HETEROLOGOUS DESENSITIZATION OF THE GLUCAGON-LIKE PEPTIDE-1 RECEPTOR BY PHORBOL ESTERS REQUIRES PHOSPHORYLATION OF THE CYTOPLASMIC TAIL AT 4 DIFFERENT SITES

Glucagon-like peptide-1 stimulates glucose-induced insulin secretion b y binding to a specific G protein-coupled receptor that activates the adenylyl cyclase pathway, We previously demonstrated that heterologous desensitization of the receptor by protein kinase C correlated with p hosphorylation in a 33-amino acid-long segment of the receptor carboxy l-terminal cytoplasmic tail. Here, we determined that the in vivo site s of phosphorylation are four serine doublets present at positions 431 /432, 441/442, 444/445, and 451/452. In vitro phosphorylation of fusio n proteins containing mutant receptor C-tails, however, indicated that whereas serines at position 431/432 were good substrates for protein kinase C (PKC), serines 444/445 and 451/452 were poor substrates, and serines 441/442 were not substrates. In addition, serine 416 was phosp horylated on fusion protein but not in intact cells. This indicated th at in vivo a different PKC isoform or a PKC-activated kinase may phosp horylate the receptor. The role of phosphorylation on receptor desensi tization was assessed using receptor mutants expressed in COS cells or Chinese hamster lung fibroblasts. Mutation of any single serine doubl et to alanines reduced the extent of phorbol ia-myristate 13-acetate-i nduced desensitization, whereas substitution of any combination of two serine doublets suppressed it. Our data thus show that the glucagon-l ike peptide-1 receptor can be phosphorylated in response to phorbol 12 -myristate 13-acetate on four different sites within the cytoplasmic t ail. Furthermore, phosphorylation of at least three sites was required for desensitization, although maximal desensitization was only achiev ed when all four sites were phosphorylated.