Regulation of Adhesion G Protein-Coupled Receptor ADGRG1 (GPR56) by Receptor Activity-Modifying Proteins 公开

Gao, Anqi (2017)

Permanent URL: https://etd.library.emory.edu/concern/etds/8c97kr169?locale=zh
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Abstract

G protein-coupled receptors (GPCRs) are a diverse superfamily of transmembrane proteins that transmit signals from the extracellular side of cells into the cytoplasm. Adhesion GPCRs are a family of receptors that are characterized by long N-termini containing adhesion-like motifs. ADGRG1 (also known as GPR56 or G1) is an adhesion GPCR that is involved in brain development. Mutations to this receptor cause a neurodevelopmental disorder known as bilateral frontoparietal polymicrogyria (BFPP). The purpose of this project was to examine receptor activity-modifying proteins (RAMPs) might regulate the activity of G1. We found that G1 physically associates with RAMP1 and RAMP3, and that a truncated constitutively-active version of G1 (ΔNT-G1) associates with RAMP1 and RAMP3 even more robustly. Co-expression with RAMPs had no significant effect on the signaling activity of G1 or ΔNT-G1 when receptor signaling to NFAT luciferase was assessed. Conversely, co-expression with RAMP3 sharply decreased ΔNT-G1 signaling to SRF luciferase, revealing a differential effect of RAMP3 on distinct signaling readouts. These findings represent the first description of RAMP interactions with adhesion GPCRs and provide a novel mechanism by which G1 activity may be regulated.

Table of Contents

Table of Contents

Abbreviations 1

I. Introduction

- Cell to cell communication 2

- G protein-coupled receptors 2

- Adhesion G protein-coupled receptors 4

- G protein-coupled receptor 56 (ADGRG1) 5

- Bilateral frontoparietal polymicrogyria (BFPP) 5

- Roles of ADGRG1 in the immune system and cancer 6

- Receptor activity-modifying proteins (RAMPs) 8

- Connections between G protein-coupled receptors and RAMPs 9

II. Materials and Methods

- Constructs 10

- Cell culture 10

- Co-Immunoprecipitation of G1 and ΔNT-G1with HA-RAMP1-3 10

- Evaluation of the effects of G1 and ΔNT-G1 interactions with HA-RAMP1-3 11

on signaling through G1 via NFAT and SRF-luciferase gene reporter assays

- Evaluation of G1, ΔNT-G1, and HA-RAMP3 surface expression via biotinylation 12

- Western blot 13

- Stripping of Western blot membranes 14

- Statistical analysis 15

III. Results

- G1 physically interacts with RAMP1 and RAMP3 in HEK-293T cells 15

- NFAT-luciferase assay 16

- SRF-luciferase assay 17

- Interactions between G1 and ΔNT-G1 with RAMP3 decreases surface expression 18

of G1 and RAMP3 increases surface expression of ΔNT-G1

- Interactions between G1 and ΔNT-G1 with RAMP3 increases RAMP3 surface expression 18

IV. Discussion and Conclusions 19

VI. Figures

- Figure 1 24

- Figure 2 25

- Figure 3 26

- Figure 4 27

- Figure 5 28

- Figure 6 29

- Figure 7 30

- Figure 8 31

VII. References 32

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