Novel Regulatory Mechanisms of D1 Dopamine Receptor Maturation and Internalization by Michael Ming Chuen Kong A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Pharmacology University of Toronto © Copyright by Michael Ming Chuen Kong (2008) Novel Regulatory Mechanisms of D1 Dopamine Receptor Maturation and Internalization Michael Ming Chuen Kong Degree of Doctor of Philosophy, 2008 Department of Pharmacology University of Toronto ABSTRACT Dopamine is the most abundant catecholamine neurotransmitter in the mammalian brain and controls various physiological processes. The D1 dopamine receptor (D1DR) is the predominant dopamine receptor in the brain and traditionally couples to stimulatory G proteins, such as Gs, to activate adenylyl cyclase and generate cAMP. Although the trafficking itinerary of ER/Golgi maturation, agonist-induced internalization, and recycling/degradation are features common to many G protein-coupled receptors (GPCRs), the molecular regulation of these individual processes for the D1DR is not fully elucidated. Many GPCRs have been shown to form homo-oligomers; the work presented in this thesis explores how multimerization of D1DR has a role in regulating how these receptors are trafficked to the plasma membrane. In addition, the regulation of D1DR internalization is investigated in the context of emerging evidence highlighting the importance of lipid rafts. Using strategically designed point mutations of the D1DR, specific receptor mutants were found to intracellularly sequester the wild-type receptor by oligomerization. This level of scrutiny by the quality control machinery in the cell could be circumvented by treatment with cell permeable dopaminergic agonists, but not antagonists or inverse agonists. This finding suggests that specific conformational requirements must be achieved before full ii maturation and anterograde trafficking of the D1DR can proceed. Furthermore, it was determined that cell surface bound D1DRs could internalize through a novel clathrin independent pathway that required binding to the scaffolding protein, caveolin-1. This interaction with caveolin-1 was identified in whole rat brain and was found to require a putative caveolin binding motif in transmembrane domain 7. Palmitoylation of D1DR was found to regulate the rate of agonist-induced caveolae mediated internalization. Finally, we determined that the integrity of caveolae was important in regulating cAMP signaling through D1DR. These findings provide novel insight into the trafficking requirements of newly synthesized D1DRs as well as alternative mechanisms of regulation of receptors after agonist activation. The oligomerization of GPCRs and the localization of GPCRs in lipid rafts represent two emerging concepts important to many aspects of GPCR function. Future work aimed at integrating these overlapping processes will further our understanding of this important group of cell surface receptors. iii ACKNOWLEDGEMENTS The compilation of work embodied in this thesis could not have been possible without the help of many individuals, all of whom I am indebted to. I’d first like to thank members of the examination committee: Dr. Stephane Angers, Dr. Peter Chidiac, and Dr. Denis Grant, for a very stimulating discussion and for providing unique perspectives on this work. I’d also like to thank Dr. Reinhart Reithmeier and Dr. Bernard Schimmer for all of their guidance and advice throughout my doctoral studies. I would like to thank all members of the lab for their outstanding expertise and for providing a congenial environment to work and learn in. In particular, I’d like to thank Theresa Fan, Ahmed Hasbi, Tuan Nguyen, and George Varghese for their technical skills and for sometimes saving me from the agony of troubleshooting. I’d also like to send a special thanks to Kevin Curley, Samuel Lee, Dennis Lee, Jason Juhasz, Jennifer Ng, Ryan Rajaram, Asim Rashid, Christopher So, and Vaneeta Verma for their banter, advice, and most importantly, their friendship, all of which has made graduate school go by so quickly. To my supervisors, Dr. Susan George and Dr. Brian O’Dowd, I am grateful for their outstanding mentorship and for giving me the independence to develop as a scientist. Your confidence in my abilities is highly valued and very much appreciated. To my parents, Robert and Vivian Kong, who have been so supportive and patient throughout every aspect of my life and for providing an endless source of encouragement and love. This work is dedicated to you. And to my wife and best friend, Siu Lan Lee, for not only her patience and understanding through this endeavour but for always having an open ear and an open heart. iv PREVIOUSLY COPYRIGHTED MATERIAL Some parts of this thesis have been