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Comprehensive Model of G Protein-Coupled Receptor Regulation by Protein Kinase C: Insight from Dopamine D1 and D5 Receptor Studies

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Comprehensive Model of G Protein-Coupled Receptor Regulation by Protein Kinase C: Insight from Dopamine D1 and D5 Receptor Studies Comprehensive Model of G Protein-coupled Receptor Regulation by Protein Kinase C: Insight from Dopamine D1 and D5 Receptor Studies. By Bianca Plouffe This thesis is submitted to the Faculty of Graduate and Postdoctoral Studies as a partial fulfillment of the Ph.D. program in Neuroscience July 2011 Department of Cellular and Molecular Medicine Faculty of Medicine University of Ottawa © Bianca Plouffe, Ottawa, Canada, 2012 Abstract Dopamine receptors belong to the G protein-coupled receptor (GPCR) superfamily and are classified into two families: D1-like (D1R and D5R) and D2-like (D2R, D3R and D4R), based on their ability to stimulate or inhibit adenylyl cyclase (AC). Classically, GPCRs (including D2R and D3R) are desensitized by the activation of the serine/threonine protein kinase C (PKC) upon phorbol-12-myristate-13-acetate (PMA) treatment. Previous studies demonstrate that while human D5R (hD5R) is also strongly desensitized upon PMA treatment, the human D1R (hD1R) undergo a robust PMA- induced sensitization. The aim of this PhD thesis was to explore how the canonical PKC- or phorbol ester-linked pathway can control the responsiveness of two similar GPCRs like hD1R and hD5R in an opposite fashion. Our data indicate that hD1R sensitization and hD5R desensitization are not mediated by a direct modulation of AC activity by PKC. Using a chimeric approach, we identified the third intracellular loop (IL3) as the key structural determinant controlling in an opposite manner the PMA-mediated regulation of hD1R and hD5R. To delineate the potential PKC phosphorylation sites, a series of mutation of serine (Ser) and threonine (Thr) located into IL3 of hD1R and hD5R were used. No hD1R mutation decreased the PMA-mediated sensitization. This suggests that hD1R phosphorylation is not required for PMA-induced sensitization. In contrast, our results indicate that PMA-mediated hD5R desensitization occurs through a hierarchical phosphorylation of Ser260, Ser261, Ser271 and Ser274. Notably, these hD5R mutants exhibited a PMA-induced sensitization, reminiscent of the PMA-induced hD1R sensitization. Additionally, using short hairpin RNAs (shRNAs), we showed that PKCε is ii the potentiating PKC while the desensitizing isoform is δ. Overall, our work suggests the presence or absence of specific Ser residues on IL3 of hD1-like receptors dictate if phosphorylation-dependent desensitization (through PKCδ) or phosphorylation- independent potentiation (via PKCε) will occur. iii Table of Contents Abstract……………………………………………………………………………………ii Table of Contents…………………………………………………………………………iv List of Tables……………………………………………………………………………..ix List of Figures…………………………………………………………………………....xii List of Abbreviations……………………………………………………………………xvi Acknowledgements…………………………………………………………………...…xxi Thesis Format…………………………………………………………………………..xxii CHAPTER 1: GENERAL INTRODUCTION………………………………………....1 1. G protein-coupled receptors……………………………………………………….2 1.1. GPCR classification…………………………………………………………..4 1.2. Heterotrimeric G proteins…………………………………………………….5 1.3. GPCR activation………………………………………………………………6 1.3.1. Heterotrimeric G proteins cycle…………………………………….....6 1.3.2. Extended ternary complex model………………………………………8 1.3.3. Main GPCR-mediated pathways……………………………………….9 2. Regulation of GPCR responsiveness…………………………………………….11 2.1. Homologous regulation……………………………………………………...12 2.2. Heterologous regulation……………………………………………………..14 3. Protein kinase C (PKC)…………………………………………………………..16 3.1. PKC classification…………………………………………………………...16 3.2. PKC maturation……………………………………………………………...19 3.3. PKC activation………………………………………………………………20 iv 3.4. PKC inhibition………………………………………………………………23 3.5. Non PKC phorbol ester/DAG receptors……………………………………..25 3.6. PKC knockout mice and human diseases……………………………………27 3.6.1. Conventional PKC isoforms…………………………………………27 3.6.2. Novel PKC isoforms…………………………………………………29 4. Dopaminergic system…………………………………………………………….32 4.1. Dopaminergic synapse………………………………………………………32 4.2. Dopaminergic neuronal pathways…………………………………………...34 4.3. Dopaminergic receptor classification………………………………………..37 4.3.1. D1-like receptors……………………………………………………..37 4.3.2. D2-like receptors……………………………………………………..38 4.4. Dopaminergic receptor signaling……………………………………………40 4.4.1. D1-like receptor signaling pathways………………………………...40 4.4.1.1. Modulation of ion channels by D1-like receptors……………44 4.4.1.2. Modulation of ligand-gated ion channels by D1-like receptors……………………………………………..44 4.4.2. D2-like receptor signaling pathways………………………………...46 4.4.2.1. Modulation of ion channels by D2-like receptors……………48 4.4.2.2. Modulation of ligand-gated ion