the primary antibodies overnight at 4ºC. After

three washes with PBS-Tween20, the samples were incubated with secondary anit-rat and anti-

mouse antibodies for 2 hours at room temperature. After three washes, the glass coverslips were

mounted on slides using a mounting solution (Dako, Carpinteria, CA, USA) and the images were

acquired using a confocal Fluoview Olympus microscope (FV 1000). All images were acquired

in sequential mode to minimize any bleed-through.

2.7 Neuronal Cultures- Neuronal cultures derived from post natal day 1 rodent striatum were

trypsinized in HBSS with 0.25% trypsin and 0.05% DNase (Sigma) at 37ºC, and then washed 3

times in HBSS with 12 mM MgSO4. Cells were dissociated in Dulbeco’s Modified Eagle

Medium with 2 mM glutamine and 10% FBS and plated at 2x105 cells per poly-L-lysine coated

well (Sigma 50 μg/ml). After 24 h the media was replaced with neurobasal medium with 50X

B27 supplement and 2 mM glutamine (Invitrogen). After three days in culture, cytosine

arabinoside was added (5 μM) to inhibit glial cell proliferation. Half of the medium was changed

every three days. The neurons were in culture for 7-14 days before experiments were performed.

Cell viability was tested with trypan blue (0.4%) (Invitrogen) exclusion and indicated 2% cell death during this time period.

58 2.8 Immunocytochemistry of Cultured Neurons- Neurons that were grown on glass coverslips

in 24 well plates were washed twice in PBS and then fixed in 4% paraformaldehyde for 30 min at 37oC. The primary antibodies used were rat anti-D1 (Sigma, 1:400), and rabbit anti-D2

(Millipore, Billerica, Massachusetts, USA, 1:400). The secondary antibodies used were anti-rat

conjugated to fluorophore Alexa Fluor 568 and anti-rabbit conjugated to fluorophore Alexa Fluor

647 (Invitrogen 1:500). Paraformaldehyde-fixed neurons were incubated with the primary antibodies overnight at 4ºC. After three washes with PBS-Tween20, the samples were incubated with secondary anti-rat and anti-rabbit antibodies for 2 hours at room temperature. After three washes, the glass coverslips were mounted on slides using a mounting solution and the images were acquired using a confocal Fluoview Olympus microscope (FV 1000). All images were acquired in sequential mode to minimize any bleed-through. For GRK2 studies, the neurons were

pretreated with 100 nM dopamine, SKF 83959, or SKF 83822 for 5 min and then washed twice

with PBS before fixing with paraformaldehyde. The primary antibody used was rabbit anti-

GRK2 (Sigma, 1:200) and the secondary antibody used was anti-rabbit conjugated to

fluorophore Alexa Fluor 350 (Invitrogen 1:500).

2.9 Confocal Microscopy FRET- Paraformaldehyde-fixed striatal neurons from rat brain were

incubated for 24 hours at 4°C with primary antibodies highly specific to D1 and D2 receptors

(Lee et al., 2004), and the species-specific secondary antibodies conjugated to Alexa 568 and

Alexa 647 dyes, respectively. The primary antibodies have been shown to be highly specific to

D1 or D2 receptors using HEK 293T cells expressing individual D1, D2, D3, D4 or D5 receptors

(Lee et al., 2004). They were further validated by immunohistochemistry showing lack of

reactivity in striatum slices from D1-/- and D2-/- mice. Anti-D2-Alexa 350 and anti-D1-Alexa 488 were used as the donor and acceptor dipoles, respectively. An Olympus Fluoview FV 1000 laser

59 scanning confocal microscope with a 60X/1.4 NA objective was used to obtain the images. A

Krypton laser at 405 nm and an Argon laser at 488 nm were used to excite the donor and

acceptor, respectively. The emissions were collected at 430 and 530 nm LP filter. Other FRET

pairs (488-568 and 568-647) were tested and showed comparable results. Each FRET analysis

was performed using eleven images and calculated using an algorithm (Chen, 2005). The

corrected FRET (cFRET) images were then generated based on the described algorithm, in

which: cFRET = UFRET – ASBT – DSBT, where UFRET is uncorrected FRET and ASBT and

DSBT are the acceptor and the donor spectral bleed-through signals. Small regions of interest

(ROI), using the same images and software, were used to estimate the rate of energy transfer

efficiency (E) and the distance (r) between the donor (D) and the acceptor (A) molecules in

accordance with the following equation: Efficiency: E = 1 - IDA / [IDA+ pFRET*((ψdd / ψaa )*

(Qd/Qa))], where IDA is the donor image in the presence of acceptor, ψdd and ψaa are collection efficiencies in the donor and acceptor channels, Qd and Qa are the quantum yields. E is proportional to the 6th power of the distance (r) separating the FRET pair. r = Ro [(1/E) – 1]1/6,

Ro is Förster’s distance.

2.10 Measurement of the Calcium Signal in Primary Striatal Neurons- Calcium mobilization was measured in neonatal neurons in culture using cameleon YC6.1 (Truong, et al., 2001), an engineered calcium indicator based on the conformational change of a calmodulin (CaM) peptide flanked by two fluorophores, cyan fluorescent protein (CFP) and yellow fuorescent protein (

YFP). An increase in calcium binding to CaM leads to a decrease in the distance separating the two flanking proteins, CFP and YFP, and results in a measurable energy transfer. The cameleon was a generous gift from Dr. M. Ikura, University of Toronto. The striatal neuronal cultures were

transfected with cameleon YC6.1 using a combination of Effectene (Qiagen, Mississauga, ON,

60 Canada) and ExgenTM500 (Fermentas, Burlington, ON, Canada) which resulted in a transfection

efficiency of 40-70%. Briefly, 2.7 μg of cameleon YC.6.1 cDNA was mixed with Buffer EC

(Effectene kit), 150 mM NaCl, and Exgen reagent and enhancer and incubated at room

temperature for 5 min. Subsequently, Effectene reagent was added and mixed. Ten minutes later,

6 ml of culture media was added to the mixture, which was split into 24 wells. Treatment with

trypan blue (0.4%) indicated that cell death was ~ 10% after transfection. The experiments were

then performed in these live neurons 48 hours post transfection with cameleon in the absence of

extracellular calcium. Using a single excitation wavelength at 405 nm, which solely excites CFP,

images and fluorescence emissions data for both CFP and YFP were collected and energy

transfer was calculated (Chen, 2005). Activation of the calcium signal was measured following

treatment with either 100 nM dopamine, SKF 83959, or SKF 83822. The background signal was

subtracted from the values obtained after drug injection. For desensitization studies, the neurons

were pre-treated with agonists in HBSS for 30 min and washed off with the same media before

calcium measurement. For inhibitor studies, the neurons were pretreated with 10 μM SQ22536

or H-89, for 30 min before agonist pretreatment. For siRNA experiments, the neurons were

transfected with either GRK2 siRNA or a non-silencing siRNA in cultured medium using

HiPerFect transfection reagent (Qiagen), as per the manufacturer’s instructions. Briefly, 3 μl

HiPerFect reagent was mixed with siRNA diluted in culture medium without serum and

incubated for 10 min at room temperature. The mixture was then added drop wise onto the

neurons in each well. The siRNA final concentration was 40 nM. Experiments were performed

48 hours post siRNA transfection. The effect of siRNA transfection was assessed by Western

blotting analysis. Average knock down of protein expression in these experiments was 70% of

basal levels.

61 2.11 Immunoprecipitation- HEK 293T cells stably expressing both the HA-D1 receptor and

Flag-D2 receptor were transfected with 2 μg GRK2 cDNA using Lipofectamine 2000. 48 hours post transfection, the cells were treated with 1 μM dopamine, SKF 83959 or SKF 83822 in advanced minimum essential medium for 5 min at 37 oC and then washed twice with ice cold

PBS. The cells were collected and centrifuged at 2000 RPM for 10 min at 4oC to obtain a pellet.

Cell lysates were prepared by disruption with a polytron homogenizer (Kinematica, Basel,

Switzerland) in ice cold lysis buffer containing protease inhibitors. Lysates were centrifuged at

800 RPM for 10 min and the supernatant was collected. Membrane fractions were prepared by

centrifuging the supernatant at 13000 RPM for 20 min. The resultant pellet was solubilized with

NP-40 for 2 hrs at 4oC and then centrifuged at 15000g for 20 min. The supernatant was collected

for protein determination by the Bradford assay (BioRad). 500 μg of total cell lysate was used for immunoprecipitation. After pre clearing for 30 min with 10μl protein G agarose beads

(Sigma), the lysates were incubated overnight with 75 μl of anti-FLAG M2-Agarose (Sigma) at

4ºC to immunoprecipitate the FLAG-D2 receptor. Immuno-complexes were collected and washed 4 times followed by an overnight incubation with 100 μg of a FLAG peptide at 4ºC to displace the anti-FLAG antibody from the D2 receptor. The proteins were resolved by gel electrophoresis. Immunodetection of the FLAG-D2 receptor from immunoprecipitates was detected with mouse anti-FLAG antibody (1:1000) (Sigma) and HA-D1 receptor and GRK2 immunoreactivity were detected with rat anti-HA antibody (1:1000) (Roche) and rabbit anti-

GRK2 antibody (1:1000) (Sigma), respectively.

62 2.12 BRET Assay- To detect an agonist induced interaction between the D1-D2 receptor heteromer and GRK2, BRET studies were performed on HEK 293T cells transfected with 1 μg cDNA of Renilla luciferase-D1 (Rluc-D1) receptor, FLAG-D2 receptor, EGFP-GRK2 or empty vector EGFP. Cells were seeded into 96 well plates at a density of 105 cells/well for 24 hours and then treated with 1 μM dopamine, SFK 83959, SKF 83822 in HBSS for 1 to 10 min at 37 ºC.

After the induction of Rluc-mediated light emission by the addition of 5μM of the substrate coelenterazine h (Discoverx, San Diego, CA, USA) emission was measured using a plate-reader spectrofluorometer (Victor3, Perkin-Elmer) at wavelengths 480 and 535 nm, corresponding to the maxima of the emission spectra for Rluc and EGFP, respectively. The Net BRET ratio was calculated by subtracting the background BRET signal obtained from Rluc-D1 receptor in the presence of empty vector, EGFP from the BRET signal obtained from Rluc-D1 receptor in the presence of EGFP-GRK2 as defined by the following calculation: [(emission at 535 nm)/

(emission at 480] – Cf where Cf corresponds to (emission at 535)/(emission at 480) from cells expressing the Rluc-D1 receptor and EGFP.

2.13 SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting- Harvested HEK 293T cells were solubilized in lysis buffer + a protease and phosphatase inhibitor, sonicated for ~ 30 sec on ice and protein estimation was determined using the Bradford assay (BioRad). The same procedure was performed for the neurons except they were solubilized in RIPA buffer (1M Tris,

1M NaCl, Na-Deoxycholate, Igepal CA-630, 0.5M EDTA, 500 mM MgCl2). Samples were prepared in 2X sample buffer (0.5M Tris HCl, glycerol, 10% SDS, 1% Bromophenol blue, β-

Mercaptoethanol) and boiled for 3 min. Samples were separated on pre-cast 10% polyacrylamide gels (Invitrogen) for 2 hrs at 128V. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Amersham Biosciences, Piscataway, NJ, USA) at 33 V for 2.5 hrs and blocked for 1

63 hour in 5% skim milk powder and then incubated with the appropriate primary antibody overnight at 4ºC. Rabbit anti-GRK2 antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA) was used at 1:1000 dilution to assess overexpression of GRK2 and 1:600 to measure knockdown of GRK2. Rabbit anti-GAPDH (Abcam, Cambridge, MA, USA) was used at 1:7500 dilution.

Blots were rinsed 3 times with TBS-tween and incubated with horseradish peroxidase- conjugated goat anti-rabbit secondary antibody (1:4000) (Santa Cruz biotechnology, Santa Cruz,

CA) for 2 hrs at room temperature. Immunoreactivity was detected by enhanced chemiluminescence (ECL) using an ECL plus kit (Amersham, Biosciences).

