Graduate Theses, Dissertations, and Problem Reports 2003 Molecular mechanisms of G protein-receptor coupling Hongzheng Ma West Virginia University Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Ma, Hongzheng, "Molecular mechanisms of G protein-receptor coupling" (2003). Graduate Theses, Dissertations, and Problem Reports. 1878. https://researchrepository.wvu.edu/etd/1878 This Dissertation is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Dissertation in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you must obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Dissertation has been accepted for inclusion in WVU Graduate Theses, Dissertations, and Problem Reports collection by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. Molecular Mechanisms of G Protein-Receptor Coupling Hongzheng Ma Dissertation submitted to the School of Medicine at West Virginia University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmacology & Toxicology Approved by Stephen G. Graber, Ph.D. (Chair) Daniel C. Flynn, Ph.D. James E. Mahaney, Ph.D. Jeannine S. Strobl, Ph.D. William F. Wonderlin, Ph.D. Department of Biochemistry & Molecular Pharmacology School of Medicine West Virginia University Morgantown, West Virginia 2003 Keywords: G-protein, GPCR, Receptor coupling, Muscarinic, AGS3, Sf9 cells. UMI Number: 3132956 ________________________________________________________ UMI Microform 3132956 Copyright 2003 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ____________________________________________________________ ProQuest Information and Learning Company 300 North Zeeb Road PO Box 1346 Ann Arbor, MI 48106-1346 ABSTRACT Molecular Mechanisms of G Protein-Receptor Coupling Hongzheng Ma A variety of extracellular signals are transmitted into the cell interior by interactions with a superfamily of heptahelical cell surface receptors. Heterotrimeric G proteins mediate the signal transduction by coupling these receptors to intracellular effector proteins. The molecular mechanisms in G protein-receptor coupling processes are still not completely understood. In this project, two aspects of G protein-receptor coupling were examined: 1) the G protein-coupling properties of the five human muscarinic acetylcholine receptors (mAChRs); 2) a novel regulatory mechanism by a newly identified G protein signal regulator, Activator of G-protein Signaling 3 (AGS3). To study the regulation of G protein-receptor coupling, an Sf9 cell membrane-based in vitro reconstitution system was used, in which purified G protein heterotrimers were reconstituted with individually expressed membrane receptors and their coupling was assessed with radioligand binding assays. Functional G protein coupling was successfully established for M1, M2, M4 and M5 mAChRs in urea-extracted Sf9 cell membranes. Under the same conditions, M3 mAChRs failed to couple with purified G proteins, indicating they may have a unique G protein signaling mechanism. Within the odd- or even- numbered mAChR groups similar apparent affinities for G protein interactions were observed, however, the odd-numbered mAChRs exhibited higher affinity for G protein heterotrimers than did the even-numbered mAChRs. Differences were also observed among the individual receptor subtypes in their affinity states for the agonist, Oxotremorine-M. Studies on AGS3 revealed that cytosolic AGS3, but not membrane- associated AGS3, can interfere with receptor-Gi protein coupling. Cytosolic AGS3 can remove Giα subunits from the plasma membrane and sequester them in the cytosol. Each of the four AGS3 GPR (G protein regulatory) domains was able to interfere with receptor-Gi protein coupling; however, individual domains were less effective than the full-length GPR domain. None of the GPR domains distinguish among the three Giα subunits but they all interact more weakly with Goα subunits. These studies demonstrate that five mAChRs have distinct G protein- coupling behaviors in an identical membrane environment and that AGS3 may down-regulate G protein signaling by interfering with receptor coupling. These findings contribute to the understanding of the mechanism and regulation of G protein-receptor coupling. DEDICATIONS To my dear wife, Min Wang and my lovely daughter, Grace. iii ACKNOWLEDGEMENTS I would like to thank everyone who have helped and inspired me over these five years. Special thanks to my advisor, Dr. Stephen Graber, for his great guidance, unconditional support, encouragement and patience. He not only