Signaling and Regulation of the Polymicrogyria-associated receptor GPR56: A Model Biochemical Study of the Adhesion G Protein-coupled Receptor Family Open Access

Abstract

Abstract
Signaling and Regulation of the Polymicrogyria-associated
receptor GPR56: A Model Biochemical Study of the Adhesion
G Protein-coupled Receptor Family
The G protein-coupled receptor (GPCR) superfamily represents the largest library
of pharmaceutical drug targets. This is mostly due to their large number, surface
expression, signal amplification, and connection to human disease. GPR56 is a member
of the adhesion GPCR subfamily and mutations to this receptor have been shown to cause
cortical developmental defects leading to bilateral frontoparietal polymicrogyria. In this
dissertation, we sought to biochemically decipher the G protein dependent signaling
pathways of the receptor, as well as the mechanism of activation. Moreover, we searched
for novel protein-protein interaction that could regulate receptor function. Our studies
showed that GPR56 signals through Gα12/13 to activate Rho and β-catenin. We also
performed truncation studies on the large heavily glycosylated cleave N-terminus (NT)
and discovered that it remains non-covalently associated to the seven transmembrane
(7TM) region of the GPCR. Moreover, association to the 7TM region antagonized
GPR56-dependent activation, and removal of the NT showed evidence of a constitutively
active receptor. Furthermore, we found that the GPR56-NT is
capable of homophilic trans-trans interactions that enhance receptor signaling activity.
Finally, we showed novel protein-protein interactions invovled in GPR56 biology. Based on
these data, we propose a novel general mechanism of activation for the adhesion GPCR
family where the NT antagonizes receptor activation and removal by large NT binding
partners can alleviate this inhibitory influence. These studies offer a template for the
decoding of other adhesion GPCR signaling pathways and activation mechanisms, in
which all our considered orphan receptors to this point.

Signaling and Regulation of the Polymicrogyria-associated
receptor GPR56: A Model Biochemical Study of the Adhesion
G Protein-coupled Receptor Family

By

Kevin James Paavola
B.S., Carnegie-Mellon University, 2005
Advisor: Randy A. Hall, Ph.D.
A dissertation submitted to the Faculty of the
James T. Laney School of Graduate Studies of Emory University
In partial fulfillment of the requirements for the degree of
Doctor of Philosophy in
Graduate Division of Biological and Biomedical Sciences
Program in Molecular and Systems Pharmacology
2012

Table of Contents

Table of Contents

CHAPTER I: Introduction……………………………………………………………….1

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

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

1.3 Clinical relevance of G protein-coupled receptors……………………………….…6

1.4 Orphan G protein-coupled receptors……………………………………………….11

1.5 Adhesion family G protein-coupled receptors……………………………………..13

1.6 Adhesion G protein-coupled receptor structure……………………………………14

1.7 Adhesion GPCR family ligands……………………………………………………17

1.8 G protein dependent signaling of the adhesion family GPCR……………………..25

1.9 Physiological importance of adhesion G protein-coupled receptors........................27

1.10 Human diseases associated with mutations to adhesion GPCRs…...…………….29

1.10.1 VLGR1 and Ushers Syndrome.........................................................................29

1.10.2 Bilateral frontoparietal polymicrogyria and GPR56………………………..30

1.11 Aim of Dissertation research……………………………………………………...33

CHAPTER II: The N-terminus of GPR56 Controls Receptor Signaling Activity……..37

2.1 Introduction………………………………………………………………………...38

2.2 Experimental Procedure……………………………………………………………...39

2.3 Results………………………………………………………………………………..44

2.3.1 GPR56 couples to Gα12/13 to activate tcf/β-catenin through Rho………………….44

2.3.2 GPR56 is processed into two fragments that remain associated

at the cell surface…………………………………………………………………….......47

2.3.3 Sequential truncations of the GPR56 N-terminus results in reduction of total Rho levels……………………………………………………………………………………..51

2.3.4 Removal of the GPR56-NT enhances GPR56-mediated stimulation of

Rho activity and induces receptor ubiquitination and Cyr61 expression……………….54

2.3.5 Truncation of the GPR56-NT enhances receptor interactions with β-arrestin2…..58

2.3.6 Over-expression of GPR56 ΔNT induces cell death that can be rescued by

co-expression of β-arrestin2……………………………………………………………..62

2.4 Discussion……………………………………………………………………………65

CHAPTER III: GPR56 N-termini Undergo Homophilic Trans-interactions to Positively Regulate Receptor-dependent Signaling…………………………………………………69

3.1 Introduction………….……………………………………………………………….70

3.2 Experimental Procedure……….……………………………………………………..72

3.3 Results………………….…………………………………………………………….76

3.3.1 The N-termini of GPR56 are capable of homophilic trans-trans interactions…….76

3.3.2 GPR56 trans-trans N-terminal interactions enhance receptor signaling….……….81

3.3.3 A region between amino acids 258 - 288 is necessary to mediate interactions between GPR56 N-termini………………………………………………………………81

3.3.4 GPR56 N-terminal trans-interactions functionally inhibit glioma cell migration...84

3.4 Discussion…………………………………………………………………………...91

CHAPTER IV: Protein-Protein Interactions Regulate GPR56 Biology………………...93

4.1 Introduction………………………………………………………………………….94

4.2 Experimental Procedure……………………………………………………………..97

4.3 Results………………………………………………………………………………102

4.3.1 GPR56 interacts with PDZ proteins MAGI-3 and CFTR-associated ligand……..102

4.3.2 Collagen III antagonizes GPR56 signaling in HEK293 cells…………………….105

4.3.3 GPR56 localizes to primary cilia & interacts with the cilial protein β-arrestin2...106

4.4 Discussion…………………………………………………………………………..112

CHAPTER V: Further Discussion and Future Directions……………………...………115

5.1 Signaling and mechanism of activation for GPR56………………………………..116

5.2 A possible conserved mechanism of activation for the adhesion GPCR family…..117

5.3 Complexity and multiplicity of adhesion GPCR biology……………………….…119

5.4 Potential drug development strategies for targeting GPR56………………….…...120

5.5 Future directions: From GPR56-dependent Rho activation to the inhibition of cellular migration……………………………………………………………………………....123

5.6Future Directions: GPR56 as a target for anti-cancer therapeutics…………….….126

5.7Future Directions: Targeting GPR56 in treating neurodegenerative diseases….…128

5.8Concluding thoughts………………………………………………………………129

References…………………………………………………………………….……….131

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	Biology, Neuroscience Biology, Molecular Biology, Cell
Keyword	Adhesion GPCR GPR56 G protein-coupled receptor orphan GPCR Polymicrogyria
Committee Chair / Thesis Advisor	Hall, Randy A, Emory University
Committee Members	Miller, Gary W, Emory University Hepler, John R, Emory University Mao, Zixu, Emory University

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