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

Molecular Metabolism

卷:53

Guo C. Huang¹  Peter M. Jones¹  Jordan Cheng²  Anastasia Arvaniti³  Shanta J. Persaud⁴  David J. Hodson⁴  Johannes Broichhagen⁴  Oladapo E. Olaniru⁴  Julia Ast⁵  Patricio Atanes⁶  Aileen J.F. King⁶ 

[1] Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, Birmingham, UK;

[2] Corresponding author.;

[3] Imaging Sciences, 4th floor Lambeth Wing, St Thomas' Hospital, London, SE1 7EH, UK;

[4] Department of Diabetes, School of Life Course Sciences, King's College London, Guy's Campus, London SE1 1UL, UK;

[5] Department of Imaging Chemistry and Biology, School of Biomedical Engineering &

[6] Institute of Metabolism and Systems Research (IMSR), Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham, Birmingham, UK;

关键词: GPR56;    SNAP-tag;    Trafficking;    Islets;    Apoptosis;    CRISPR-Cas9;   

DOI  :  

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown