Regulation of Adhesion G Protein-Coupled Receptor ADGRG1 (GPR56) by Receptor Activity-Modifying Proteins Öffentlichkeit
Gao, Anqi (2017)
Published
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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