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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 beta-cells and in MIN6 beta-cells where GPR56 had been deleted by CRISPR-Cas9 gene editing. Insulin secretion, changes in intracellular calcium, and beta-cell apoptosis were determined by radioimmunoassay, single-cell calcium microfluorimetry, and measuring caspase 3/7 activities, respectively, in MIN6 beta-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 beta-cells did not affect insulin secretion. However, it was associated with reduced beta-cell apoptosis, while the deletion of GPR56 made MIN6 beta-cells more susceptible to cytokine-induced apoptosis. P7 induced a rapid increase in the intracellular calcium in MIN6 beta-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 beta-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 beta-cells against apoptosis, offering a potential therapeutic target to maintain beta-cell mass in type 2 diabetes.

Original publication

DOI

10.1016/j.molmet.2021.101285

Type

Journal article

Journal

Mol Metab

Publication Date

2021

Volume

53

Keywords

*Apoptosis *CRISPR-Cas9 *gpr56 *Islets *SNAP-tag *Trafficking