2.14 Statistical Analysis- All pharmacological data were analyzed using the computer program

GraphPad Prism version 3.00 for Windows. Saturation binding curves were analyzed by nonlinear regression analysis for the determination of dissociation constants (Kd) and the density of receptors (Bmax). Data from multiple experiments were averaged and expressed as the means

± Standard Error of the Mean (SEM). Statistical significance at the p<0.05 level is denoted with

* and was determined using the unpaired Student’s t test or one way ANOVA followed by

Tukeys post hoc test.

64 3 RESULTS

3.1 Activation of the D1-D2 Receptor Heteromer Mediated Calcium Signal in HEK293T

Cells- To study the effects of the agonists on the D1-D2 receptor heteromer mediated calcium

signal, we first confirmed the abilities of each agonist to activate the intracellular calcium signal

in a HEK 293T stable cell line co-expressing D1 and D2 receptors (Lee et al., 2004) in the

presence of EGTA, an extracellular calcium chelator. The cells expressed D1 and D2 receptors in

a 1:1 ratio with final receptor densities of each that were approximately 0.8 pmol/mg protein.

The addition of either dopamine, SKF 83959, the agonist that selectively triggers

phosphoinositide hydrolysis, or SKF 81297, the agonist which activates both adenylyl cyclase

and phosphoinositide turnover, stimulated a robust calcium signal that peaked within 20-40 sec

of agonist activation and declined within 120 sec, compared to vehicle as shown in a representative tracing (Fig 3-1A) or the peak heights of the calcium signals (Fig 3-1B). The addition of SKF 83822, the agonist that has been shown to only activate adenylyl cyclase, did not elicit a significant calcium signal (Fig 3-1A,B).

3.2 Desensitization of the D1-D2 Receptor Heteromer Mediated Calcium Signal in

HEK293T Cells- To investigate calcium signal desensitization, the HEK 293T cells were pre- treated for 5 min with increasing concentrations of each agonist, from 10-11 to 10-4 M, then was

washed off followed by subsequent activation with 10 µM dopamine. All three agonists by prior exposure were able to significantly desensitize the calcium signal to dopamine activation (60.1 ±

2.8% reduction for SKF 81297 (n=3), 74.4 ± 2.2% reduction for SKF 83959 (n=5), and 62.8 ±

2.8% reduction for SKF 83822 (n=8)).

AA 100000 Dopamine 90000 80000 70000 83959 60000

AFU 50000 40000 Drug Addition 81297 30000

20000 Vehicle 10000 0 83822 0 20 40 60 80 100 Time (seconds) B 125000

100000 *

75000 * AFU * 50000 25000 0 e 9 in 22 m a p 8395 81297 838 ehicle o V D

FIGURE 3-1. Specificity of dopamine receptor agonists activating the D1-D2 receptor heteromer calcium signal in HEK 293T cells stably expressing the D1 and D2 receptors. (A) Representative tracings displaying changes in fluorescence corresponding to changes in intracellular calcium levels on treatment of D1 and D2 receptors with 1μM concentrations of dopamine, SKF 83959, SKF 81297, SKF 83822 or vehicle. A.F.U. = absolute fluorescence units. (B) Peak heights of agonist induced calcium release through activation of the D1-D2 receptor heteromer. Values shown are the means ± S.E.M. of n=3 experiments. A significant difference from vehicle is denoted by * = p < 0.05.

SKF 83959 was the most potent in desensitizing the signal (EC50 = 29.1 ± 0.5nM), followed by

SKF 83822 (EC50 = 54.7 ± 1.2nM), and the least potent was SKF 81297 (EC50 = 130.9 ± 4.1nM)

(Fig 3-2A-D). The potency of the agonists to induce desensitization and the extent of desensitization of the calcium signal after agonist exposure for 10 or 30 min was similar to that seen after agonist exposure for 5 min (Fig 3-3 and 3-4 A-C). The experiments were performed in the presence of 250 µM EGTA, indicating the calcium signal was from intracellular calcium stores. To demonstrate that depletion of intracellular calcium stores by prolonged agonist treatment was not the mediator of this calcium signal desensitization, endogenously expressed purinergic receptors, which also use the Gq protein and PLC as a means to generate calcium release through intracellular stores (Schachter et al., 1996), were activated with 10 µM ATP after dopamine agonist pre-treatment in the presence of EGTA. No significant difference in the extent of the ATP mediated calcium signal was observed after pre-treatment with SKF 83959 (for 30 min) compared to control as determined by their peak heights, suggesting that calcium stores were not significantly depleted after dopamine agonist pre-treatment (Fig 3-5). To demonstrate that the desensitization of the calcium signal was not due to residual agonist persistently occupying the ligand binding pocket of the receptor, saturation binding studies were carried out after the HEK 293T cells co-expressing D1 and D2 receptors were treated with 100 nM SKF

83959 for 30 min and then washed off. The Bmax and Kd values for [3H]SCH 23390 binding

were 0.892 ± 0.17 pmol/mg protein and 277 ± 9.6 pM for the control cells not pre-treated with

agonist and 0.875 ± 0.13 pmol/mg protein and 308 ± 11 pM (n=4), for cells pre-treated with SKF

83959. These results indicated that there was no persistent occupancy of the ligand binding

pocket after agonist wash off.

A 100000 90000 Control 80000 70000 60000

AFU 50000 83959 40000 Pretreatment 30000 20000 10000 0 0 20 40 60 80 100 Time (seconds)

B 75

25 Peak Height Peak Height

of of Reduction Reduction % 0

-25 -12 -11-10 -9 -8 -7 -6 -5 -4 -3 -2 Log [SKF 81297 (M)]

C 100

50 25 Peak Height

% Reduction of Reduction % 0

-25 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 Log [SKF 83959 (M)] D 75 50

25 Peak Height

% Reduction of 0

-25 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 Log [SKF 83822 (M)]

FIGURE 3-2. The D1-D2 receptor heteromer mediated calcium signal is desensitized by prior treatment with dopamine agonists for 5 min. (A) Representative calcium tracings displaying the calcium signal activated by 10 μM dopamine (control) and after pre-treatment with 1μM SKF 83959 for 5 min in HEK 293T cells stably expressing D1 and D2 receptors. Dose response curves demonstrating the percentage reduction in peak calcium levels after pre-treatment with increasing concentrations of SKF 81297 (B), SKF 83959 (C), or SKF 83822 (D), from 10-11 M to 10-4 M for 5 min, washed, and activated with 10 μM dopamine. Values shown are the means ± S.E.M. of n=3-6 experiments.

A 100

25 Peak Height of Reduction % 0 -25 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 Log [SKF 81297(M)]

B 100 75 50

Peak Height

% Reduction of Reduction % 0

-25 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 Log [SKF 83959(M)]

C 100 75

25 Peak Height

% Reduction of Reduction % 0

-25 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 Log [SKF 83822 (M)]

FIGURE 3-3. The D1-D2 receptor heteromer mediated calcium signal is desensitized by prior treatment with dopamine agonists for 10 min. Dose response curves demonstrating the percentage reduction in peak calcium levels after pre-treatment with increasing concentrations of SKF 81297 (A), SKF 83959 (B), or SKF 83822 (C), from 10-11 M to 10-4 M for 10 min, washed, and activated with 10 μM dopamine. Values shown are the means ± S.E.M. of n=3-6 experiments.

A 100 75

50 Peak Height Peak Height

% Reduction of of 25

0 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 Log [SKF 81297 (M)] B 100

25 Peak Height of Reduction % 0 -25 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 Log [SKF 83959 (M)]

C 100

50 25 Peak Height

% Reduction of Reduction % 0

-25 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 Log [SKF 83822 (M)]

FIGURE 3-4. The D1-D2 receptor heteromer mediated calcium signal is desensitized by prior treatment with dopamine agonists for 30 min. Dose response curves demonstrating the percentage reduction in peak calcium levels after pre-treatment with increasing concentrations of SKF 81297 (A), SKF 83959 (B), or SKF 83822 (C), from 10-11 M to 10-4 M for 30 min, washed, and activated with 10 μM dopamine. Values shown are the means ± S.E.M. of n=5-8 experiments.

150000

100000

50000

ATP Induced AFU Induced ATP

SKF 83959 - +

FIGURE 3-5. The ATP induced calcium signal is not reduced by prior treatment with the agonist, SKF 83959. There was no significant difference in the extent of the ATP mediated calcium signal after pre-treatment with SKF 83959 for 30 min compared to control as determined by their peak heights. A.F.U. = absolute fluorescence units. Values shown are the means ± S.E.M. of n=3 experiments.

71 Since SKF 83822 was not able to significantly activate the calcium signal, but still led to its

desensitization, the involvement of a heterologous mechanism involving the adenylyl cyclase pathway was investigated. When cells were pre-treated with SKF 83822 in the absence and presence of the adenylyl cyclase inhibitor, SQ 22536, there was no significant difference in the extent of the calcium signal, suggesting the desensitization mediated by SKF 83822 did not involve adenylyl cyclase (Fig 3-6). Taken together, these results demonstrated that although SKF

83959 and SKF 83822 have differential abilities to activate the D1-D2 receptor heteromer mediated calcium signal, both were able to elicit significant calcium signal desensitization.

3.2.1 Desensitization through selective occupancy of the D1 receptor- Because SKF 81297 and SKF 83959 act as full agonists for the D1 receptor and partial agonists for the D2 receptor within the D1-D2 receptor complex (Rashid et al., 2007), the desensitization observed could

potentially be mediated by occupancy of both receptors. To investigate whether both D1 and D2 receptors were involved in the desensitization of the signal, a D2 selective antagonist, raclopride,

10 µM, was added with SKF 81297, SKF 83959 or SKF 83822 pre-treatment for 30 min and

then washed off followed by activation with 10 µM dopamine. No significant difference in the extent of desensitization caused by any of the agonists was observed in the presence of

raclopride (Fig 3-7A-C). However, the desensitization induced by each drug was abolished by

pre-treatment with the D1 antagonist, SCH 23390, 10 µM. These results suggested that the

desensitization elicited by each agonist occurred through selective occupancy of the D1 receptor.

Peak Height Peak 25 % Reduction of

0 + + SKF83822 SQ 22536 - +

FIGURE 3-6. Effect of the adenylyl cyclase inhibitor, SQ 22536, on the SKF 83822 induced desensitization of the D1-D2 receptor heteromer mediated calcium signal. Data represent the percent reduction in peak calcium levels after pre-treatment with 1 μM SKF 83822 in the absence and presence of SQ 22536 (500 μM). Values shown are the means ± S.E.M. of n=4 experiments.

A 100

75 * * 50

25 Dopamine Induced %Fluoresence Peak 0 SKF 81297 - + + + SCH 23390 - - - + Raclopride - - + -

B 100

75 * * 50

25 Dopamine Induced Dopamine % Peak Fluorescence Peak % 0 SKF83959 - + ++ SCH23390 - - - + Raclopride - - + -

C 100

75 * * 50

25 Dopamine Induced Dopamine

Fluorescence % Peak 0 SKF83822 - +++ SCH23390 - - - + Raclopride - - + -

FIGURE 3-7. Effect of dopamine receptor antagonists on the desensitization of the D1-D2 receptor heteromer mediated calcium signal in HEK 293T cells stably expressing D1 and D2 receptors. Data were represented as the percentage of peak fluorescence of the dopamine induced calcium signal and values are the means ± S.E.M. of the numbers shown in brackets. Desensitization of the calcium signal elicited by dopamine following pre-treatment with 1μM SKF 81297 (A), 1μM SKF 83959 (B) or 1μM SKF 83822 (C) for 30 min without or with pre- treatment with 10 μM raclopride or 10 μM SCH 23390 (n=4-6).

74 To further confirm the observed desensitization was through occupancy of the D1 receptor rather

than the D2 receptor, the cells were pre-treated with both a D1 agonist and the D2 agonist,

quinpirole, for 30 min and then washed off followed by a subsequent challenge with dopamine.

No significant difference in the extent of desensitization was observed, suggesting that occupancy of the D1 and D2 receptors concurrently did not enhance desensitization of the signal

(Fig 3-8A-C). Pre-treatment with quinpirole alone did not induce any significant desensitization.

3.3 Activation of the D1-D2 Receptor Mediated Calcium Signal in Primary Striatal

Neurons- D1 and D2 receptors were mainly expressed at the cell surface and on proximal neurites with a high degree of colocalization in postnatal striatal neurons as determined by immunocytochemistry (Fig 3-9A). Localization of D2 receptors was also observed in the cytosol.

