Glucagon-like peptide-1 receptor C-terminal tail phosphorylation determines signalling responses from pancreatic β-cells

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that regulates glucose homeostasis by promoting cAMP production and insulin secretion in pancreatic {beta}-cells. Although receptor phosphorylation is known to influence GLP-1R signalling, its functional relevance in {beta}-cells remains incompletely understood. Here, we investigated the role of three C-terminal serine doublets (Ser441/442, Ser444/445, Ser451/452) in the human receptor using site-directed mutagenesis in INS-1 832/3 {beta}-cells deleted for endogenous GLP-1R expression. GLP-1 stimulation induced serine phosphorylation of wild-type GLP-1R, which was abolished in all individual doublet as well as triple doublet phospho-null mutants. Basal phosphorylation was detected in wild-type receptors under unstimulated conditions, suggesting dynamic cycling between phosphorylated and unphosphorylated states. All mutant receptors trafficked normally to the plasma membrane, but the triple alanine doublet (AA) mutant exhibited reduced GLP-1-induced internalisation and significantly decreased plasma membrane diffusion under vehicle conditions, indicating altered receptor dynamics even in the absence of agonist. Importantly, the Ser451/452 doublet was required for GRK2 recruitment to the active GLP-1R. Functionally, the triple AA mutant showed impaired GLP-1-induced cAMP generation with minimal change in {beta}-arrestin 2 recruitment, resulting in biased signalling. The cAMP defect was partially rescued by phosphodiesterase 4 (PDE4) inhibition with rolipram, implicating altered PDE engagement in the defective cAMP response. These findings demonstrate that both basal and agonist-induced GLP-1R phosphorylation are critical for receptor signalling in {beta}-cells, revealing a previously unappreciated role for basal phosphorylation in shaping receptor behaviour and establishing a positive link between GLP-1R phosphorylation and {beta}-cell signalling competence.