TY - JOUR
T1 - SNAP-tag-enabled super-resolution imaging reveals constitutive and agonist-dependent trafficking of GPR56 in pancreatic β-cells
AU - Olaniru, Oladapo E.
AU - Cheng, Jordan
AU - Ast, Julia
AU - Arvaniti, Anastasia
AU - Atanes, Patricio
AU - Huang, Guo C.
AU - King, Aileen J.F.
AU - Jones, Peter M.
AU - Broichhagen, Johannes
AU - Hodson, David J.
AU - Persaud, Shanta J.
N1 - 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.
PY - 2021/11
Y1 - 2021/11
N2 - 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.
AB - 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.
KW - Apoptosis
KW - CRISPR-Cas9
KW - GPR56
KW - Islets
KW - SNAP-tag
KW - Trafficking
UR - http://www.scopus.com/inward/record.url?scp=85111022727&partnerID=8YFLogxK
U2 - 10.1016/j.molmet.2021.101285
DO - 10.1016/j.molmet.2021.101285
M3 - Article
C2 - 34224919
AN - SCOPUS:85111022727
SN - 2212-8778
VL - 53
JO - Molecular Metabolism
JF - Molecular Metabolism
M1 - 101285
ER -