channels by D2-like receptors……………………………………………..49 4.5. D1-like receptor knockout mice……………………………………………..50 5. Regulation of dopaminergic receptors by PKC………………………………….53 5.1. Regulation of D2-like receptor activity by PKC…………………………….53 v 5.2. Evidence of D1R potentiation by PKC in striatum………………………….56 6. Rationale, hypotheses and objectives……………………………………………58 CHAPTER 2: MANUSCRIPT 1: The third intracellular loop of D1 and D5 dopaminergic receptors dictates their subtype-specific PKC-induced sensitization and desensitization in a receptor conformation-dependent manner………………...62 Statement of author contributions………………………………………………………..64 Abstract…………………………………………………………………………………..65 Introduction………………………………………………………………………………67 Materials and methods…………………………………………………………………...70 Results and discussion…………………………………………………………………...74 Conclusions………………………………………………………………………………98 Research highlights of chapter 2 – manuscript 1……………………………………..….99 CHAPTER 3: MANUSCRIPT 2: Serine and Threonine Clusters in the Third Intracellular Loop of Dopamine D1-like Receptors Modulate Agonist Binding and Activation Properties………………………………………………………………….100 Statement of author contributions………………………………………………………102 Abstract…………………………………………………………………………………103 Introduction……………………………………………………………………………..105 Material and Methods…………………………………………………………………..110 Results…………………………………………………………………………………..122 Discussion………………………………………………………………………………146 Research highlights of chapter 3 – manuscript 2…………………………………….....152 vi CHAPTER 4: MANUSCRIPT 3: Molecular mechanisms underlying the opposite PKC-induced regulation of dopamine D1 and D5 receptor coupling to cAMP pathway ………………………………………………………………………………..153 Statement of author contributions………………………………………………………155 Summary………………………………………………………………………………..156 Introduction……………………………………………………………………………..158 Experimental procedures……………………………………………………………….162 Results………………………………………………………………………………..…170 Discussion……………………………………………………………………………....201 Conclusions……………………………………………………………………………..206 Research highlights of chapter 4 – manuscript 3……………………………….………207 CHAPTER 5: MANSCRIPT 4: Role of novel PKC isoforms in dopamine D1 and D5 receptor signaling ……………………………………………………………………..208 Statement of author contributions………………………………………………………210 Summary………………………………………………………………………………..211 Introduction……………………………………………………………………………..213 Experimental procedures……………………………………………………………….217 Results and discussion………………………………………………………………….223 Conclusions……………………………………………………………………………..245 Research highlights of chapter 5 – manuscript 4……………………………………….246 CHAPTER 6: GENERAL DISCUSSION…………………………………………...247 1. General working model of PKC-mediated regulation of D1-like receptors……248 2. PMA-mediated potentiation of hD1R through PKC activation………….……..251 vii 2.1. Possible players………………………………………………………...252 2.1.1. GRK……………………………………………………….....252 2.1.2. RGS…..………………………………………………………253 2.1.3. AGS….….….….….….….….….….….….….….….….….…254 2.2. Possible mechanism……………………………………………..….….255 3. Physiological relevance of PKC-mediated regulation of D1R and D5R…….....256 3.1. D1R in striatum for motor control…………………………………......256 3.2. D1R and D5R in subthalamic nucleus for awareness modulation……..259 3.3. D1R and D5R in hippocampus for learning and memory……………..263 3.4. D1R evolution and emergence of consciousness?.…………………….266 4. Conclusions and future studies………………………………………………....267 CHAPTER 7: REFERENCES………………………………………………………..271 viii List of Tables CHAPTER 2 Table 1. Best-fitted parameters for dose-response curves of DA in HEK293 cells transfected with hD1R, hD1-CTD5, hD5R and hD5-CTD1 in the absence or presence of PMA.…………………………………………………………….....81 Table 2. Best-fitted parameters for dose-response curves of DA in HEK293 cells transfected with hD1R, hD1-TRD5, hD5R and hD5-TRD1 in the absence or presence of PMA.………………………………………………………...……..85 Table 3. Best-fitted parameters for dose-response curves of DA in HEK293 cells transfected with hD1R, hD1-IL3D5, hD5R and hD5-IL3D1 in the absence and presence of PMA.………………………………………………………..……...88 Table 4. Best-fitted parameters for dose-response curves of DA in HEK293 cells transfected with hD1R, hD1-IL3TRD5, hD5R and hD5-IL3TRD1 in the absence and presence of PMA……………………………………………………..…….93 CHAPTER 3 Table S1. Sequences of oligonucleotide primers for the construction of hD1R single-point mutations.……………………………………………………………..……..117 Table S2. Sequences of oligonucleotide primers for the construction of hD5R single-point mutations.………………………………………………………………..…..119 Table 1. Equilibrium dissociation constants (Kd) and maximal binding capacity (Bmax) 3 values of [ H]-SCH23390 for human wild type and mutant D1-like receptors.125 Table 2. Equilibrium dissociation constant values (Ki) of unlabeled dopaminergic ligands for human wild type and mutant D1-like receptors ………………………......129 ix Table 3. Best-fitted EC50 and Emax values of dopamine in cells expressing human
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