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Modulation of G Protein-Coupled Receptor Intracellular Trafficking and Signal Transduction

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Western University Scholarship@Western Electronic Thesis and Dissertation Repository 4-25-2012 12:00 AM Modulation of G Protein-Coupled Receptor Intracellular Trafficking and Signal Transduction Jessica L. Esseltine The University of Western Ontario Supervisor Stephen SG Ferguson The University of Western Ontario Graduate Program in Physiology A thesis submitted in partial fulfillment of the equirr ements for the degree in Doctor of Philosophy © Jessica L. Esseltine 2012 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Cell Biology Commons Recommended Citation Esseltine, Jessica L., "Modulation of G Protein-Coupled Receptor Intracellular Trafficking and Signal Transduction" (2012). Electronic Thesis and Dissertation Repository. 473. https://ir.lib.uwo.ca/etd/473 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. MODULATION OF G PROTEIN-COUPLED RECEPTOR INTRACELLULAR TRAFFICKING AND SIGNAL TRANSDUCTION (Spine Title: GPCR Intracellular Trafficking and Signal Transduction) (Thesis format: integrated article) by Jessica Lynn Esseltine Graduate Program in Physiology Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy School of Graduate and Postdoctoral Studies The University of Western Ontario London, Ontario, Canada April, 2012 © Jessica Lynn Esseltine 2012 Abstract The seven transmembrane-spanning G Protein-coupled Receptor (GPCR) super family is the largest family of cell-surface receptors, comprising greater than 650 members. GPCRs represent the primary targets of most therapeutic drugs. Desensitization, endocytosis and recycling are major mechanisms of receptor regulation and intracellular trafficking of GPCRs is linked to the Rab family of small G proteins. In the present study, we examined whether multiple Rab GTPase regulate receptor trafficking through endosomal cellular compartments as a consequence of their direct association with GPCRs. We find that Rab4, Rab7 and Rab11 all bind to the last 10 amino acid residues of the angiotensin II Type 1 (AT1R) carboxyl-terminal tail. We show that the Rab GTPases compete with one another for receptor binding and that Rab4 effectively displaces Rab11 from the receptor. In contrast, Rab11 overexpression does not prevent Rab4 binding to the AT1R. Overexpression of wild-type Rab4, but not Rab11, facilitates AT1R dephosphorylation, and a constitutively active Rab4- Q67L mutant reduces AT1R desensitization and promotes AT1R resensitization. We also find that Rab8, a RabGTPase involved in the regulation of secretory/recycling vesicles, modulation of the actin cytoskeleton and cell polarity, interacts with the carboxyl-terminal tail of metabotropic glutamate receptor 1 (mGluR1a) and attenuates mGluR1a signalling and endocytosis in a protein kinase C-dependent manner. Finally, we have examined several previously uncharacterised but naturally occurring mutaions in mGluR1a that have been associated with cancer that may alter mGluR1a signalling. We find that mutations found within the ligand binding domain of mGluR1a result in both decreased cell surface expression and basal inositol 1,4,5, trisphosphate formation and bias mGluR1a signalling via the ERK1/2 pathway. Additional mGluR1a mutations localized to the mGluR1a glutamate iii binding site, intracellular regulatory domains and Homer binding site also result in changes in mGluR1a subcellular localization, signalling and cell morphology. Taken together, these results indicate that GPCR signalling is significantly modulated by the association of intracellular regulatory proteins that can be influenced by receptor structure. iv Key Words G Protein-Coupled Receptors Angiotensin II Type 1 Receptor Metabotropic Glutamate Receptor Heterotrimeric G Protein Desensitization Receptor Signalling Phosphorylation Endocytosis Resensitization Plasma Membrane Recycling Intracellular Membrane Compartments Receptor Trafficking Rab GTPase Small G Proteins Receptor Carboxyl-Terminal Tail Domains Human Embryonic Kidney 293 Cells Hippocampal Neurons v Co-Authorship Data presented in Chapter 2 are published elsewhere. Data presented in Chapters 3 and 4 have been submitted for publication. Ms. Lianne Dale and Dr. Ana Cristina Magalhaes assisted me in creating the Rab4 mutant cDNA constructs used in chapter 2. Mutant AT1R constructs used in chapter 2 were created by me. All mGluR1 mutant cDNA constructs used in chapter 4 were created by the Oncology group at Eli Lilly and Company and provided to me through collaboration with Dr. Melinda Willard and Dr. Thomas Barber. All other constructs were previously generated by the Ferguson