Confocal FRET analysis of the natively expressed dopamine D1 and D2 receptors demonstrated a relative distance of 5-7 nm (50-70Å) in localized membrane microdomains, thus indicating a physical interaction between the natively expressed D1 and D2 receptors. FRET efficiency (E) ranged from 0.1-0.5, with a higher efficiency in the soma and proximal dendrites and lower in distal processes as shown in a representative figure (Fig 3-9A). Cameleon was transfected into the striatal neurons and was used as an indicator for the calcium signal. The majority of cameleon was localized in the cell bodies as well as a small amount in the dendritic processes

(Fig 3-9B). In the absence of extracellular calcium, addition of either 100 nM dopamine or SKF

83959 to the striatal neurons led to rapid increases in cameleon FRET, corresponding to a rise in intracellular calcium, as shown in a representative tracing (Fig 3-9C) or the peak heights of the calcium signals (Fig 3-9D).

A 100

75 * * 50

25 Dopamine Induced % Fluoresence Peak

0 - - SKF 81297 + + Quinpirole - - + +

B 100

50 * *

25 Dopamine Induced Dopamine % Peak Fluorescence % Peak 0 SKF 83959 - + + - Quinpirole - - + +

C 100

50 * *

25 Dopamine Induced Dopamine % Peak Fluorescence 0 SKF 83822 - + + - Quinpirole - - + +

FIGURE 3-8. Effect of D2 receptor agonist, quinpirole, on the desensitization of the D1-D2 receptor heteromer mediated calcium signal in HEK 293T cells stably expressing D1 and D2 receptors. Data were represented as the percentage of peak fluorescence of the dopamine induced calcium signal and values are the means ± S.E.M. of the numbers shown in brackets. Desensitization of the calcium signal elicited by dopamine following pre-treatment with 1 μM SKF 81297 (A), 1 μM SKF 83959 (B), or 1 μM SKF 83822 (C) in the absence and presence of 1 μM quinpirole or quinpirole treatment alone (n=3-4). A significant difference from control is denoted by * = p < 0.05.

A B D1 R D2 R

MERGED FRETc

C 0.25 D 27cells Dopamine 0.25 * 0.20 0.20 20cells 0.15 83959 * 0.15 0.10 83822 0.10 NET FRET 0.05 NET FRET 32cells 0.00 0.05 -0.05 0.00 0 10 20 30 40 50 60 70 59 Time (Seconds) 822 839 83 F opamine K KF D S S

FIGURE 3-9. Specificity of dopamine receptor agonists activating the D1-D2 receptor heteromer calcium signal in primary striatal neurons. (A) Immunocytochemistry showing endogenously expressed dopamine D1 and D2 receptor colocalization (merged) and interaction (corrected FRET = FRETc). The inset shows the calibration for FRET efficiency. (B) Striatal neurons displaying the presence of transfected cameleon (blue). (C) Representative tracings of cameleon FRET resulting from intracellular calcium release from D1-D2 receptor heteromer activation by 100 nM dopamine or SKF 83959 but not SKF 83822. The arrow indicates the time point of drug addition. (D) Peak heights of agonist induced cameleon FRET corresponding to calcium release through activation of the D1-D2 receptor heteromer. The results shown represent the means ± S.E.M. of values from the number of cells shown. A significant difference from SKF 83822 is denoted by * = p < 0.05.

77 The FRET signal peaked within 10 sec of agonist activation and declined within 50 sec. In contrast, induction of calcium mobilization by SKF 83822 was minimal (Fig 3-9C,D).

3.4 Desensitization of the D1-D2 Receptor Mediated Calcium Signal in Primary Striatal

Neurons- For desensitization studies, the neurons were pre-treated with 100 nM SKF 83959 or

SKF 83822 for 30 min and then washed off, followed by subsequent activation with 100 nM dopamine. Pre-treatment with either SKF 83959 or SKF 83822 led to a significant attenuation of the calcium signal peak heights (50.9 ± 0 .01% of control for SKF 83959 and 48.8 ± 0.01% of control for SKF 83822) (Fig 3-10A). The extent of desensitization was not significantly different when the neurons were pre-treated with SKF 83822 in the presence or absence of either the adenylyl cyclase inhibitor, SQ 22536, or the protein kinase A (PKA) inhibitor, H-89, suggesting a heterologous mechanism involving the adenylyl cyclase or cAMP pathway was not responsible for the signal attenuation by SKF 83822 (Fig 3-10B). Pre-treatment with either inhibitor alone did not result in any significant difference in the calcium signal from that elicited by dopamine.

Since it was shown that SKF 83959 can occupy both the D1 and D2 receptors within the

D1-D2 receptor complex (Rashid et al., 2007), the desensitization observed may be mediated by occupancy of both receptors. To prevent agonist occupancy of the D2 receptor, a D2 selective antagonist, raclopride 10µM was added with SKF 83959 or SKF 83822 pre-treatment for 30 min and then washed off followed by activation with 100 nM dopamine. No significant difference in the extent of desensitization with either SKF 83959 or SKF 83822 was observed in the presence of raclopride (Fig 3-11A and B). However, SKF 83959 and SKF 83822 mediated desensitization was abolished by pre-treatment with the D1 antagonist, SCH 23390 10 µM.

A 0.25 38cells 0.20 0.15 52cells 49cells 0.10 * * Net FRET Net 0.05 Dopamine Induced Dopamine

0.00 SKF 83959 - + - SKF 83822 - - +

B 0.25 30cells 41cells 47cells 0.20 45cells 0.15 57cells 47cells * 0.10 * Net FRETNet

0.05 Dopamine Induced Dopamine 0.00 SKF 83822 - + + + - - SQ 22536 - - + - + - H-89 - - - + - +

FIGURE 3-10. The D1-D2 receptor heteromer mediated calcium signal is desensitized in striatal neurons by prior treatment with dopamine agonists for 30 min. (A) Peak FRET levels corresponding to rises in intracellular calcium by activation with 100 nM dopamine and after pre-treatment with 100 nM SKF 83959 or 100 nM SKF 83822 for 30 min. (B) Peak FRET levels corresponding to rises in intracellular calcium by activation with 100 nM dopamine and after pre-treatment with 100 nM SKF 83822 without and with 10 μΜ SQ 22536 or 10 μΜ H-89, or SQ 22536 alone or H-89 alone. The results shown represent the means ± S.E.M. of values from the number of cells shown. A significant difference from control (no pre-treatment) is denoted by * = p < 0.05.

AB 0.25 0.25 49cells 46cells 33cells 46cells 0.20 0.20

0.15 0.15 53cells 50cells 50cells 48cells * * 0.10 0.10 * Net FRET * Net FRET 0.05 0.05

Induced Dopamine Induced Dopamine 0.00 0.00 SKF 83959 - + + + SKF 83822 - + + + Raclopride - - + - Raclopride - - + - SCH 23390 - - - + SCH 23390 - - - +

FIGURE 3-11. Effect of dopamine receptor antagonists on the desensitization of the D1-D2 receptor heteromer mediated calcium signal in striatal neurons. Peak FRET levels corresponding to rises in intracellular calcium by activation with 100 nM dopamine and after pre-treatment with 100 nM SKF 83959 (A) or 100 nM SKF 83822 (B) without and with 10 μM raclopride or 10 μM SCH 23390. The results shown represent the means ± S.E.M. of values from the number of cells shown. A significant difference from control (no pre-treatment) is denoted by * = p < 0.05.

80 These results suggested that the desensitization elicited by each agonist in these neurons

occurred through selective occupancy of the D1 receptor.

3.5 Role of GRK2 in Regulating the D1-D2 Receptor Heteromer Mediated Calcium Signal-

To analyze the mechanism by which GRK2 mediated the D1-D2 receptor heteromer calcium signal desensitization, GRK2 was transiently transfected into the HEK 293T D1-D2 receptor heteromer stable cell line in increasing concentrations which led to a progressive attenuation of the calcium signal when activated with dopamine (Fig 3-12A, bars 3-6 and Fig 3-12B). No significant decrease in this calcium signal was observed when cells were transfected with the empty vector pcDNA3. The addition of ATP to the GRK2 transfected cells to activate endogenously expressed purinergic receptors that couple to Gq, also demonstrated a progressive decline in the ATP mediated calcium signal, indicating the GRK2 was active in these cells (Fig

3-12C, bars 3-6).

3.5.1 Evaluation of GRK2 functional domains in regulating the D1-D2 receptor heteromer mediated calcium signal- The GRK2 crystal structure confirms that it is composed of three functional domains: an amino terminal RGS domain, a central protein kinase domain, and a carboxyl-terminal, Gβγ PH domain (Lodowski et al., 2003). To determine whether the calcium signal attenuation was due to catalytic or RGS activity, GRK2 mutant constructs were transiently transfected into the HEK 293T D1-D2 receptor heteromer stable cell line.

81 A 100

75 * 50 *

25 *

Dopamine Induced Dopamine * % Fluorescence Peak 0 ol .5 5 8 1.3 2 GRK2 Plasmid cDNA [μg] contr pcDNA3

B 5 .3 . 0 1 2 5 8 μg cDNA

_ 80 GRK2

_ 36 GAPDH

100

75 * 50 *

ATP Induced ATP * 25 % Fluorescence Peak 0 l o 3 3 5 8 tr A 1. 2.5 n N GRK2 Plasmid cDNA [μg] co pcD

FIGURE 3-12. Increased expression of GRK 2 led to a concentration dependent decrease of the D1-D2 receptor heteromer activated calcium signal. Data represent the percentage of peak fluorescence of the agonist induced calcium signal and values are the means ± S.E.M. of the numbers shown in brackets. (A) Activation of the D1-D2 receptor mediated calcium signal with 10 μM dopamine in HEK 293T cells without or with increasing expression of GRK2 (n=4). (B) Immunoblot demonstrating the increasing expression of GRK2 in D1-D2 receptor expressing cells transfected with GRK2 cDNA. GAPDH immunoreactivity was used as a control for protein loading. (C) Activation of the endogenous purinergic receptor mediated calcium signal with 10 μM ATP in HEK 293T cells without or with increasing expression of GRK2 (n=3-4). A significant difference from control is denoted by * = p < 0.05.

82 Transfection of the catalytically inactive GRK2 (GRK2-K220R), previously shown as being

capable of acting in a dominant negative manner to reverse desensitization of some GPCRs

(Claing et al., 2002; Ferguson, 2001; Krupnick and Benovic, 1998), led to a partial but not

complete restoration of the calcium signal following transfection of 2.5 µg or 5 µg cDNA, in

comparison to wild type GRK2 (Fig 3-13A bars 3,4 and 6,7 and Fig 3-13B lanes 3 and 6). Since

expression of the catalytically inactive GRK2 only led to a partial recovery of the calcium signal, the involvement of the RGS domain of GRK2 was investigated. Expression of a GRK2 mutant

(GRK2-D110A), that lacks the ability to interact with Gq (Sterne-Marr et al., 2003), also led to a partial but not complete restoration of the signal following transfection of 2.5 µg or 5 µg cDNA in comparison to wildtype GRK2 (Fig 3-13A bars 3,5 and 6,8 and Fig 3-13B lanes 4 and 7). Co- expression of a GRK2 double point mutant (GRK2-R106A/K220R) that lacked both catalytic and RGS function led to full restoration of the calcium signal following transfection of 1µg cDNA and partial restoration following transfection of 2.5 µg and 5 µg cDNA (Fig 3-13C, bars

3-5 and Fig 3-13D). Taken together, these results indicated that both the RGS and catalytic domains of GRK2 played a role in inhibiting D1-D2 receptor heteromer signalling after activation.

The involvement of GRK2 and its mutant constructs in the desensitization of the dopamine induced calcium signal after agonist pre-treatment was also investigated. Increased expression of GRK2 led to a significant increase in desensitization of the dopamine induced signal after pre-treatment with either SKF 83959 or SKF 83822 for 30 min (Fig 3-14A).

Expression of either GRK2-K220R or GRK2-D110A did not lead to any significant changes in the level of desensitization after pre-treatment with either agonist (Fig 3-14B).

