Conserved residues in the extracellular loop 2 regulate Stachel-mediated activation of ADGRG2

Abanoub A Gad,Pedram Azimzadeh,Nariman Balenga

doi:10.1038/s41598-021-93577-y

Abanoub A Gad, Pedram Azimzadeh + Show 1 more

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https://doi.org/10.1038/s41598-021-93577-y

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Journal: Scientific Reports	Publication Date: Jul 7, 2021
Citations: 4	License type: open-access

Affiliation: University of Maryland, Baltimore

Abstract

Cleavage and dissociation of a large N-terminal fragment and the consequent unmasking of a short sequence (Stachel) remaining on the N-terminus have been proposed as mechanisms of activation of some members of the adhesion G protein-coupled receptor (aGPCR) family. However, the identity of residues that play a role in the activation of aGPCRs by the cognate Stachel remains largely unknown. Protein sequence alignments revealed a conserved stretch of residues in the extracellular loop 2 (ECL2) of all 33 members of the aGPCR family. ADGRG2, an orphan aGPCR, plays a major role in male fertility, Ewing sarcoma cell proliferation, and parathyroid cell function. We used ADGRG2 as a model aGPCR and generated mutants of the conserved residues in the ECL2 via site-directed mutagenesis. We show that tryptophan and isoleucine in the ECL2 are essential for receptor stability and surface expression in the HEK293 cells. By adjusting the receptor surface expression levels, we show that mutation of these residues of ECL2 ablates the Stachel-mediated activation of multiple signaling pathways of ADGRG2. This study provides a novel understanding of the role of the ECL2 in Stachel-mediated signaling and degradation of ADGRG2, which may lay the foundation for the rational design of therapeutics to target aGPCRs.

Highlights

G protein-coupled receptors (GPCRs) play fundamental roles in various cellular processes such as proliferation, metabolism, hormone secretion, contraction, and n eurotransmission[1]
Our understanding of the mechanisms of activation of adhesion G protein-coupled receptor (aGPCR) has substantially increased over the last decade
We know that binding of an extracellular molecular partner to the N-terminal fragment (NTF) of ADGRG1 dissociates it from the C-terminal fragment (CTF) and unmasks a Stachel sequence, which subsequently activates ADGRG114,19,35

Summary

Introduction

G protein-coupled receptors (GPCRs) play fundamental roles in various cellular processes such as proliferation, metabolism, hormone secretion, contraction, and n eurotransmission[1]. The signaling pathways of GPCRs via G proteins and β-arrestins, and their trafficking trajectories have been studied for several d ecades[2,3] This in combination with recent advances in our understanding of GPCR structures has formed a strong foundation for rational drug design to target this largest superfamily of surface r eceptors[4,5]. NTF-dissociating molecular partners can potentially unmask the Stachel sequence for its binding to the cognate aGPCR These studies exploited two main tools to reveal this mechanism: (a) NTFtruncated aGPCRs that show constitutive activation of G proteins; (b) synthetic peptides resembling the Stachel sequence that activate a GPCRs13,14. Baltimore Street, Room 10‐027, Scientific Reports | (2021) 11:14060 These studies show that aGPCRs use various structural segments, motifs, and the Stachel peptide to engage distinct signaling m odes[18]. The binding site of Stachel remains unknown among aGPCRs

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