Lab. I performed all experiments in Chapter 2 with the assistance of Ms. Lianne Dale in both the experimental design and troubleshooting. In Chapter 3 Dr. Fabiola M. Ribeiro contributed Figure 3.4A and 3.5A. Drs. Thomas Barber and Melinda D. Willard of Eli Lilly and Company contributed Figure 4.7 and 4.8B in Chapter 4. vi Dedication For William, of course! vii Acknowledgements Steve, thank you for your support, financially and otherwise during my degree. Thank you for the four years I spent as a teaching assistant for phys 3140 and it meant a great deal to me to give your last lecture this year. Thank you for allowing me to travel during my degree, especially for Australia. You provided an excellent atmosphere to foster independent thinking and research, and I owe much of my scientific competence to that atmosphere. I appreciate you recommending me for your collaboration with Eli Lilly and Company—I have gained valuable connections and resources because of that. Lianne, how can I convey the impact you have had these past five years! Thank you for answering my gazillion questions, for patiently walking me through troubleshooting, endless scientific talks, analyzing and interpreting data, teaching me how to think critically and objectively. Mostly, thank you for being my friend, for laughing with me, and listening. Thank you for sticking up for me and for your unconditional support and advice, especially through the rough times. Ana, thank you for your advice and guidance. Thank you for keeping me safe in Brazil and wow-thank you for taking me on a family vacation with you! I also appreciate your unconditional love and support as well. You are a brilliant and dedicated scientist and I know you will excel at whatever you do. Steve and Jane, thank you for writing endless letters of reference for me. Because of the great things you said in them, I was very successful in securing scholarships. And Jane, thank you also for allowing me to give two of your lectures - I really enjoy teaching and found them to be a treat. Thank you to all the other members of my lab, for teaching me new things every day. viii TABLE OF CONTENTS ABSTRACT ..................................................................................................................... III KEY WORDS .................................................................................................................... V CO-AUTHORSHIP ........................................................................................................ VI ACKNOWLEDGEMENTS ........................................................................................ VIII LIST OF FIGURES ....................................................................................................... XV LIST OF ABBREVIATIONS .................................................................................... XVII CHAPTER 1 ....................................................................................................................... 1 INTRODUCTION.............................................................................................................. 1 1.1. THE G PROTEIN-COUPLED RECEPTOR SUPERFAMILY ........................ 2 1.2. RECEPTOR SIGNALLING .............................................................................. 2 1.2.1. G Protein-Dependent Signal Transduction Paradigm ..................... 2 1.2.2. G Protein-Independent and Non-Classical Signal Transduction ..... 4 1.2.3. Agonists and Functional Agonism .................................................. 5 1.2.4. Receptor Desensitization ................................................................. 6 1.2.4.1. Receptor Phosphorylation ............................................................... 7 1.2.2.4. Second-Messenger-Dependent Protein Kinase ............................... 9 1.2.4.2. G Protein-Coupled Receptor Kinases .............................................. 9 1.2.4.3. Arrestin .......................................................................................... 10 1.2.4.4. Phosphorylation-Independent Desensitization .............................. 12 1.2.4.5. GPCR Down-Regulation ............................................................... 12 1.2.4.6. GPCR Proteolytic Degradation ..................................................... 13 1.2.4.7. Transcriptional Downregulation .................................................... 14 1.3. GPCR TRAFFICKING .................................................................................... 14 1.3.1. ER/Golgi to Plasma membrane ..................................................... 14 1.3.2. Receptor Internalization ...............................................................
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