2.5μg 5μg A B R A 2 R A 100 k 2 0 0 0 0 c K 2 1 K 2 1 o R 2 1 R 2 1 2.5μg5μg M G K D G K D 75 * 50 * * 80_

25 * Dopamine Induced % PeakFluorescence

K2 0R K2 0R 36_ ntrol R GAPDH o 22 110A 22 110A c G K D GR K D pcDNA3 C D 100 0 1 2.5 5 μg cDNA

75 * * _ R106A-K220R-GRK2 50 80

25 Dopamine Induced Dopamine

% Peak Fluorescence 0 l _ o 3 1 5 36 GAPDH A 2.5 ntr N o C R106A/K220R pcD Plasmid cDNA [μg]

FIGURE 3-13. Effect of catalytic domain mutated or RGS domain mutated GRK2 on the D1-D2 receptor heteromer mediated calcium signal. Data represent the percentage of peak fluorescence of the dopamine induced calcium signal and values are the means ± S.E.M. of the numbers shown in brackets. (A) Activation of the D1-D2 receptor heteromer mediated calcium signal by 10 μM dopamine in HEK 293T cells without or with expression of 2.5 μg or 5 μg cDNA for GRK2 (bars 3 and 6), GRK2-K220R (bars 4 and 7) or GRK2 D110A (bars 4 and 8) (n=3-5). (B) Immunoblot demonstrating the increasing expression of GRK2 and mutated constructs in D1-D2 receptor expressing cells. GAPDH immunoreactivity was used as a control for protein loading. (C) Activation of the D1-D2 receptor heteromer mediated calcium signal with 10 μM dopamine in cells without or with increasing expression of GRK2-R106A/K220R (n=3). (D) Immunoblot demonstrating the increasing expression of GRK2-R106A/K220R. GAPDH immunoreactivity was used as a control for protein loading. A significant difference from control is denoted by * = p < 0.05.

A 100

75 SKF 83959 SKF 83822

50 * * 25

Induced Dopamine % Peak Fluorescence % Peak 0 GRK2 - - + - +

B 100

75 SKF 83959 SKF 83822

Dopamine Induced Dopamine 25 Fluoresence Peak %

0 GRK2 K220R - - + - - + - GRK2 D110A - - - + - - +

FIGURE 3-14. Effect of GRK2, catalytic domain mutated or RGS domain mutated GRK2 on the D1-D2 receptor heteromer mediated calcium signal after agonist pre-treatment with dopamine agonists for 30 min. Data represent the percentage of peak fluorescence of the dopamine induced calcium signal and values are the means ± S.E.M. of the numbers shown in brackets. (A) Activation of the D1-D2 receptor heteromer mediated calcium signal by 10 μM dopamine in HEK 293T cells following pre-treatment with 1 μM SKF 83959 or SKF 83822 without or with expression of 1.3 μg cDNA for GRK2 (n=3-4). (B) Activation of the D1-D2 receptor heteromer mediated calcium signal by 10 μM dopamine in cells following pre-treatment with 1 μM SKF 83959 or SKF 83822 without or with expression of 1.3 μg cDNA for GRK2-K220R or GRK2 D110A (n=3-5). A significant difference from control is denoted by * = p < 0.05.

85 3.5.2 Knockdown of GRK2 in HEK293T cells and striatal neurons- To further validate the role of GRK2 in desensitization of the calcium signal, endogenous GRK2 was attenuated with siRNA in both the D1-D2 receptor heteromer stable cell line as well as in striatal neurons.

Transfection of siRNA to silence GRK2 in the D1-D2 receptor heteromer expressing cells resulted in significant recovery of the calcium signal after pre-treatment with either SKF 83959 or SKF 83822 for 30 min (Fig 3-15A). Furthermore, the knockdown of endogenous GRK2 led to a significantly higher dopamine induced calcium signal in the absence of agonist pre-treatment in two of out of the three experiments performed, indicating the contribution of physiological levels of endogenous GRK2 in regulating the peak height of the dopamine induced calcium signal (3-

15C). The siRNA mediated reduction in expression of GRK2 levels was confirmed by Western blotting (Fig 3-15B). Knockdown of endogenous GRK2 in the striatal neurons also resulted in significant recovery of the dopamine induced calcium signal after exposure to either SKF 83959 or SKF 83822 for 30 min (Fig 3-16A). The siRNA mediated reduction in expression of GRK2 levels in the striatal neurons was confirmed by Western blotting (Fig 3-16B).

3.5.3 D1-D2 receptor heteromer interaction with GRK2- To examine whether the agonists induced recruitment of GRK2 to the D1-D2 receptor heteromer, we performed immunocytochemistry on striatal neurons. Pre-treatment of the striatal neurons with 100 nM dopamine, SKF 83959 or SKF 83822 for 5 min each led to a similar extent of GRK2 relocalization in a punctuate distribution, indicating GRK2 reactivity induced by these agonists

(Fig 3-17).

86 A SKF 83959 SKF 83822 100

75 * * 50

25 Dopamine Induced Dopamine % Peak Fluorescence % 0 GRK2 siRNA - + - +

_ B siRNA +

80 _ GRK2

_ 36 GAPDH

C 100000

75000

50000 AFU

25000 Dopamine Induced

0 GRK2 siRNA - + FIGURE 3-15. Decreased expression of GRK2 by siRNA led to significant recovery of the D1- D2 receptor heteromer mediated calcium signal after pre-treatment with either SKF 83959 or SKF 83822 in the HEK 293T D1-D2 receptor heteromer stable cell line. Data represent the percentage of peak fluorescence (A and B) or AFU (C) of the dopamine induced calcium signal and values are the means ± S.E.M. of the numbers shown in brackets. A.F.U. = absolute fluorescence units. (A) Desensitization of the calcium signal elicited by dopamine following pre- treatment with 1μM SKF 83959 or 1μM SKF 83822 for 30 min after expression of either non silencing (-) or GRK2 siRNA (+) (n=3). (B) Immunoblot demonstrating the decreased expression of GRK2. GAPDH immunoreactivity was used as a control for protein loading. (C) Dopamine induced calcium signal after expression of either non silencing (-) or GRK2 siRNA (+) (n=2).

A B

100 SKF 83959 SKF 83822 _ siRNA_ + 75 80 GRK2 * * 50

36_ GAPDH % Net FRET % Net 25 Dopamine Induced

0 - GRK2 siRNA + - +

FIGURE 3-16. Decreased expression of GRK2 by siRNA led to significant recovery of the D1- D2 receptor heteromer mediated calcium signal after pre-treatment with either SKF 83959 or SKF 83822 in striatal neurons. Data represent the percentage of peak FRET levels of the dopamine induced calcium signal. Peak FRET levels are the means ± S.E.M. from a range of 35 to 60 neurons from a total of 3 experiments. (A) Desensitization of the calcium signal elicited by dopamine following pre-treatment with 100 nM SKF 83959 or 100 nM SKF 83822 for 30 min after expression of either non silencing (-) or GRK2 siRNA (+). (B) Immunoblot demonstrating the decreased expression of GRK2 in striatal neurons. GAPDH immunoreactivity was used as a control for protein loading. A significant difference from control (no-pre-treatment) is denoted by * = p < 0.05.

Control

10μM Dopamine

SKF 83959

SKF 83822

FIGURE 3-17. Immunocytochemistry of striatal neurons in culture showing endogenously expressed GRK2 localization before (control) and after exposure to either 100 nM dopamine, SKF 83959 or SKF 83822 for 5 min. Arrow indicates distinct punctate.

89 To determine whether GRK2 physically interacted with the D1-D2 receptor heteromeric complex, we performed co-immunoprecipitation studies. After 5 min agonist treatment of HEK

293T cells stably expressing both the HA-D1 and FLAG-D2 receptors, the FLAG-D2 receptor was immunoprecipitated from a P2 membrane preparation. In addition to the D1 receptor at

55kDa, immunoblotting of this preparation revealed a band corresponding to GRK2 at 80kDa for each agonist treatment that was denser than the control band (no agonist treatment) (Fig 3-

18), suggesting an agonist induced increase in the physical association between the D1-D2 receptor heteromeric complex and GRK2. To measure the extent of GRK2 recruitment, the

GRK2 immunoblot was normalized to the amount of FLAG-D2 receptor immunoprecipitated for vehicle and each agonist treatment. There was 350%, 270%, and 168% increases in GRK2 precipitation with the FLAG-D2 receptor compared to control for treatments with dopamine,

SKF 83959 and SKF 83822, respectively.

Moreover, to lend further support to the notion of a direct agonist induced interaction between the D1-D2 receptor heteromer and GRK2, a BRET assay was performed with HEK

293T cells transfected with Rluc-D1 receptor, D2 receptor and GFP-GRK2, or GFP cDNAs.

Treatment with 1 μM dopamine, SKF 83822, or SKF 83959 for 1 min led to a rapid rise in the

BRET ratio between Rluc-D1 receptor and GFP-GRK2 in comparison to control (no agonist treatment) (Fig 3-19A) and declined within 10 min of agonist exposure (Fig 3-19B). This indicated a rapid agonist induced association (proximity <100Å) between Rluc-D1 receptor and

GFP-GRK2.

IP: FLAG-D2

l l o o r r t t n n SKF 83959 SKF 83959 SKF 83822 Co Dopamine Dopamine SKF 83959 SKF 83822 Co Dopamine Dopamine

IB: HA-D1 - 55 IB: GRK2 - 80

FIGURE 3-18. Co-immunoprecipitation of HA-D1 and GRK2 with FLAG-D2 receptor from P2 membranes expressing FLAG-D2 receptor, HA-D1 receptor and GRK2 after the HEK 293T cells were treated with vehicle (control), 1 μM dopamine, SKF 83959, or SKF 83822 for 5 min. IP=Immunoprecipitation with FLAG antibody. IB=Immunoblot with HA or GRK2 antibody.

A 0.05 * 0.04 * 0.03 *

0.02

Ratio BRET 0.01

0.00 l 9 2 5 2 ine 9 8 3 8 Contro F83 F opam K K D S S

B 0.05 0.04 0.03 0.02

BRET Ratio BRET 0.01

0.00

-0.01 9 ol ne 5 822 mi 3 Contr F8 opa KF839 K D S S

FIGURE 3-19. BRET detection of Rluc-D1 and GFP-GRK2 interaction after either 1 min treatment (A) or 10 min treatment (B) with vehicle (control), 1 μM dopamine, SKF 83959, or SKF 83822. Values shown are the means ± S.E.M. of n=3 experiments. A significant difference from control is denoted by * = p < 0.05.

92 3.6 Resensitization of the D1-D2 Receptor Heteromer Mediated Calcium Signal in

HEK293T Cells- To study resensitization of the calcium signal, the HEK 293T cells co- expressing D1 and D2 receptors were pre-treated with 1 μM dopamine or SKF 83959 for 30 min, then was washed off followed by incubation at 37oC for up to 3 hrs before activation of the calcium signal with dopamine. The desensitized response significantly resensitized within 1.5 hr and 3 hr of dopamine removal but not SKF 83959 removal (Fig 3-20 A and B). To determine if protein synthesis played a role in the recovery of the desensitized response, the protein synthesis inhibitor, cycloheximide, was added to the cells during the 3 hr incubation after dopamine and

SKF 83959 wash off. No significant difference in the level of the calcium signal was observed in the presence and absence of cycloheximide, indicating protein synthesis was not necessary for recovery of the signal (Fig 3-20 A and B).

3.7 Internalization and Recycling of D1 and D2 receptors following treatment with dopamine or SKF 83959- To investigate the intracellular trafficking of D1 and D2 receptors following agonist treatment, immunocytochemistry was performed with the HEK 293T cell line stably expressing the D1 and D2 receptors. In the absence of agonist treatment, the D1 receptor was localized primarily to the cell surface and the D2 receptor was expressed both intracellularly and at the cell surface (Fig 3-21). To study internalization of the receptors, the cells were treated with 1 μM dopamine or SKF 83959 for 30 min. Treatment with either agonist induced significant internalization of both the D1 and D2 receptors (Fig 3-21 and 3-22). To determine whether the receptors recycled back to the cell surface, the cells were monitored either 1.5 hr or

3 hr after agonist wash off. For dopamine treated cells, the D1 and D2 receptor reappeared at the cell surface within 1.5 hr and 3 hr of dopamine removal (Fig 3-21), suggesting receptors treated with dopamine could recycle.

