INTERNALIZATION AND HOMOLOGOUS DESENSITIZATION OF THE GLP-1 RECEPTOR DEPEND ON PHOSPHORYLATION OF THE RECEPTOR CARBOXYL TAIL AT THE SAME 3 SITES

Homologous desensitization and internalization of the GLP-1 receptor c orrelate with phosphorylation of the receptor in a 33-amino acid segme nt of the cytoplasmic tail. Here, we identify the sites of phosphoryla tion as being three serine doublets located at positions 441/442, 444/ 445, and 451/452. The role of phosphorylation on homologous desensitiz ation was assessed after stable expression in fibroblasts of the wild type or of mutant receptors in which phosphorylation sites were change d in various combinations to alanines. We showed that desensitization, as measured by a decrease in the maximal production of cAMP after a f irst exposure of the cells to GLP-1, was strictly dependent on phospho rylation. Furthermore, the number of phosphorylation sites correlated with the extent of desensitization with no, intermediate, or maximal d esensitization observed in the presence of one, two, or three phosphor ylation sites, respectively. Internalization of the receptor-ligand co mplex was assessed by measuring the rate of internalization of bound [ I-125]GLP-1 or the redistribution of the receptor to an endosomal comp artment after agonist binding. Our data demonstrate that internalizati on was prevented in the absence of receptor phosphorylation and that i ntermediate rates of endocytosis were obtained with receptors containi ng one or two phosphorylation sites. Thus, homologous desensitization and internalization require phosphorylation of the receptor at the sam e three sites. However, the differential quantitative impairment of th ese two processes in the single and double mutants suggests different molecular mechanisms controlling desensitization and internalization.