reproduced in whole or in part from the following sources with permission: Gouldson PR, Higgs C, Smith RE, Dean MK, Gkoutos GV, Reynolds CA 2000. Dimerization and domain swapping in G protein-coupled receptors: a computational study. Neuropsychopharmacology, Oct;23(4 Suppl):S60-77. Copyright © by Macmillan Publishers. Bouvier M 2001. Oligomerization of G protein-coupled transmitter receptors. Nature Reviews Neuroscience, Apr; 2(4):274-86. Copyright © by Macmillan Publishers. Pierce KL, Premont RT, Lefkowitz RJ. 2002. Seven-transmembrane receptors. Nature Reviews Molecular Cell Biology, Sept:3(9):639-50. Copyright © by Macmillan Publishers. Fotiadis D, Liang Y, Filipek S, Saperstein DA, Engel A, Palczewski K. 2004. The G protein-coupled receptor rhodopsin in the native membrane. FEBS Letters, Apr 30; 564(3):281-8. Copyright © by Elsevier Limited. Kong MM, So CH, O’Dowd BF, and George SR. The Role of Oligomerization in G Protein-Coupled Receptor Maturation, The G Protein-Coupled Receptors Handbook; Humana Press, 2005. Copyright © by Springer Science and Business Media. Kong MM, O’Dowd BF, and George SR. The Oligomerization of G Protein-Coupled Receptors, The Cell Biology of Addiction; Cold Spring Harbor Press, 2005. Copyright © by Cold Spring Harbor Laboratory Press So CH, Varghese G, Curley KJ, Kong MM, Alijaniaram M, Ji X, Nguyen T, O'Dowd BF, and George SR. 2005. D1 and D2 dopamine receptors form heterooligomers and cointernalize after selective activation of either receptor. Molecular Pharmacology, Sept; 68(3): 568-78. Copyright © 2005 by the American Society for Pharmacology and Experimental Therapeutics. O'Dowd BF, Ji X, Alijaniaram M, Rajaram RD, Kong MM, Rashid A, Nguyen T, and George SR. 2005. Dopamine receptor oligomerization visualized in living cells. Journal of Biological Chemistry, Nov 4; 280 (44): 37225-35. Copyright © 2005 by the American Society for Biochemistry and Molecular Biology. Kong MM, Fan T, Varghese G, O’Dowd BF, and George SR. 2006. Agonist-induced cell surface trafficking of an intracellularly sequestered D1 dopamine receptor homo-oligomer. Molecular Pharmacology, Jul; 70(1): 78-89. Copyright © 2005 by the American Society for Pharmacology and Experimental Therapeutics. Rashid AJ, So CH, Kong MM, Furtak T, Cheng R, O’Dowd BF,and George SR. 2006. A D1-D2 dopamine receptor heterooligomer with unique pharmacology is coupled to rapid activation of Gq/11 in the striatum. Proceedings of the National Academy of Sciences, Jan 9; 104(2):654-9. Copyright © 2007 by the National Academy of Sciences v Parton RG and Simons K. 2007. The multiple faces of caveolae. Nature Reviews Molecular Cell Biology, Mar:8(3):185-94. Copyright © by Macmillan Publishers. Kong MM, Hasbi A, Mattocks M, Fan T, O’Dowd BF, George SR. Regulation of D1 Dopamine Receptor Trafficking and Signaling by Caveolin-1. Molecular Pharmacology in press. Copyright © 2007 by the American Society for Pharmacology and Experimental Therapeutics vi TABLE OF CONTENTS ABSTRACT ii ACKNOWLEDGEMENTS iv PREVIOUSLY COPYRIGHTED MATERIAL v LIST OF REFEREED AND NON-REFEREED PUBLICATIONS 1 LIST OF ABSTRACTS 2 ABBREVIATIONS 4 LIST OF FIGURES 8 LIST OF TABLES 11 1 GENERAL INTRODUCTION 14 1.1 G Protein-Coupled Receptor Overview 14 1.2 Traditional Classification of GPCRs 16 1.3 G protein and Effector Signaling 19 1.4 Biosynthesis of GPCRs 20 1.4.1 Molecular Chaperones and Anterograde Trafficking 21 1.5 Receptor Activation and Desensitization 22 1.6 Internalization of GPCRs 25 1.6.1 Post Endocytic Sorting 26 1.7 Palmitoylation of GPCRs 27 1.8 Dopamine Receptor Subfamily 29 1.8.1 D1 Dopamine Receptor Structure 30 1.8.2 Pharmacology of the D1 Dopamine Receptor 32 1.8.3 Signal Transduction of the D1 Dopamine Receptor 33 1.9 Dopamine Neurotransmission in the Brain 35 1.10 Anatomical Distribution of the D1 Dopamine Receptor 36 1.10.1 Physiological Role of D1 Dopamine Receptors 37 1.11 Introduction to G Protein-Coupled Receptor Oligomerization 39 1.12 Evidence for G Protein-Coupled Receptor Oligomerization 40 1.12.1 Receptor Complementation 40 1.12.2 Co-immunoprecipitation 41 1.12.3 Novel Pharmacology 44 1.12.4 Receptor Trafficking Studies 45 1.12.5 Resonance Energy Transfer 46 1.12.6 Atomic Force Microscopy 49 1.12.7 X-ray Crystallography 50 1.13 Structural Features of G Protein-Coupled Receptor Oligomers 50 1.13.1 Disulphide Bonds 51 1.13.2 Transmembrane Interactions 52 vii 1.13.3 Intracellular and Extracellular Domain Interactions 53 1.14 Mechanisms of Agonist-Induced Activation of G Protein-Coupled Receptor Oligomers 54 1.15 G Protein-Coupled Receptor Oligomers In Native Brain Tissue 56 1.16 Introduction to Lipid Rafts 58 1.17 Platforms for Signaling Complex Assembly 59 1.17.1 Nitric Oxide Synthase Signaling Pathway 61 1.17.2