A 100

75 *

Dopamine Induced 25 % Peak Fluoresence

0 Pretreatment None Dopamine Dopamine Dopamine Dopamine Recovery Time 0 hr 0 hr 1.5 hr 3 hr 3 hr Cycloheximide +

B 100

75 * * * *

Dopamine Induced 25 % Peak Fluoresence

0 Pretreatment None 83959 83959 83959 83959

Recovery Time 0 hr 0 hr 1.5 hr 3 hr 3 hr Cycloheximide +

FIGURE 3-20. Resensitization of the D1-D2 receptor heteromer mediated calcium signal in HEK 293T cells stably expressing D1 and D2 receptors. Data were represented as the percentage of peak fluorescence of the dopamine induced calcium signal. Cells were pre-treated with 1 μM dopamine (A) or SKF 83959 (B) for 30 min and the calcium signal was activated either immediately, 1.5 hr, or 3 hr after agonist wash off in the absence and presence of cycloheximide (35 μM). Values shown are the means ± S.E.M. of n=3-6 experiments. A significant difference from control (no pretreatment) is denoted by * = p < 0.05.

D1 D2 Merged

Control

Dopamine 30min Treatment

1.5hr Recovery

3hr Recovery

FIGURE 3-21. Trafficking of D1 and D2 receptors after treatment with dopamine in HEK 293T cells stably expressing D1 and D2 receptors. Immunocytochemistry displaying D1 (green), D2 (red), or both receptors (merged) before and after 30 min treatment with 1 μM dopamine as well as 1.5hr and 3hr after dopamine wash off. Images shown are representatives from a range of 93 to 143 cell images.

D1 D2 Merged

Control

SKF 83959 30min Treatment

1.5hr Recovery

3hr Recovery

FIGURE 3-22.Trafficking of D1 and D2 receptors after treatment with SKF 83959 in HEK 293T cells stably expressing D1 and D2 receptors. Immunocytochemistry displaying D1 (green), D2 (red), or both receptors (merged) before and after 30 min treatment with 1 μM SKF 83959 as well as 1.5hr and 3hr after SKF 83959 wash off. Images shown are representatives from a range of 112 to 167 cell images.

96 In contrast, in cells treated with SKF 83959, the D1 or D2 receptor did not reappear at the cell

surface within 1.5 hr or 3 hr of agonist removal (Fig 3-22), suggesting SKF 83959 treatment did

not result in recycling of these receptors. Taken together, these results suggest that trafficking of

the D1-D2 receptor heteromer is determined by specific ligand exposure.

To quantify D1 and D2 receptor internalization, radioligand binding assays using HEK

293T whole cells were performed with [3H]SCH 23390 or [3H]Raclopride to measure whole cell surface binding of either the D1 or D2 receptor, respectively. To ensure that [3H]SCH 23390 or

[3H]Raclopride measured cell surface receptors, the assay was conducted at 4ºC and specific

binding to cell surface receptors was calculated by the displacement of radioligand by 100 μM

dopamine. Since dopamine does not cross the cell membrane it would only displace radioligand

from receptors on the cell surface. Non-specific binding accounted for approximately 5% of total

bound ligand. Treatment with SKF 83959 for 5 min or 30 min resulted in significant

internalization of the D1 receptor in cells expressing the D1 receptor alone (68.5 ± 12.8% for 5

min and 75.6 ± 3.3% for 30 min) or cells expressing both the D1 and D2 receptors (67.5 ± 17.5%

for 5 min and 67.7 ± 7.7% for 30 min), indicating maximum D1 receptor internalization occurred

within 5 min of agonist stimulation (Fig 3-23). Treatment with dopamine for 30 min also resulted

internalization of the D1 receptor in cells expressing the D1 receptor alone (26.2 ± 5.0%) or cells

expressing both the D1 and D2 receptors (20.6 ± 2.9%), but it was significantly less than what

was observed with SKF 83959 (Fig 3-23). Treatment with either agonist also resulted in

internalization of the D2 receptor co-expressed with the D1 receptor, but results were

inconsistent and therefore are not reported here.

100 SKF 83959 5 min 30 min

50 Dopamine 30 min

%Receptor D1 * 25 * Internalization of Control of Internalization

0 D1 D1-D2 D1 D1-D2 D1 D1-D2

FIGURE 3-23. Agonist induced internalization of the D1 receptor in HEK 293T cells expressing the D1 receptor alone or co-expressing both the D1 and D2 receptors. Cells were treated with 100 nM SKF 83959 for 5 min or 30 min or 1μM dopamine for 30 min. Percentage internalization represents the loss of [3H]SCH 23390 binding from the cell surface of intact cells after agonist treatment compared with vehicle treated controls. Values shown are the means ± S.E.M. of n=3- 6 experiments. A significant difference from treatment with SKF 83959 is denoted by * = p < 0.05.

98 4 DISCUSSION

4.1 Desensitization of the D1-D2 receptor heteromer mediated calcium signal

The discovery of the D1-D2 receptor heteromer Gq-mediated calcium signal is important

since by this mechanism activation of dopamine receptors is directly linked to calcium signaling.

Given that there is localization of the D1-D2 receptor heteromer throughout the basal ganglia, it

is essential to determine how this complex and its signal are regulated. Termination of a signal

by receptor desensitization is an important component of GPCR signaling. The first study

investigating the desensitization of the D1-D2 receptor complex was reported in 2007 (So et al.,

2007). This report demonstrated that the D1-D2 receptor heteromer mediated calcium signal

rapidly desensitized within 2 minutes by prior exposure to dopamine or selective D1 or D2

agonists in heterologous cells. The desensitization was triggered by agonist occupancy of either

receptor subtype, even though co-occupancy of both receptors was necessary for generation of

the calcium signal. Additionally, desensitization of the signal was suggested to occur before the complex was recruited into vesicles by the endocytic machinery since preventing internalization of the complex did not decrease the extent of signal desensitization. Furthermore, the desensitization occurred specifically through a homologous mechanism and was independent of intracellular calcium store depletion, suggesting the calcium signal desensitization might occur at the level of the receptor complex. To study potential mediators of D1-D2 receptor heteromer calcium signal desensitization, several second messenger kinases were tested, but only GRKs 2 or 3 were shown to play a role (So et al., 2007).

My studies have demonstrated the desensitization of the D1-D2 receptor heteromer mediated calcium signal by agonists that occupied the D1 receptor binding pocket regardless of

99 whether they activated the D1-D2 receptor heteromer induced calcium signal. This

desensitization was shown to be mediated by at least two functional domains of GRK2.

All three agonists with high affinities for the D1 receptor were compared for their ability

to stimulate intracellular calcium release through the D1-D2 receptor heterooligomer. While SKF

83959 and SKF 81297 generated robust, almost equivalent calcium signals, SKF 83822 was

unable to generate a significant calcium signal in either the D1-D2 receptor heteromer expressing

cells or in striatal neurons. We have previously demonstrated that the D1-D2 receptor heteromer

is coupled to Gq which could be activated by SKF 83959 but not by SKF 83822, as shown by

S35GTPγS incorporation into Gq (Rashid et al., 2007). Desensitization of the D1-D2 receptor

heteromer mediated calcium signal occurred not only by exposure to SKF 83959 and SKF 81297

but also by SKF 83822 to a level comparable with the other agonists. This suggested that D1-D2

receptor heteromer activation was not a prerequisite for its desensitization and that a ligand

occupying the binding pocket of one constituent receptor with high affinity could result in signal

desensitization. A heterologous mechanism involving the adenylyl cyclase pathway was not

responsible for this observed desensitization since inhibition of adenylyl cyclase or PKA did not

significantly change the attenuation of the signal by SKF 83822 in striatal neurons. These results

suggested that receptor occupancy by the agonist and its associated conformational changes may

be sufficient for desensitization of the signal without Gq protein activation. Biochemical and

biophysical data suggest that different ligands can indeed induce and/or stabilize subsets of the

multiple active conformations of a receptor (Swaminath et al., 2005; Vilardaga et al., 2005). It is

possible that occupancy of the receptor by SKF 83822 leads to a stabilization of the receptor into

a conformation where it becomes a target for kinases, arrestins or endocytic machinery without

Gq protein activation. In fact, several ligands that recruit arrestin and/or induce receptor

internalization without stimulating G protein signaling have been identified. Receptors for which

100 this has been shown include the angiotensin II type 1 receptor (Wei et al., 2003), β2- adrenoreceptor (Shenoy et al., 2006), cholecystokinin receptor (Roettger et al., 1997), V2- vasopressin receptor (Ren et al., 2005) and type 1 parathyroid hormone receptor (Gesty-Palmer et al., 2006). However, as opposed to these homooligomeric complexes, agonist induced structural changes within the D1-D2 receptor heterooligomer likely exhibit an increased level of complexity as a result of the presence of two distinct dopamine receptor subtypes within a single heteromeric complex, and hence an increased potential for multiple conformational states.

Alternatively, SKF 83822 exposure may have led to activation of GPCR pathways mediated by other G proteins other than Gq such as Gα12/13 which could have potentially played a role in the desensitization of the calcium signal.

Desensitization of the calcium signal occurred independent of calcium storage capacity and exogenous calcium entry, suggesting that there was no impairment of intracellular calcium kinetics by agonist activation and that desensitization occurred at the receptor level. However, the involvement of other calcium homeostatic pathways that may have been affected by D1-D2 receptor heteromer activation could not be ruled out. For example, plasma membrane calcium-

ATPases (PMCAs) are a family of calcium pumps that function to maintain low concentrations of cytosolic calcium by responding to elevations in calcium following calcium release from intracellular organelles or after the influx of extracellular calcium (Di Leva et al., 2008). These

PMCAs could potentially have been upregulated by D1-D2 receptor heteromer activation and therefore played a role in the observed decreased calcium signal after agonist pre-treatment.

To investigate a possible mechanism responsible for D1-D2 receptor heteromer mediated calcium signal desensitization at the receptor level, GRK2 and its mutant constructs were utilized. Increased expression of GRK2 led to a concentration dependent decrease of the calcium signal and knockdown of GRK2 by siRNA led to an increase in the calcium signal. The GRK2

101 constructs that were catalytically inactive or RGS mutated each led to a partial reversal of the

GRK2 calcium signal effect suggesting that both GRK2 domains were involved and thus GRK2 had a dual role in mediating calcium signal desensitization. This is the first demonstration of

GRK2 having a bifunctional role in regulating a GPCR heterooligomer where the constituent receptor homooligomers are not coupled to the Gq protein.

The lowest concentration of GRK2-R106A/K220R, which lacked both the catalytic and

RGS functions, led to a near complete recovery of the calcium signal, providing further evidence that both GRK2 domains were involved in the calcium signal regulation. However, expression of

GRK2-R106A/K220R at higher concentrations was not able to completely restore the calcium signal. Although GRK2-R106A/K220R contains a mutation within its RGS domain resulting in prevention of Gq binding it is possible that it still retains the ability to bind the D1-D2 receptor heteromeric complex. Indeed, a recent study has demonstrated that GRK2 and several of its mutants including the catalytically inactive and RGS mutated GRK2 constructs were able to co- immunoprecipitate with the D2 receptor homooligomer (Namkung et al., 2009b). A similar phenomenon has been demonstrated for the metabotropic glutamate receptor 1 (mGluR1) where

GRK2 mutants impaired in Gq binding could still bind avidly to the mGluR1 receptor (Dhami et al., 2004). Thus, it can be postulated that the failure of higher concentrations of GRK2-

R106A/K220R to completely restore calcium signaling could be attributed to steric hindrance of receptor-Gq coupling caused by GRK2-R106A/K220R binding to the D1-D2 receptor heteromeric complex. My results are consistent with what has been reported for the Gq coupled

H1 histamine receptor, where although lower concentrations of the GRK2 double mutant resulted in little inhibition of agonist induced inositol phosphate production, higher concentrations showed a partial inhibitory effect (Iwata et al., 2005). Thus, it is possible that a

102 similar process occurred in the present system where excessive binding of GRK2-R106A/K220R to the D1-D2 receptor heteromer resulted in the attenuation of inherent receptor Gq interactions.

