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SNAP-tag-enabled super-resolution imaging reveals constitutive and agonist-dependent trafficking of GPR56 in pancreatic β-cells

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Oladapo E. Olaniru, Jordan Cheng, Julia Ast, Anastasia Arvaniti, Patricio Atanes, Guo C. Huang, Aileen J.F. King, Peter M. Jones, Johannes Broichhagen, David J. Hodson, Shanta J. Persaud

Original languageEnglish
Article number101285
JournalMolecular Metabolism
PublishedNov 2021

Bibliographical note

Funding Information: This research was supported by Diabetes UK (grant number 17/0005600). D.J.H. was supported by MRC (MR/N00275X/1 and MR/S025618/1) and Diabetes UK (17/0005681) project grants and funding from the European Research Council ( ERC ) under the European Union's Horizon 2020 research and innovation programme (Starting Grant 715884 to D.J.H.). We thank Professor Xianhua Piao (University of California) for critically reviewing the manuscript. Publisher Copyright: © 2021 The Author(s) Copyright: Copyright 2021 Elsevier B.V., All rights reserved.

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Objective: Members of the adhesion G protein-coupled receptor (aGPCR) subfamily are important actors in metabolic processes, with GPR56 (ADGRG1) emerging as a possible target for type 2 diabetes therapy. GPR56 can be activated by collagen III, its endogenous ligand, and by a synthetic seven amino-acid peptide (TYFAVLM; P7) contained within the GPR56 Stachel sequence. However, the mechanisms regulating GPR56 trafficking dynamics and agonist activities are not yet clear. Methods: Here, we introduced SNAPf-tag into the N-terminal segment of GPR56 to monitor GPR56 cellular activity in situ. Confocal and super-resolution microscopy were used to investigate the trafficking pattern of GPR56 in native MIN6 β-cells and in MIN6 β-cells where GPR56 had been deleted by CRISPR-Cas9 gene editing. Insulin secretion, changes in intracellular calcium, and β-cell apoptosis were determined by radioimmunoassay, single-cell calcium microfluorimetry, and measuring caspase 3/7 activities, respectively, in MIN6 β-cells and human islets. Results: SNAP-tag labelling indicated that GPR56 predominantly underwent constitutive internalisation in the absence of an exogenous agonist, unlike GLP-1R. Collagen III further stimulated GPR56 internalisation, whereas P7 was without significant effect. The overexpression of GPR56 in MIN6 β-cells did not affect insulin secretion. However, it was associated with reduced β-cell apoptosis, while the deletion of GPR56 made MIN6 β-cells more susceptible to cytokine-induced apoptosis. P7 induced a rapid increase in the intracellular calcium in MIN6 β-cells (in a GPR56-dependent manner) and human islets, and it also caused a sustained and reversible increase in insulin secretion from human islets. Collagen III protected human islets from cytokine-induced apoptosis, while P7 was without significant effect. Conclusions: These data indicate that GPR56 exhibits both agonist-dependent and -independent trafficking in β-cells and suggest that while GPR56 undergoes constitutive signalling, it can also respond to its ligands when required. We have also identified that constitutive and agonist-dependent GPR56 activation is coupled to protect β-cells against apoptosis, offering a potential therapeutic target to maintain β-cell mass in type 2 diabetes.

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