Integrationof Heterotrimeric G Protein Signaling by the Regulator of G Protein Signaling 14 (RGS14): Independent Regulation of Gα Signaling by the RGS Domain and GPR Motif Public

Abstract

The Regulators of G protein Signaling (RGS) proteins are key modulators of G protein-coupled receptor and heterotrimeric G protein (Gαβγ) signaling events. One RGS protein, RGS14, is a key suppressor of synaptic plasticity, learning, and memory in the hippocampus. Like all RGS proteins, RGS14 contains an RGS domain that binds active Gα-GTP and catalyzes hydrolysis of GTP to GDP, thus acting as a GTPase activating protein (GAP). RGS14-mediated GAP activity accelerates the deactivation of Gαi/o proteins to terminate signaling by Gα and Gβγ subunits. RGS14 also contains a second Gα interaction site, a G Protein Regulatory (GPR) motif, which selectively binds inactive Gαi1/3-GDP. The GPR motif and Gβγ bind to the same site on Gα, thus RGS14 and Gβγ binding are mutually exclusive. Gαi1/3-GDP recruits RGS14 to the plasma membrane via the GPR motif to form a stable GPR:Gα-GDP complex devoid of Gβγ. Here I investigated the roles of the RGS14 RGS domain and GPR motif in regulating heterotrimeric G protein signaling. My studies highlight RGS14 as a structurally dynamic protein that is stabilized upon binding Gα at either the RGS domain or the GPR motif. Upon binding Gα through the GPR motif, RGS14 is allosterically stabilized at the RGS domain. Additionally, my studies demonstrate that despite sharing an overlapping binding site with Gβγ on Gα, RGS14 does not interfere with heterotrimer formation. Moreover, while RGS14 is capable of binding two distinct forms of Gα, my findings suggest RGS14 can functionally engage two Gα proteins simultaneously. I show that a preformed RGS14:Gαi1-GDP complex exhibits full capacity to stimulate the GTPase activity of a second Gαo-GTP protein in vitro and in a cellular context. Together, these studies demonstrate that despite engaging two G proteins simultaneously, the RGS domain and GPR motif function independently. Mechanistically, I propose RGS14 forms a stable complex at the plasma membrane with Gαi1-GDP through its GPR motif where the RGS domain is free to regulate a second G protein signaling event. My findings suggest that, in hippocampal neurons, native RGS14 serves as a dynamic multifunctional scaffolding protein that mediates unconventional G protein signaling events underlying synaptic plasticity.

Table of Contents

Chapter 1: Introduction 1

1.1 G Protein-Coupled Receptors 1

1.2 Heterotrimeric G Proteins 3

1.3 Regulators of G Protein Signaling 5

1.4 Non-Canonical G Protein Signaling 10

1.5 G Protein Regulatory Motif Proteins 12

1.6 Regulator of G Protein Signaling 14 (RGS14) 15

1.7 Overall Hypothesis and Objective of this Research 19

Chapter 2: Protein Purification Methods to Purify RGS14 and RGS14:Gα Complexes

2.1 Introduction 24

2.2 Materials 28

2.3 Methods 32

2.4 Notes 36

Chapter 3: Bioluminescence Resonance Energy Transfer to Detect Protein-Protein Interactions in Live Cells

3.1 Introduction 42

3.2 Materials 45

3.3 Methods 47

3.4 Notes 51

Chapter 4: Integration of G Protein Alpha (Gα) Signaling by the Regulator of G Protein Signaling 14 (RGS14)

4.1 Introduction 55

4.2 Experimental Procedures 57

4.3 Results 61

4.4 Discussion 78

Chapter 5: RGS14 Regulates the Lifetime of Gα-GTP Signaling but does not Prolong Gβγ Signaling Following Receptor Activation in Live Cells

5.1 Introduction 89

5.2 Experimental Procedures 91

5.3 Results 93

5.4 Discussion 110

Chapter 6: Discussion

6.1 Introduction 117

6.2 RGS14 is a Dynamic Scaffolding Protein 117

6.3 RGS14 Binds Two G proteins Simultaneously 118

6.4 Binding of Gα at the GPR Motif Does Not Prevent RGS GAP Function 119

6.5 Binding of Gα at the GPR Motif Does Not Alter RGS GAP Function in Live Cells 119

6.6 RGS14 Does Not Alter G Protein Heterotrimer Formation 120

6.7 Working Model 121

6.8 Future Directions 122

6.9 Concluding Remarks 133

References 135

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry Health Sciences, Pharmacology
Mot-clé	GPR G protein GPCR G Protein Regulatory RGS Regulator of G Protein Signaling
Committee Chair / Thesis Advisor	Hepler, John R, Emory University
Committee Members	Hall, Randy A, Emory University Kukar, Thomas, Emory University Ortlund, Eric, Emory University

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