The involvement of GRK2 in calcium signal desensitization after agonist pre-treatment with either SKF 83959 or SKF 83822 was confirmed by both increasing and decreasing the expression of GRK2 in D1-D2 receptor heteromer expressing cells as well as by significant knockdown of endogenous GRK2 in primary culture neurons. Since SKF 83822 does not activate the calcium signal but has structural similarity to SKF 83959, these results suggest that

GRK2 may be involved in receptor occupancy mediated calcium desensitization by either SKF

83822 or SKF 83959. Accordingly, agonist induced recruitment of GRK2 to the D1-D2 receptor heteromer was suggested by the redistribution of GRK2 to the cell periphery as well as significant co-immunoprecipitation of both GRK2 and the D1 receptor with the D2 receptor after agonist treatment. Although the co-immunoprecipitation data indicated that SKF 83822 resulted in less GRK2 recruitment than dopamine or SKF 83959, all three agonists still elicited equivalent levels of desensitization of the D1-D2 receptor heteromer mediated calcium signal. Furthermore, there was a significant BRET signal between the D1 receptor and GRK2 in the presence of the

D2 receptor, indicating a physical association between the two proteins which was enhanced by all three agonists.

Expression of either GRK2-K220R or GRK2-D110A did not attenuate the desensitization of the calcium signal elicited by exposure to either agonist. In contrast, in the absence of agonist pre-treatment the expression of either GRK2 construct each led to a partial reversal of the GRK2 attenuated calcium signal. It is possible that the presence of agonist resulted in a greater recruitment of endogenous GRK2 to the D1-D2 receptor heteromeric complex in comparison to the basal state, resulting in increased signal attenuation and therefore may have masked any calcium signal recovery effects by the GRK2 mutant constructs. Agonist induced recruitment of

103 GRK2 to the D1-D2 receptor heteromer was suggested by immunocytochemistry, co- immunoprecipitation and BRET assays.

Alternatively, other GRK2 independent mechanisms may also be involved in the calcium signal attenuation. For example, agonist exposure may induce the formation of a stable receptor conformation that prevents restoration of the calcium signal or the receptor complex may have internalized or have been disrupted after agonist pre-treatment and thus may have resulted in a decreased dopamine induced calcium signal even in the presence of the GRK2 mutant constructs.

While GRK2 mediated desensitization has been reported for both D1 and D2 receptor homooligomers, only the catalytic activity of GRK2 has been indicated to be important for the

D1 receptor (Jackson et al., 2002; Kim et al., 2004; Lamey et al., 2002). For D2 receptor regulation, a recent study has provided evidence to suggest a role for the GRK2 carboxyl- terminal, Gβγ pleckstrin homology domain in addition to catalytic function (Namkung et al.,

2009b). Given this evidence, it is possible that the GRK2 Gβγ pleckstrin homology domain may also be involved in D1-D2 receptor heteromer regulation and this will be important to investigate. Interestingly, the GRK2 RGS domain was not involved in suppressing D2 receptor homooligomer signaling (Namkung et al., 2009b). Thus, my results indicated that D1-D2 receptor heterooligomer signaling regulation by GRK2 was distinct from D1 and D2 receptor homooligomer regulation, where this dual function involving both the catalytic and RGS domains of GRK2 in inhibiting signaling has not been reported. Additionally, adenylyl cyclase activity regulated by D1 and D2 receptor homooligomers is maximal within 15 to 30 min, as shown by measuring cAMP using competition radioimmuno assays or column chromatography

(Ryman-Rasmussen et al., 2005; Tong et al., 2001), whereas the D1-D2 receptor heteromer mediated calcium signal peaks within seconds of activation. Thus, the rapid nature of this calcium signal may require a quick and robust quenching mechanism that cannot be controlled

104 by the GRK2 catalytic activity alone and therefore regulation by both the catalytic and RGS domains within GRK2 may favor the prompt termination of the D1-D2 receptor heteromer mediated signal.

Signaling through the D1-D2 receptor heteromeric complex involves activation of the

Gq/calcium pathway. The ability for D1-D2 receptor heteromer to also signal through the

Gs/cAMP pathway cannot be determined since the D1-D2 receptor heteromer response cannot be completely separated from the D1 receptor homooligomer response in heterologous cells and therefore it is impossible to separate out the differences between the D1 receptor homooligomer

Gs/cAMP pathway from a potential D1-D2 receptor heteromer activation of the Gs/cAMP pathway. However, our lab has previously demonstrated that the agonist SKF 83959, which selectively activates the D1-D2 receptor heteromer, did not activate Gs/olf in striatal membranes, but activated Gq as shown by incorporation of S35GTPγS (Rashid et al., 2007).

Another potential mechanism for decreased calcium signaling after agonist exposure

could be that agonist occupancy of the receptors triggers the D1-D2 receptor complex to break

apart, thus disrupting and turning off the signal. Although a majority of studies indicate that cell

surface homooligomers remain intact after agonist activation (Babcock et al., 2003; Dinger et al.,

2003; Lee et al., 2000), agonist-dependent dissociation of GPCR oligomers has been suggested

to occur for certain receptors (Cheng and Miller, 2001; Gines et al., 2000). In fact, we recently

demonstrated that long term dopamine exposure disrupted D1-D2 receptor heterooligomer

formation after activation in heterologous cells (O’Dowd et al., 2011) and therefore short term agonist treatments used in the present experiments could also potentially result in a similar disruption of the heterooligomer.

105 In summary, I have demonstrated that desensitization of the D1-D2 receptor heteromer mediated calcium signal occurs by D1 receptor occupancy with or without signal activation and that GRK2 plays a role in regulating the signal response with at least two distinct functions. My results provide evidence for an entirely novel mechanism for dopamine D1-D2 receptor heteromer desensitization that is distinct from mechanisms that have been reported for D1 or D2 receptor homooligomers.

4.2 Internalization of the D1-D2 receptor heteromer and resensitization of the associated calcium signal

I have demonstrated that the D1-D2 receptor heteromer internalized by pre-treatment with either dopamine or SKF 83959, which was accompanied by desensitization of the calcium signal, but recycling of the receptors and resensitization of the calcium signal only occurred by exposure to dopamine and not SKF 83959.

Differential agonist exposure to the D1-D2 receptor heteromer resulted in different regulatory pathways for this heteromeric complex. First, although treatment with either dopamine or SKF 83959 resulted in internalization of the D1-D2 receptor heteromer, results from radioligand binding assays indicated that SKF 83959 exposure led to significantly more D1 receptor internalization than dopamine pre-treatment in both HEK 293T cells expressing D1 receptors alone and HEK 293T cells co-expressing D1 and D2 receptors. Second, D1 and D2 receptor recycling to the plasma membrane was observed after dopamine removal but not after

SKF 83959 removal even after a prolonged recovery time. Third, resensitization of the calcium signal was observed after dopamine wash-off but not after SKF 83959 removal; the protein synthesis inhibitor, cycloheximide, had no effect on the recovery of the calcium signal,

106 indicating protein synthesis was not necessary for resensitization. Taken together, these results suggest that trafficking of the D1-D2 receptor heteromer and resensitization of its associated calcium signal are delineated by particular agonist exposure rather than by a defined property of the D1-D2 receptor heteromer.

At the basal level without agonist exposure, D1 receptors were predominantly at the cell surface, whereas D2 receptors were localized both intracellularly and at the cell surface, which is a particular characteristic of the D2 receptor that has been well documented previously

(Fishburn et al., 1995; Prou et al., 2001; Takeuchi and Fukunaga, 2003). The internalized D1 and

D2 receptors returned back to basal localization after dopamine removal. However, a significant proportion of the confocal images also demonstrated almost complete recycling of D2 receptors to the cell surface with no D2 receptors being detected intracellularly. This suggests that internalized D2 receptors may have formed homooligomers with existing intracellular D2 receptors, resulting in recycling of both the agonist internalized D2 receptors and D2 receptors preexisting in the cytoplasm.

Following endocytosis, D1 receptor homooligomers are reported to predominantly recycle back to the plasma membrane (Bartlett et al., 2005; Dumartin et al., 1998; Jackson et al.,

2002; Lamey et al., 2002; Martin-Negrier et al., 2006; Vargas and Von Zastrow, 2004; Vickery and von Zastrow, 1999), but D2 receptor homooligomers are documented to predominantly degrade after dopamine exposure in cells and in neurons (Bartlett et al., 2005). Therefore, these results suggest that the dopamine induced D2 receptor trafficking profile is not conserved upon heterooligomerization with the D1 receptor, since D1-D2 receptor heteromer expression resulted in D2 receptor recycling. However, it is also important to note that the immunocytochemistry experiments were not performed in the presence of cycloheximide and therefore the involvement of D2 receptor synthesis cannot be completely ruled out. Yet, protein synthesis was not

107 responsible for resensitization of the calcium signal, thus suggesting recycling of the receptors

occurred in the absence of protein synthesis. The results also suggest that the D1 receptor

homooligomer recycling response is not conserved upon heteromerization with the D2 receptor

after SKF 83959 exposure, since D1-D2 receptor heteromer expression resulted in a lack of D1

receptor recycling after SKF 83959 pre-treatment. Thus, the D1-D2 receptor heterooligomer appears to adopt the recycling characteristics of the D1 receptor after dopamine exposure and the

properties of the D2 receptor after SKF 83959 exposure.

Although the results demonstrate that both D1 and D2 receptors internalized in response

to dopamine or SKF 83959 pre-treatment, it is not clear if these receptors remained together or

separated once internalized. Additionally, if the D1-D2 receptor heterooligomer did separate into

its constituent receptors after agonist exposure it is not clear if the receptors reformed into a

heterooligomer once they returned to the cell surface after dopamine exposure. Separation of the

internalized D1 and D2 receptors and then reformation into a heterooligomer at the cell surface

after recycling is a possibility since our lab has recently demonstrated that long term dopamine

exposure (4h) can disrupt D1-D2 receptor heterooligomer formation after activation in

heterologous cells and the receptors can reform into a complex at the cell surface (O'Dowd et al.,

2011). Furthermore, if the D1-D2 receptor heterooligomer did separate once internalized,

reformation at the cell surface would be expected since there was complete resensitization of the

D1-D2 receptor heterooligomer mediated calcium signal after dopamine exposure. However,

agonist treatments were tested up to 30 min in my experiments and therefore may not have

resulted in a similar heterooligomer disruption that was observed after long term agonist

exposure. Moreover, separation and reformation characteristics of this heteromer could depend

on specific ligand exposure and therefore could be different after dopamine vs. SKF 83859

exposure.

108 Differences in receptor recycling back to the plasma membrane and resensitization of the calcium signal after specific agonist exposure may be due to a differential interaction with arrestins. Dopamine exposure may allow the D1-D2 receptor heterooligomer to behave with typical Class A GPCR characteristics. Class A receptors do not internalize with the arrestin protein since it dissociates from the receptor at or near the plasma membrane, allowing for dephosphorylation in endosomes and rapid recycling back to the plasma membrane resulting in resensitization of the signal. In contrast, SKF 83959 exposure may cause the D1-D2 receptor heteromeric complex to behave with typical Class B GPCR characteristics. These GPCRs form a stable complex with arrestins, internalize with the arrestin protein bound and have been shown to either recycle slowly, be retained in the endosomal compartment or traffic to lysosomes for degradation (Oakley et al., 1999; Oakley et al., 2001; Oakley et al., 2000). While this differential interaction has been demonstrated for different GPCRs, the ability of different ligands to dictate a Class A or Class B response for the same receptor has only recently been demonstrated (Lee et al., 2010), but has never been shown for a receptor heterooligomer. It is believed that the interaction of arrestin with specific phosphorylated residues in the receptor’s carboxyl-terminal

tail is responsible for determining class A or class B characteristics with specific clusters of

residues absent in class A and present in class B GPCRs (Oakley et al., 2001). Exposure to either

dopamine or SKF 83959 may lead to differences in the extent of phosphorylation of the specific

subset of residues in the carboxyl tail of the D1 and/or D2 receptors resulting in changes in the

strength of the arrestin interaction with the receptors.

In summary, I have demonstrated that recycling of ligand induced internalized D1 and D2 receptors and resensitization the D1-D2 receptor heteromer mediated calcium signal occurs after

exposure to dopamine but not SKF 83959. This is the first demonstration of a ligand dictating the

resensitization response of a desensitized signal mediated through a receptor heteromer.

