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

Aims/hypothesis: 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 enable monitoring of 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 by 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 exogenous agonist, unlike GLP-1R. Collagen III further stimulated GPR56 internalisation, whereas P7 was without significant effect. Overexpression of GPR56 in MIN6 β-cells did not affect insulin secretion. However, it was associated with reduced β-cell apoptosis, while deletion of GPR56 made MIN6 β-cells more susceptible to cytokine-induced apoptosis. P7 induced a rapid increase in intracellular calcium in MIN6 β-cells, in a GPR56-dependent manner, and also in 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/interpretation: 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 protection of β-cells against apoptosis, thus offering a potential therapeutic target to maintain β-cell mass in type 2 diabetes.