Agonist-induced internalisation of the glucagon-like peptide-1 receptor is mediated by the G alpha(q) pathway

The glucagon-like peptide-1 receptor (GLP-1R) is a G-protein-coupled receptor (GPCR) and an important target in the treatment of type 2 diabetes mellitus (T2DM). Upon stimulation with agonist, the GLP-1R signals through both G alpha(s) and G alpha(q) coupled pathways to stimulate insulin secretion. The agonist-induced GLP-1R internalisation has recently been shown to be important for insulin secretion. However, the molecular mechanisms underlying GLP-1R internalisation remain unknown. The aim of this study was to determine the role of GLP-1R downstream signalling pathways in its internalisation. Agonist-induced human GLP-1R (hGLP-1R) internalisation and activity were examined using a number of techniques including immunoblotting, ELISA, immunofluorescence and luciferase assays to determine cAMP production, intracellular Ca2+ accumulation and ERK phosphorylation. Agonist-induced hGLP-1R internalisation is dependent on caveolin-1 and dynamin. Inhibition of the G alpha(q) pathway but not the G alpha(s), pathway affected hGLP-1R internalisation. Consistent with this, hGLP-1R mutant T149M and small-molecule agonists (compound 2 and compound B), which activate only the G alpha(s) pathway, failed to induce internalisation of the receptor. Chemical inhibitors of the G alpha(q) pathway, PKC and ERK phosphorylation significantly reduced agonist-induced hGLP-1R internalisation. These inhibitors also suppressed agonist-induced ERK1/2 phosphorylation demonstrating that the phosphorylated ERK acts downstream of the G alpha(q) pathway in the hGLP-1R internalisation. In summary, agonist-induced hGLP-1R internalisation is mediated by the G alpha(q) pathway. The internalised hGLP-1R stimulates insulin secretion from pancreatic beta-cells, indicating the importance of GLP-1 internalisation for insulin secretion. (C) 2014 Elsevier Inc. All rights reserved.