109 4.3 Related Studies

This section describes two publications that are related to the D1-D2 receptor heterooligomer to

which I contributed.

1. So CH, Verma V, O’Dowd BF, George SR (2007). Desensitization of the dopamine D1 and D2 receptor heterooligomer mediated calcium signal by agonist occupancy of either receptor. Mol Pharmacol 72:450-62.

Summary:

This was the first study that demonstrated rapid desensitization of the D1-D2 receptor

heterooligomer mediated calcium signal by pre-treatment with dopamine or selective D1 or D2

receptor agonists. The efficacy, potency and rate of signal desensitization differed between

agonists that selectively occupied the D1 or D2 receptors or both receptors simultaneously.

Several second messenger kinases were tested for a role in this desensitization, but only GRK2

and GRK3 were demonstrated to be important.

2. So CH, Verma V, Alijaniaram M, Cheng R, Rashid AJ, O'Dowd BF, George SR (2009). Calcium signaling by dopamine D5 receptor and D5-D2 receptor heterooligomers occurs by a mechanism distinct from that for dopamine D1-D2 receptor heterooligomers. Mol Pharmacol 75: 843-54.

Summary:

Since the D5 receptor has structural and sequence homology with the D1 receptor, this report

investigated if the D5 receptor could also interact with the D2 receptor to mediate a calcium signal. The results demonstrated that D5 and D2 receptors did indeed form heterooligomers and co-activation resulted in a calcium signal that was Gq and PLC mediated. However, in contrast

110 to what was observed for D1 receptors, which activate robust calcium mobilization only within a

complex with D2 receptors, an extensive Gq mediated calcium signal was triggered by D5

receptors expressed alone and this was attenuated by heterooligomer formation with D2 receptors. Additionally, the D5 receptor homooligomer and D5-D2 receptor heteromer mediated

calcium signals were critically dependent on extracellular calcium stores in addition to

intracellular stores, unlike what is observed for D1-D2 receptor heteromers which only trigger

intracellular calcium release.

My contributions to these publications were performing some of the calcium signaling assays

and aiding in the optimization of experiments and analysis of the experimental results.

4.4 Novel Findings and General Conclusions

Because D1-D2 receptor complexes are linked to calcium signaling and exist in vivo, it is

of major importance then to determine their regulation. Although much is known about the

mechanisms mediating D1 and D2 receptor homooligomers, the regulation of the D1-D2

receptor heteromer was largely unexplored when I began my PhD studies. My work has

identified some of the regulatory properties of the D1-D2 receptor heterooligomer and

demonstrated mechanisms that are distinct from its constituent receptors. A summary of the

novel findings and general conclusions generated by my work is provided below.

1. Desensitization of the D1-D2 receptor heteromer mediated calcium signal occurs by D1

receptor occupancy with or without signal activation.

111 By performing calcium mobilization assays in both heterologous cells and cultured striatal

neurons, my studies have demonstrated that agonists occupying the D1 receptor binding pocket

regardless of whether they activated the D1-D2 receptor heteromer mediated calcium signal

resulted in significant desensitization of the signal. These results indicated that D1-D2 receptor

heteromer activation was not a prerequisite for desensitization of its signal and occupancy of the

receptor binding pocket with its associated conformational changes could result in decreased signaling. This desensitization was mediated through selective occupancy of the D1 receptor since reduction of the calcium signal was unchanged in the presence of the D2 antagonist, raclopride, but abolished in the presence of the D1 antagonist, SCH 23390. Moreover,

pretreatment with both a D1 agonist and D2 agonist did not result in a further degree of

desensitization of the calcium signal in comparison to a D1 agonist alone, confirming the

desensitization was elicited through the D1 receptor.

The discrete signaling effects of SKF 83822 could have significant implications for

dopaminergic signaling in vivo, since this agonist could result in activation of D1 receptor

homooligomer signaling while simultaneously attenuating D1-D2 receptor heteromeric signaling

and therefore alter the general tone of dopamine transmission.

Pharmacological modification of dopaminergic signaling is used as a common

therapeutic tool in the treatment of many dopamine related diseases, but there are still currently

many undesirable side effects associated with these agents. Therefore, research still continues to

thrive in order to discover novel therapies with less unwanted side effects. Research development

in agonists that exhibit functional selectivity is a relatively new area of research and refers to

agonists that can selectively modify signal transduction pathways through a single receptor

isoform, depending on the effector pathway coupled to that receptor (Ryman-Rasmussen et al.,

2007). Given that occupancy of the D1 receptor by SKF 83822 within the D1-D2 receptor

112 heterooligomer induces GRK2 recruitment, this suggests that arrestins may be involved in the

regulation and these arrestins may also possibly lead to their own signaling pathway, as has been shown for other GPCR homooligomers (Gesty-Palmer et al., 2006; Ren et al., 2005; Shenoy et al., 2006). Thus, my results suggest that SKF 83822 could potentially be functionally selective as

an antagonist for the D1-D2 receptor heterooligomer since it attenuates the associated calcium

signal but may lead to a simultaneous arrestin mediated signal as a potential agonist of this pathway.

Overall, these results add to the complexity of GPCR heteromeric signaling by demonstrating the potential for unique conformations within a heterooligomer by selective agonists that can lead to altered signaling pathways, different from the constituent receptor homooligomers.

2. GRK2 regulates the D1-D2 receptor heteromer by distinct functions.

Calcium mobilization assays in heterologous cells and cultured striatal neurons demonstrated that GRK2 had a dual role in regulating the D1-D2 receptor heteromer mediated calcium signal.

The contribution of both catalytic and Gq binding functions of GRK2 in regulating D1-D2 receptor heterooligomer signaling not only showed the significance of different multi-function domains within GRK2, but also demonstrated unique D1-D2 receptor heterooligomer regulation in comparison to its constituent receptors, findings which support the potential for distinct regulation of other GPCR heterooligomers by GRK2 as well.

The attenuation of the D1-D2 receptor heteromer mediated calcium signal by GRK2 with multiple functions adds to the complexity of how this receptor complex is regulated in vivo. In

113 addition to providing a quick quenching mechanism, dual functioning by GRK2 could also enable different levels of attenuation of the calcium signal by potentially only using one functional domain as opposed to both; regulation by a single function could also lead to changes in the trafficking and responsiveness of the D1-D2 receptor complex. For example, a reduction in signaling caused solely by Gq binding functions without catalytic action suggests the endocytic machinery normally associated with phosphorylation, such as arrestins and clathrin, may not be involved, thus resulting in changes of the D1-D2 receptor heterooligomer internalization profile.

3. Agonist exposure to the D1-D2 receptor heteromer resulted in recruitment of GRK2 and direct interaction with GRK2.

Results from Immunocytochemistry, co-immunoprecipitation, and BRET assays suggested that pretreatment with selective agonists which occupied the D1 or both D1 and D2 receptor binding pockets regardless of whether they activated the D1-D2 receptor heteromer mediated calcium signal could lead to an increased GRK2 interaction with the D1-D2 receptor heteromeric complex. These findings suggest that agonist occupancy of receptors with the D1-D2 receptor heteroolgiomer is sufficient to induce desensitization through a GRK2 mediated mechanism.

4. The D1-D2 receptor heteromeric complex internalized after dopamine exposure and the associated calcium signal fully resensitized.

By performing calcium mobilization assays and immunocytochemistry, I have demonstrated that the D1-D2 receptor heteromer mediated calcium signal not only desensitized after exposure to

114 dopamine, but also that the receptor complex internalized followed by receptor recycling back to

the plasma membrane allowing for resensitization of the signal.

Given that the dopamine induced D2 receptor degradative fate is not conserved upon

heteromerization with the D1 receptor, where it appears to recycle rather than degrade, could

have implications with regards to dopaminergic signaling in vivo. Dopamine exposure would

presumably result in differential trafficking of the D2 receptor within the D1-D2 receptor

heteromer vs. the D2 receptor expressed alone leading to changes in dopamine transmission

throughout the brain. The potential long term consequences on dopamine receptor transmission

would depend on the extent of D1-D2 receptor heteromer activation vs. D2 receptor

homooligomer activation within discrete brain regions. For example, repeated dopamine

activation of this receptor complex in the nucleus accumbens, a brain region that demonstrated

the highest percentage of D1-D2 receptor heterooligomer expression in the adult rat (Perreault et

al., 2010), could possibly lead to a shift from D2 receptor homooligomer signaling to D1-D2

receptor heteromeric signaling over time, since the dopamine activated receptor heteromer would

seemingly recycle resulting in a rapidly restored signal, in constrast to D2 receptor

homooligomers that would presumably not recycle and therefore restoration of signaling would

take much longer.

5. The D1-D2 receptor complex internalized after SKF 83959 exposure, but the receptors did not recycle back to the cell surface.

In contrast to dopamine, it appears that SKF 83959 exposure does not lead to recycling of the

D1-D2 receptor heteromer or resensitization of the signal even after a prolonged recovery time.

These results suggest that exposure to this drug, which selectively activates the D1-D2 receptor

115 heteromer, in vivo would lead to changes in the balance of endogenous dopaminergic signaling

since D1 and D2 receptor homooligomers would presumably be unaffected by SKF 83959 but

trafficking and responsiveness of the D1-D2 receptor heteromer would be decreased, thus

leading to long term changes in dopamine transmission. Additionally, the ability for SKF 83959

exposure to possibly lead to decreased D1-D2 receptor heteromer signaling over time, suggests

that it could potentially be used as a pharmacological tool in order to discern how this D1-D2

receptor heteromer mediated signal contributes to neuronal function in vivo.

4.5 Significance and Future Studies

Since there is localization of the D1-D2 receptor heteromer throughout the basal ganglia

(Perreault et al., 2010), furthering the understanding of its activation and regulatory mechanisms will not only increase our knowledge of how this complex behaves but also may enhance our understanding of neuropsychiatric diseases in which this receptor heterooligomer may play role.

The functional implications of this unique signaling complex are only beginning to be understood, but already suggest a role for this D1-D2 receptor heterooligomer in neuropsychiatric disorders, such as schizophrenia. Schizophrenia has been linked to increased dopamine transmission and abnormal calcium signaling has been proposed to constitute the central unifying factor that is responsible for the psychopathology of this disorder (Lidow, 2003).

This D1-D2 receptor heteromer directly links dopamine receptors to calcium signaling providing a potential mechanism to bridge these two streams of evidence. Additionally, activation of the

D1-D2 receptor heteromer resulted in increased CaMKII activation as well as BDNF expression

(Hasbi et al., 2009; Rashid et al., 2007), two proteins that have been linked to schizophrenia

(Carlino et al., 2011; Jindal et al., 2010; Novak and Seeman, 2010; Weickert et al., 2003; Wong

116 et al., 2010). Furthermore, there was an increased proportion of the D1-D2 receptor heteromer in the agonist detected high affinity state that was observed either in rat striatum after chronic treatment with amphetamine or in human schizophrenia brain basal ganglia (Perreault et al.,

2010). The D1-D2 receptor heteromer has also been suggested to be involved in the pathology of depression, since coupling between D1 and D2 receptors was increased in postmortem brain of subjects suffering from depression and disruption of the D1-D2 receptor complex elicited antidepressant like effects in rats (Pei et al., 2010).

The potential role for this D1-D2 receptor complex in disorders such as schizophrenia suggest it would be of clinical relevance to eventually design drugs that specifically target D1-

D2 receptor heterooligomers in order to aid in the treatment of schizophrenia and/or other neuropsychiatric disorders. Determining precisely how D1-D2 receptor heteromer intracellular signaling is regulated will not only increase our understanding of the available repertoire of dopamine receptor signaling pathways but also enable investigation of how these signaling pathways may be disrupted in pathogenic states such as schizophrenia. However, further research is still required to understand the full complement of D1-D2 receptor heteromeric regulation. A summary of potential future studies that can be performed is provided below.

Future Studies

1. Desensitization mechanisms associated with the D1-D2 receptor heteromer mediated calcium signal.

My studies have demonstrated that agonists occupying the D1 receptor binding pocket regardless of whether they activated the D1-D2 receptor heteromer-mediated calcium signal resulted in significant desensitization of the signal and recruitment of GRK2. However, further studies to

117 elucidate mechanisms involved in regulating the D1-D2 receptor heteromeric complex and its associated calcium signal could be done.

