Multi-modal Signaling and Regulation of the Adhesion G Protein-coupled Receptor ADGRG1 (GPR56) Pubblico

Abstract

G protein-coupled receptors (GPCRs) are important drug targets due to their tissue expression profiles and wide-ranging involvement in human physiology. The adhesion GPCRs (aGPCRs) comprise a subfamily of GPCRs that have been implicated in a number of human diseases but still remain mysterious in many ways. ADGRG1 (G1 or GPR56) is an aGPCR of special interest because loss-of-function mutations to the receptor underlie a devastating human neurological disease called bilateral frontoparietal polymicrogyria (BFPP). A characteristic feature of aGPCRs, including G1, is their ability to autocatalytically cleave at the first transmembrane domain. Multiple aGPCRs have been shown to exhibit significantly increased constitutive activity when truncated to the point of cleavage (ΔNT), and a general model of aGPCR activation has been proposed in which the new post-cleavage N-terminal stalk directly stimulates receptor activity. We tested whether G1 adheres to this ‘cryptic agonist' model by engineering a mutant version of the receptor that lacks the entire NT including the stalk (G1-SL) and broadly assessing receptor signaling. G1-SL displayed robust activity in several assays (including activation of NFAT-luciferase and βarrestin recruitment) but lost the ability to activate SRF-luciferase, a classic measure of Gα12/13-mediated activity. We also examined the effects of two different BFPP-inducing extracellular loop mutations (R565W & L640R) on multi-modal signaling by both full-length (FL) and ΔNT versions of G1. Similar to stalk deletion, the disease-associated mutations ablated receptor-mediated SRF activation but had no effect on receptor-mediated NFAT activation. Given these differential signaling results, we sought to further elucidate G1-mediated signaling to NFAT and found that it does not involves Gαq/11 or βarrestins but rather involves liberation of Gβγ subunits and activation of calcium channels. These data support a model in which G1 is capable of at least two distinct modes of signaling: stalk-dependent and stalk-independent, with the downstream intermediates being distinct for the two modes of signaling. The findings presented in this dissertation improve understanding of G1 signaling and regulation, and make significant contributions to the larger debate on the mechanisms of aGPCR activation. By providing insights into the fundamental biology of G1, these studies set the stage for future drug development efforts aimed at G1 and other aGPCRs.

Table of Contents

Chapter 1: Introduction………………………………………………...………..…...............................................................................................................................................….…1

1.1 G protein-coupled receptors……………………………………………….…..............................................................................................................................................…..…..2

1.2 G protein-coupled receptor signaling…………………………….……….….…..................................................................................................................................................…4

1.3 Regulation of G protein-dependent signaling…………………………………............................................................................................................................................…….....6

1.4 G protein-independent signaling by G protein-coupled receptors……………............................................................................................................................................…….….8

1.5 Regulation of G protein-coupled receptor interacting proteins………...............................................………................................................................................................…..….9

1.6 G protein-coupled receptors are outstanding drug targets…..………………................................................…..….................................................................................................10

1.7 Adhesion G protein-coupled receptors………….......………………................................................……….……..............................................................................................…10

1.8 Adhesion G protein-coupled receptor structure……………….………………….............................................................................................................................................…...11

1.9 Evidence of G protein-mediated signaling by Adhesion G protein-coupled receptors…………..………..............................................................................................………….14

1.10 Adhesion G protein-coupled receptor ligands……………...…………..……................................................…..............................................................................................…..17

1.11 Adhesion G protein-coupled receptors in human disease…..………………….................................................................................................................................................….23

1.12 Adhesion G protein-coupled receptor models of activation……......……….................................................................................................................................................…….25

1.13 Adhesion G protein-coupled receptor N-termini as sensors of mechanical forces…………………………...................................................................................................…...27

1.14 Adhesion G protein-coupled receptor associations with signaling proteins other than G proteins……….........................................................................................................…28

1.15 Dissertation Aims…………………………………………………………...............................................................................................................................................……….29

Chapter 2: Stalk-dependent and Stalk-independent signaling by ADGRG1……..............................................................................................................................................…........36

2.1 Introduction…………………………………………………..………..............................................................................................................................................……….……..37

2.2 Experimental Procedures……………………………………………………….............................................................................................................................................…….39

2.3 Results…………………………………………..……………….……………..............................................................................................................................................……..43

2.3.1 ADGRG1 autoproteolysis is not necessary for signaling activity………..............................................................................................................................................……...…43

2.3.2 Stalk-less ADGRG1 retains activity in some signaling assays but not others…………………………………………………………………………….…......................…....47

2.3.3 Stalk-less ADGRG1 exhibits robust β-Arrestin association and ubiquitination, two correlates of enhanced GPCR activity……………………..……….………..……...…..56

2.4 Discussion…………......................................……………………..............................................................................................................................................…………….....…60

Chapter 3: Disease-Associated Extracellular Loop Mutations Differentially Regulate Signaling Pathways Downstream of ADGRG1……….................................………….…….64

3.1 Introduction………………………………………………………………………..............................................................................................................................................…..65

3.2 Experimental Procedures………………………………….............................................................................................................................................………………….…….…67

3.3 Results…………………………………………………………………………...............................................................................................................................................…….71

3.3.1 BFPP-causing mutations R565W & L640R differentially affect surface expression of full-length vs. ΔNT versions of ADGRG1………………….…….………………..…71

3.3.2 R565W & L640R mutations disrupt ADGRG1-mediated activation of SRF luciferase but not NFAT luciferase……………………………….……………….……...............77

3.3.3 ADGRG1 signaling to NFAT luciferase does not involve βarrestins or Gαq/11 but does involve Gβγ and calcium channels…………………………………………..……....79

3.4 Discussion………………………………………………………………………..............................................................................................................................................……87

Chapter 4: Discussion…………………………………………………………………...........................................................................................................................………...……91

4.1 Dissecting two qualitatively distinct modes of ADGRG1-mediated signaling…..............................................................................................................................................…...92

4.2 The allosteric antagonist model of adhesion G protein-coupled receptor activation…………………………………………………………………………...................…...…..93

4.3 Elucidating the mechanisms of stalk-independent activation of NFAT by G1ΔNT………………………………………………………………………………….....................97

4.4 Future directions in studying G1 multi-modal signaling activity………………….............................................................................................................................................…98

4.5 Therapeutic potential of G1 modulation……………………………………............…................................................................................................................................….…100

4.6 Concluding remarks………………………………………………………............................................................................................................................................………....102

References………………………………………………………………………............................................................................................................................................…….…104

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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	Health Sciences, Pharmacology Health Sciences, Pharmacy Health Sciences, Medicine and Surgery
Parola chiave	receptor structure-function G protein-coupled receptor signal transduction
Committee Chair / Thesis Advisor	Hall, Randy A, Emory University
Committee Members	Hepler, John R, Emory University Kukar, Thomas, Emory University Nahta, Rita, Emory University

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