Proteins in Receptor-Mediated Cell Signaling Melinda Dale Willard A
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Multiple Domain ‘Nexus’ Proteins in Receptor-Mediated Cell Signaling Melinda Dale Willard A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology. Chapel Hill 2006 Approved by: Advisor: Professor David P. Siderovski Reader: Professor T. Kendall Harden Reader: Professor Gary L. Johnson Reader: Professor Michael D. Schaller Reader: Professor John E. Sondek ABSTRACT Melinda Dale Willard Multiple Domain ‘Nexus’ Proteins in Receptor-Mediated Cell Signaling (Under the direction of Dr. David P. Siderovski) Signal transduction is the fundamental biological process of converting changes in extracellular information into changes in intracellular functions. It controls a wide range of cellular activities, from the release of neurotransmitters and hormones, to integrated cellular decisions of proliferation, differentiation, survival, or death. The vast majority of extracellular signaling molecules exert their cellular effects through activation of G protein-coupled receptors (GPCRs); however, the G- protein coupled paradigm is by no means the exclusive mechanism of membrane receptor signal transduction. Polypeptide ligands such as nerve growth factor act exclusively on receptor tyrosine kinase receptors (RTKs) to promote signaling. GPCRs and RTKs both form an interface between extracellular and intracellular physiology by converting hormonal signals into changes in intracellular metabolism and ultimately cell phenotype. Initially, it was thought that GPCRs and RTKs represented linear and distinct signaling pathways that converge on downstream targets to regulate cell division and gene transcription. However, activation of second messenger generating systems do not fully explain the range of effects of GPCR or RTK activation on biological processes such as differentiation and cell growth. Recent work has revealed that GPCR and RTK signaling pathways are not ii mutually exclusive of one another; in fact, they often function as partners, forming complex signaling networks through scaffold/nexus proteins. The work described herein examines the complexity of signal regulation by multifunctional nexus proteins. I showed that the activation of phospholipase C-ε by Gα12/13-coupled receptors occurs through a mechanism involving the small GTPase Rho. I demonstrated the usefulness and complexities of ‘regulators of G-protein signaling’ (RGS proteins) for discerning the Gα selectivity of GPCR signaling. Finally, I found that RGS12, in addition to regulating Gα signaling, acts as a Ras/Raf/MEK scaffold in nerve growth factor-mediated differentiation. The work presented here expands our understanding of how multiple domain proteins facilitate convergence and cross-regulation of RTK, heterotrimeric G-protein, and Ras- superfamily signaling. iii To my parents, Donna and Jim, and my sister, Jennifer, who have always encouraged me to do my best, and provided me with endless love and support. And, to Francis, my husband, who has provided unbelievable amounts of guidance, support, encouragement, and patience, and indescribable love. iv ACKNOWLEDGEMENTS I would like to extend my appreciation to my dissertation advisor, Professor David Siderovski, for all his enthusiasm, guidance, and support on my dissertation project. His energy and attention to detail have helped me to grow as a scientist in countless ways, and for this, I will always be thankful. I would also like to thank Professor T. Kendall Harden, who was like a second mentor to me. His guidance and support over my graduate training has been tremendous, and I will always be grateful to him for his encouragement and friendship. I would also like to thank the additional members of my committee, Professor Gary Johnson, Professor Michael Schaller, and Professor John Sondek for their passion and for their intellectual guidance. I would also like to extend my thanks to both the current and past members of the Siderovski lab who have made important contributions to my training. I would especially like to thank Dr. Randall Kimple, Dr. Miller Jones, Dr. Christopher McCudden, Dr. Christopher Johnston, and Mr. Adam Kimple for their patience, their encouragement, their technical support, and their friendship. I would also like to express my sincere gratitude to my husband, Dr. Francis Willard, who spent countless hours discussing my project and guiding my research. His patience, knowledge, and drive to do great science helped me to grow as a scientist in ways families v that I never imagined I could. I would also like to thank the current and past members of the Harden and Sondek labs for all their contributions to my training. I would also like to thank my colleagues at Schering-Plough Research Institute, Dr. Rick Monsma, Ms. Lisa Smith, and Dr. Joseph Hedrick, among many others, for their encouragement and their friendship before and during graduate school. Finally, I would like to thank my family and friends for their endless support and friendship throughout my graduate training. vi TABLE OF CONTENTS LIST OF TABLES…………………………………………………………………………...x LIST OF FIGURES………………………………………………………………………....xi LIST OF ABBREVIATIONS AND SYMBOLS………………………..