A) Although the coimmunoprecipitation and BRET data suggest a direct agonist induced

interaction between the D1-D2 receptor heteromer and GRK2, other techniques, such as

bimolecular fluorescence complementation, could be performed to demonstrate a more definitive

interaction between GRK2 and both the D1 and D2 receptors. By using bimolecular fluorescence

complementation assays, the D1 and D2 receptor could each be tagged with a complementary

fragment of the donor molecule, R luciferase. Interaction of the D1 and D2 receptor would allow the two complementary fragments to be brought together allowing for a functional R luciferase donor molecule that could transfer light energy to an acceptor fluorophore tagged-GRK2 protein if it was interacting with the D1-D2 receptor heteromeric complex. Furthermore, an additional control experiment to complement the current coimmunoprecipitation data could be to immunoprecipitate the D2 receptor from HEK 293T cells only expressing the D2 receptor and then blot for the GRK2 protein after SKF 83959 treatment.The absence of a band for the GRK2 protein from these D2 receptor expressing cells would help prove that GRK2 is indeed interacting with D1-D2 receptor heterooligomers, rather than D2 receptor homooligomers, in the

HEK 293T cells stably expressing D1 and D2 receptors.

B) The GRK2 catalytic domain is responsible for its phosphorylation action on receptors. Given the partial involvement of this domain in regulating the D1-D2 receptor heteromer mediated calcium signal suggests that GRK2 phosphorylation of the D1-D2 receptor complex may be involved in the regulatory process. If GRK2 phosphorylates this receptor complex, the

118 phosphorylation sites on the D1 and D2 receptors expressed alone may not be conserved upon

D1-D2 receptor heteromer expression or only one receptor within the complex may get

phosphorylated. D1-D2 receptor heteromer phosphorylation by GRK2 could be investigated by

performing phosphorylation assays using HEK 293T cells stably expressing D1 and D2 receptors

and testing for radiolabelled phosphate incorporation on D1 and/or D2 receptors. Additionally,

receptor mutagenesis studies could be performed to elucidate the potential GRK2 phosphorylation sites on the D1 and/or D2 receptors.

C) My studies have demonstrated that both the catalytic and RGS domain of GRK2 are involved

in regulating the D1-D2 receptor heterooligomer. The Gβγ domain of GRK2 has also been reported to be involved in D2 receptor homooligomer regulation (Namkung et al., 2009b). Thus, the GRK2 Gβγ PH domain may also be involved in D1-D2 receptor heteromer regulation. To investigate if the PH domain of GRK2 is involved, the calcium signal could be tested after expressing either a GRK2 mutant lacking Gβγ binding ability or just the c-terminal PH domain of GRK2 in cells.

D) Although I focused my studies on GRK2, GRK3 is also widely distributed throughout the brain, but at a much lower expression level relative to GRK2 in most brain regions (Arriza et al.,

1992; Erdtmann-Vourliotis et al., 2001). Despite this lower expression level, it could be involved in regulating the D1-D2 receptor heteromer complex since it was documented to play a role in the regulation of the D1-D2 receptor complex in heterologous cells (So et al., 2007). This role for GRK3 in regulating the D1-D2 receptor heteromer could be further explored to investigate if it regulates the complex at a physiological level and if so, the significance of its domains could

119 be determined. Similar to GRK2, the RGS domain of GRK3 has been shown to bind to Gq proteins and sequester them, therefore GRK3 could also potentially regulate the D1-D2 receptor complex with multiple domains. The importance of GRK3 could be tested using similar strategies that demonstrated the involvement of GRK2, including testing of the calcium signal after expression either wildtype GRK3, GRK3 constructs with mutations in distinct functional domains, or siRNA to knockdown endogenous GRK3.

E) Since I have demonstrated that the RGS domain of GRK2 is involved in the desensitization of the calcium signal, it is possible that RGS proteins may also mediate this desensitization. RGS proteins regulate G protein signaling by limiting the signals generated by GPCRs. They dramatically increase the rate at which Gα subunits hydrolyze GTP to GDP, a property that defines them as GTPase activating proteins (Bansal et al., 2007). It has also been shown that some RGS proteins can diminish Gα mediated signaling by functionally inhibiting Gα effector coupling, a phenomenon known as “effector antagonism”, where the RGS proteins compete with effector molecules for binding to Gα subunits making them unavailable for further signaling

(Hepler et al., 1997; Heximer et al., 1997). For example, RGS2 and RGS3 have been shown to inhibit Gq mediated signaling by effector antagonism in the absence of GTPase acceleration

(Anger et al., 2004). Therefore, it is hypothesized that RGS proteins such as RGS2 or RGS3 may function to decrease the D1-D2 receptor heteromer mediated calcium signal by binding to Gq proteins and sequestering them so that they can no longer participate in signaling. In order to test this hypothesis, the calcium signal could be tested after either over expression of RGS proteins or reduced expression by using siRNA to knock down endogenous levels.

120 2. Internalization mechanisms associated with the D1-D2 receptor heteromer complex.

By performing radioligand binding assays and using confocal imaging, I have demonstrated that

the D1-D2 receptor complex internalizes after exposure to either dopamine or SKF 83959.

However, the mechanisms involved in this internalization are still unexplored.

A) Although I have demonstrated a role for GRK2 in D1-D2 receptor heteromer mediated

calcium signaling desensitization, the involvement of the endocytic machinery, such as arrestins,

could also be investigated. In addition to a role for arrestins in the desensitization and

internalization of the D1-D2 receptor complex after exposure to either SKF 83959 or SKF

83822, arrestin recruitment could potentially lead to its own signaling pathway, such as the

MAPK signaling cascade, even in the absence of G protein activation, as has been shown for

some other GPCR homoligomers (Gesty-Palmer et al., 2006; Ren et al., 2005; Shenoy et al.,

2006; Wei et al., 2003).

It has been demonstrated that the machinery associated with internalization of GPCR

heterooligomers can vary compared to when the receptor is expressed alone (AbdAlla et al.,

2000; Terrillon et al., 2004). Therefore, it is possible that the dynamin-dependent pathway

associated with the internalization of D1 receptor homooligomers may not be conserved upon

heterooligomer formation with the D2 receptor, since D2 receptor internalization, when observed

in certain cell lines, is mediated by dynamin-independent pathways (Vickery and von Zastrow,

1999). Additionally, different subtypes of both dynamin and arrestin proteins exist and therefore could potentially differ for the D1-D2 receptor heterooligomer in comparison to its constituent homooligomers.

121 A role for proteins such as arrestins and dynamin could be examined by either expressing

wildtype forms, functionally inert mutated forms to knockout wildtype functions dominant

negatively, different subtypes, or siRNA to knockdown endogenous levels of these proteins and then testing the effects on internalization of the D1-D2 receptor heterooligomer.

B) The D1-D2 receptor heteromer may also internalize through a caveolin-mediated process since this pathway was shown to be utilized by the D1 receptor expressed alone (Kong et al.,

2007; Kong et al., 2011). The D1 receptor localized to low density caveolin-enriched membrane domains and associated with caveolin-1 in rat brain. This caveolar based mechanism for internalization of the D1 receptor may or may not be conserved upon D1-D2 receptor heterooligomer expression or may also modulate D2 receptor internalization. To determine if the

D1-D2 receptor complex can internalize through this caveolar pathway, caveolin-enriched fractions could be purified from HEK293T cells co-expressing D1 and D2 receptors by performing sucrose density gradient centrifugation and then analyzing for D1 and D2 receptor distribution within these fractions. To test for an interaction between the D1-D2 receptor heterooligomer and caveolin-1, co-immunoprecipitation and BRET assays could be done with the heterologous cells co-expressing D1 and D2 receptors. Moreover, rat striatal neurons in culture could be treated with SKF 83959 to selectively activate the D1-D2 receptor heteromeric complex at a physiological level, followed by immunocytochemistry to test for internalization of the receptor complex and co-localization between the receptors and caveolin-1.

122 3. D1-D2 receptor heteromer trafficking after agonist induced internalization and resensitization of the calcium signal.

My studies have demonstrated that specific ligand exposure determined the resensitization response of the D1-D2 receptor heteromer mediated calcium signal as well as D1 and D2 receptor recycling, but further studies are still required to fully elucidate the mechanisms for these differential responses.

A) I have demonstrated that exposure to dopamine resulted in recycling of internalized D1 and

D2 receptors and resensitization of the D1-D2 receptor heteromer mediated calcium signal whereas exposure to SKF 83959 did not result in recycling of internalized D1 and D2 receptors or resensitization of the calcium signal. This difference could be mediated by a differential interaction with arrestins where the D1-D2 receptor heterooligomer either dissociates from arrestin or forms a stable complex with it after ligand exposure. This differential interaction with arrestins is a general phenomenon that has been shown for GPCR homooligomers designating them as either Class A, where the receptors internalize without arrestin bound and recycle or

Class B, where the receptors internalize with the arrestin protein bound and do not recycle

(Oakley et al., 1999; Oakley et al., 2001; Oakley et al., 2000). Thus, dopamine pre-treatment

may result in internalization of the D1-D2 receptor complex without the arrestin protein leading

to recycling of the receptors back to the plasma membrane. In contrast, SKF 83959 pre-treatment

may result in internalization of the D1-D2 receptor complex with the arrestin protein still bound

and therefore the receptors are either retained in endosomal compartments or targeted for

degradation.

123 To test the interaction with arrestin, its translocation with D1 and D2 receptors after pre-

treatment with either dopamine or SKF 83959 could be monitored by performing

immunocytofluorescence studies in which the fate of the arrestin protein after agonist wash off

and recovery periods could be observed. Additionally, confocal microscopy FRET analysis could

be performed to detect an interaction between the D1-D2 receptor heterooligomer and arrestin

after treatment with either dopamine or SKF83959. The FRET signal could be monitored

immediately after agonist removal as well as after different recovery time points.

B) Since I have demonstrated that D1 and D2 receptor recycling is different after exposure to

either dopamine or SKF 83959, it would be of interest to investigate the trafficking of the

receptors after agonist induced internalization, especially after SKF 83959 exposure since the

receptors did not recycle back to the plasma membrane after a prolonged recovery time. One group of proteins, known as Rab proteins, are a ras super family of GTPases that control a variety of important cellular processes, such as endocytosis, trafficking, endosome fusion and exocytosis (Seachrist and Ferguson, 2003). The Rab proteins have individual isoforms that localize to the surfaces of distinct membrane bound organelles and therefore can be used as markers for different endosomes. For example, the Rab 4 protein is mainly localized in early endosomes and Rab 11 is mainly localized in perinuclear recycling endosomes and the trans-

Golgi network. It is believed that two distinct intracellular systems regulate the recycling of internalized GPCRs, where one is the Rab 4 mediated rapid recycling pathway and the other is the Rab 11 mediated slow recycling pathway or trafficking to the trans-Golgi network (Pereira-

Leal and Seabra, 2001; Pfeffer, 2003; Zerial and McBride, 2001).

124 Dopamine or SKF 83959 pretreatment could potentially target D1 and D2 receptors to different endosomes resulting in different recycling pathways. To investigate this possibility, colocalization of D1 and D2 receptors with different rab proteins, such as Rab 4 and Rab 11, after either dopamine or SKF 83959 pretreatment could be visualized through fluorescence microscopy in HEK 293T cells stably expressing both the D1 and D2 receptors. Additionally, resensitization of the calcium signal could be evaluated after expressing constitutively active or dominant negative mutants of the Rab proteins.

4.6 Concluding Remarks

My studies demonstrated that the D1-D2 receptor heterooligomer is regulated with unique characteristics from its constituent receptors and displays regulatory and trafficking properties that depend on specific ligand exposure. Dopamine receptors play a critical role in physiological functions through the transmission of extra-cellular signals to the intracellular compartments.

Given that typically, multiple intracellular signaling pathways are activated at a given time, efficient orchestrating mechanisms are required for appropriate biological outcomes and therefore defining these mechanisms for this novel D1-D2 receptor heteromer is important for advancing our knowledge of dopamine receptor signaling and regulation in brain.

125

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