has been a scientific but also a life mentor to me. His enthusiasm and optimism will always be a limitless source of inspiration for my life. I can not use words or any language to express my deep appreciation and respect for all he has done for me. Without him, I can not be myself today. I also wish to thank my dissertation committee members, Dr. Flynn, Dr. Mahaney, Dr. Strobl and Dr. Wonderlin, for their great guidance and critical remarks, which helped me to become a better scientific researcher. I would like to thank Dr. Stitzel, Dr. Reasor and Dr. van Dyke for all the help and great advice that they gave to me. Over the past five years, I was fortunate to work in a very supportive, caring and friendly group, which I wish to thank my colleagues, Janna, Lori and Karyn for creating such a wonderful working environment. I also wish to thank my English tutors, Jesse, Ann and Claudia, who gave me invaluable help in my English language skills and life experiences as well. Of course, my deepest thanks to my family for their love, support and understanding. iv TABLE OF CONTENTS ABSTRACT...……….……………………………………………………………………ii DEDICATION……………………………………………………………………………iii ACKNOWLEDGEMENTS..……………………………………………………………..iv TABLE OF CONTENTS..……………………………………………………...…………v LIST OF FIGURES ……………………………………………………………………...vi LIST OF TABLES…………………………………...…………………………………..vii LIST OF ABBREVIATIONS ………...……………………………………………….viii CHAPTER 1: INTRODUCTION ………………………………………………………...1 CHAPTER 2: METHODS.……………………………………………………………... 47 CHAPTER 3: RESULTS………………………………………………………………...69 Section 1: Human muscarinic acetylcholine receptors exhibit distinct G protein coupling behaviors in membranes from Sf9 cells………….…69 Section 2: Influence of Cytosolic AGS3 on Receptor-G Protein Coupling ……...107 CHAPTER 4: DISCUSSIONS.………...………………………...……………...……...146 BIBLIOGRAPHY………….…………………………………………………………...166 APPENDIX I: CONSTRUCTION OF CHIMERIC G PROTEIN α SUBUNITS……186 APPENDIX II: CLOSELY RELATED G PROTEIN COUPLED RECEPTORS USE MULTIPLE AND DISTINCT DOMAINS ON G PROTEIN α SUBUITS FOR SELECTIVE COUPLING………………………...200 APPENDIX III: STABILIZATION OF THE GDP-BOUND CONFORMATION OF Giα BY A PEPTIDE DERIVED FROM THE G-PROTEIN REGULATORY MOTIF OF AGS3………………………………….237 CURRICULUM VITAE …………………………………………………………….…263 APPROVAL OF THE EXAMINING COMMITTEE……………………………….…265 v LIST OF FIGURES Chapter 1&2. Figure 1. Schematic representation of Gα secondary structural domains……………5 Figure 2. The GTPase cycle of heterotrimeric G proteins ……………………………7 Figure 3: Schematic presentation of the general structure of GPCRs and terminology ………………………………………………………..….13 Figure 4. Examples of multiple GPCR/G-protein coupling ………………………...17 Figure 5. Representative experiments of characterization of receptor expression level with radioligand saturation binding assay …………………………...55 Figure 6. Effects of urea-extraction treatment on M5 mAChR-G protein coupling...56 Figure 7. Representative experiment of G protein concentration titration with radiolabeled agonist binding assay ……………………………………….62 Figure 8. Quantitation of AGS3 expressed in Sf9 cell membranes …………………64 Figure 9. Concentration- and Gβγ-dependent membrane association of Giα subunit ………………………………………………………………..65 Chapter 3. Section 1 Figure 1. Determination of high-affinity agonist binding on mAChR-containing Sf9 cell membranes stabilized by purified G proteins in reconstitution...104 Figure 2. G protein concentration dependence of high-affinity agonist binding on reconstituted mAChR membranes ..…………………………………….105 Figure 3. Inhibition of [3H]NMS binding on the reconstituted Sf9 cell Membranes containing individual mAChRs by Oxo-M………………………………106 Section 2. Figure 1. Effect of membrane expressed AGS3 on receptor-G protein coupling and association of Gi1α with the membrane……………….....140 Figure 2. Effect of cytosolic AGS3 on receptor-G protein coupling and association of Gi1 with the membrane…………………...………..141 Figure 3. Association of cytosolic AGS3 with membranes and its effect on receptor-G protein coupling…………….…………………………...142 Figure 4. Effect of cytosolic AGS3 on membrane-associated Gi1……………….143 Figure 5. Effect of cytosolic AGS3 on subcellular distribution of Gi/oα from native rat brain membranes……………………………………….144 Figure 6. Effect of GST-AGS3-GPR domains on receptor-G protein coupling….145 vi LIST OF TABLES Chapter 3. Table 1: Saturation binding parameters of five human mAChRs expressed in Sf9 cell membranes with or without urea extraction……………...…...100