The Impact of Genetic Variation on RGS and G protein Signaling in Physiology and Disease Open Access

Abstract

Regulators of G protein signaling (RGS) proteins modulate G protein-coupled receptor (GPCR) signaling by acting as negative regulators of G proteins. RGS proteins critically regulate cell physiology and pathophysiology. Each RGS protein has a distinct G protein selectivity profile that correlates to a finely tuned GPCR-G protein response. Human genetic variant information has expanded rapidly in recent years, including cancer-linked mutations in RGS proteins.  Genetic variants in RGS proteins contribute to complex polygenic human traits and pathologies by causing differences in protein function that is critical for signaling regulation and physiology. Recent genome-wide association studies (GWAS) have identified point mutations in RGS proteins that are associated with pathologies such as cancers and other diseases (e.g. RGS14 in chronic kidney disease). In this body of work, I explored a new analytical method, 3DMTR and permutation analysis, to define regions of genetic intolerance in 15 RGS proteins and prioritize which cancer-linked mutants in selected RGS proteins to test for altered function. Using complimentary cellular and biochemical approaches, RGS4, RGS10, and RGS14 were tested for effects on GPCR-Gα activation, Gα binding properties, and downstream cAMP levels. My findings show that 3DMTR identified intolerant residues that overlap with cancer-linked mutations cause phenotypic changes that negatively impact GPCR-G protein signaling and suggest that 3DMTR is a potentially useful bioinformatics tool for predicting functionally important protein residues. In addition to these findings, my studies expand our knowledge of the multifunctional signaling protein RGS14 by defining its role in regulating PTH1R-G protein signaling linked to phosphate transport in chronic kidney disease. My studies reveal that PTHR1 stimulates intracellular cAMP by coupling to Gas, as expected. However, quite unexpectedly, I also found that RGS14 stimulates intracellular calcium independent of Gaq coupling. Human RGS14 binds to the PDZ protein NHERF1. I found that RGS14 and NHERF1 each block PTH-stimulated calcium, but not cAMP accumulation,  indicating that RGS14 and NHERF1 regulate PTHR1-calcium signaling by a previously unknown mechanism. These studies expand on the fine-tuning roles of RGS14 and other RGS proteins in mediating G protein signaling and crosstalk between GPCRs and other signaling pathways linked to physiology and disease.

Table of Contents

CHAPTER 1: INTRODUCTION 1

1.1. Foundations in the GPCR-G protein field 2

1.2. Conventional GPCR and G protein signaling 5

1.3. Regulators of G protein signaling (RGS) in physiology and disease 6

1.3.1.  The R4 Family 10

1.3.2.  The R7 Family  11

1.3.3.  The RZ Family 12

1.3.4.  The R12 Family 13

1.4. Regulator of G protein signaling 14 in physiology and disease 15

1.4.1.  RGS14 is a multifunctional signaling protein 15

1.4.2.  The role of RGS14 in the brain 19

1.4.3.  The role of RGS14 outside of the brain 23

 

CHAPTER 2: FUNCTIONAL ASSESSMENT OF CANCER-LINKED MUTATIONS IN SENSITIVE REGIONS OF RGS PROTEINS PREDICTED BY 3DMTR ANALYSIS 26

2.1. Abstract 27

2.2. Introduction 29

2.3. Materials and Methods 32

2.4. Results 39

2.5. Discussion 70

2.6. Supplemental Information 76

 

CHAPTER 3: HUMAN RGS14 AND NHERF1 REGULATE PTH1R-G PROTEIN SIGNALING EVENTS LINKED TO PHOSPHATE UPTAKE IN KIDNEY 88

3.1. Abstract 89

3.2. Introduction 90

3.3. Materials and Methods 93

3.4. Results 97

3.5. Discussion 104

 

CHAPTER 4: DISCUSSION AND CONCLUDING REMARKS  108

4.1. Introduction 109

4.2. Discussion and Future Directions 111

4.3. Concluding Remarks 126

 

BIBLIOGRAPHY  127

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Molecular Biology, Physiology Biology, Genetics
Keyword	RGS14 Genetic variants RGS10 Renal phosphate uptake 3DMTR Missense Mutations RGS4 NHERF1 G Protein Coupled Receptors RGS G proteins PTH1R Regulators of G protein Signaling GPCR Parathyroid Hormone (PTH)
Committee Chair / Thesis Advisor	John R. Hepler, Emory University
Committee Members	Victor Faundez, Emory University Randy Hall, Emory University Stephen Traynelis, Emory University

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