Real-time trafficking and signaling of the glucagon-like peptide-1 receptor

•For the first time, we show real-time internalization of a family B GPCR.•GLP-1R internalizes rapidly in response to various agonists.•Ligand-induced GLP-1R internalization is confirmed in vivo in mouse pancreatic islets.•GLP-1R is a main recycling receptor.•Prolonged signaling from GLP-1R is proposed to be correlated to prolonged receptor cycling. The glucagon-like peptide-1 incretin receptor (GLP-1R) of family B G protein-coupled receptors (GPCRs) is a major drug target in type-2-diabetes due to its regulatory effect on post-prandial blood-glucose levels. The mechanism(s) controlling GLP-1R mediated signaling are far from fully understood. A fundamental mechanism controlling the signaling capacity of GPCRs is the post-endocytic trafficking of receptors between recycling and degradative fates. Here, we combined microscopy with novel real-time assays to monitor both receptor trafficking and signaling in living cells. We find that the human GLP-1R internalizes rapidly and with similar kinetics in response to equipotent concentrations of GLP-1 and the stable GLP-1 analogues exendin-4 and liraglutide. Receptor internalization was confirmed in mouse pancreatic islets. GLP-1R is shown to be a recycling receptor with faster recycling rates mediated by GLP-1 as compared to exendin-4 and liraglutide. Furthermore, a prolonged cycling of ligand-activated GLP-1Rs was observed and is suggested to be correlated with a prolonged cAMP signal.