…………………xiv Chapter 1. INTRODUCTION…………………………………………………………………….1 1.1 The Importance of Cellular Signaling………………………………………..1 1.2 Signal Transduction at the Receptor…………………………………..........1 1.2.1 Heterotrimeric G-protein Signal Transduction…………………...2 1.2.2 Receptor Tyrosine Kinase Signal Transduction…………………3 1.2.3 Integration of GPCR and RTK Signal Transduction Pathways...4 1.3 Phospholipase C and Signal Transduction……………………………........7 1.3.1 Phospholipase C-ε………………………………………………….8 1.3.2 Novel Phospholipase C Interactions…………………………….10 1.3.3 Physiological Function of Phospholipase C-ε…………………..12 1.4 RGS Proteins as Negative Regulators of GPCR Signaling……………...15 1.4.1 RGS Protein Selectivity/Determinants…………………………..16 1.5 Multidomain Containing RGS Proteins……………………………………..19 1.5.1 The D- or R12-Subfamily of RGS Proteins………………..........19 1.5.2 RGS12………………………………………………………………20 vii 1.5.3 PDZ Domain………………………………………………………..20 1.5.4 PTB Domain………………………………………………………..23 1.5.5 RGS Domain……………………………………………………….25 1.5.6 Tandem Ras-Binding Domains…………………………………..26 1.5.7 GoLoco Motif……………………………………………………….28 1.5.8 Drosophila loco…………………………………………………….30 1.6 Mitogen-Activated Protein Kinases…………………………………………31 1.6.1 MAPK Cascades and the Importance of Scaffold Proteins…...32 1.6.2 Mammalian MAPK Cascade Scaffolds………………………….33 1.6.3 Nerve Growth Factor and TrkA…………………………………..36 1.6.4 PC12 Cells…………………………………………………………38 2. Gα12/13- AND RHO-DEPENDENT ACTIVATION OF PHOSPHOLIPASE C-ε BY LYSOPHOSPHATIDIC ACID AND THROMBIN RECEPTORS………….48 2.1 Abstract……………………………………………………………………….48 2.2 Introduction…………………………………………………………………...49 2.3 Materials and Methods……………………………………………………...50 2.4 Results………………………………………………………………………..53 2.5 Discussion………………………………………………………………….…59 2.6 Acknowledgments……………………………………………………………64 2.7 Footnotes……………………………………………………………………..64 3. APPLICATION OF RGS BOX PROTEINS TO EVALUATE G-PROTEIN SELECTIVITY IN RECEPTOR-PROMOTED SIGNALING……………………74 3.1 Abstract……………………………………………………………………….74 3.2 Introduction…………………………………………………………………...74 viii 3.3 Methodology………………………………………………………………….76 3.4 Materials and Methods………………………………………………………77 3.5 Background…………………………………………………………………..81 3.6 Application……………………………………………………………………83 3.7 Concluding Remarks………………………………………………………...88 3.8 Acknowledgments…………………………………………………………...89 4. SELECTIVE ROLE FOR RGS12 AS A RAS/RAF/MEK SCAFFOLD IN NERVE GROWTH FACTOR-MEDIATED DIFFERENTIATION……………...98 4.1 Abstract…………………………………………………………………….....98 4.2 Introduction…………………………………………………………………...98 4.3 Results………………………………………………………………………101 4.4 Discussion…………………………………………………………………..110 4.5 Materials and Methods…………………………………………………….115 4.6 Acknowledgments………………………………………………………….121 5. FUTURE DIRECTIONS………………………………………………………….140 REFERENCES……………………………………………………………………157 ix LIST OF TABLES Table 1.1 Selected mammalian scaffold proteins…………………………………….47 4.1 Plasmid sources…………………………………………………………….138 4.2 Antibody sources……………………………………………………………139 x LIST OF FIGURES Figure 1.1 Standard model of the GDP/GTP cycle governing activation of heterotrimeric G protein-coupled receptor (GPCR) signaling pathways………………………………………………………………………40 1.2 Receptor tyrosine kinase activation………………………………………...41 1.3 Domain architecture of mammalian phospholipase C (PLC) family members……………………………………………………………………….42 1.4 Schematic of the varied multidomain architectures of RGS family proteins………………………………………………………………………...43 1.5 The multidomain RGS protein RGS12 and its known interactors……….44 1.6 Mammalian MAPK signaling pathways…………………………………….45 1.7 PC12 signaling pathways involving the MAPK cascade………………….46 2.1 PLC-ε-dependent stimulation of inositol phosphate accumulation by endogenous LPA and thrombin receptors in COS-7 cells……………….65 2.2 PLC-ε-dependence of LPA-stimulated [3H]inositol phosphate accumulation in cells expressing empty vector, LPA1, LPA2, or LPA3 receptor………………………………………………………………………..66 2.3 Differential effects of PLC-ε on agonist-stimulated [3H]inositol phosphate accumulation in cells expressing the PAR1 receptor versus the M1 muscarinic cholinergic or P2Y2 receptor…………………………………...67 2.4 Differential effects of RGS proteins on LPA1 receptor- and PAR1 receptor- versus M1 muscarinic receptor-promoted [3H]inositol phosphate accumulation in PLC-ε-expressing