The cysteine of the cytoplasmic tail of glucose-dependent insulinotropic peptide receptor mediates its chronic desensitization and down-regulation.

The glucose-dependent insulinotropic peptide receptor (GIP-R) is a member of G-protein-coupled, seven transmembrane-spanning receptors. Recent studies have shown that elevated serum GIP level in diabetic patients may induce chronic desensitization of the GIP-R, and that this mechanism could contribute to impaired insulin secretion. The cellular basis of down-regulation and chronic desensitization of GIP-R is unclear. To explore the role of the carboxyl terminus of the GIP-R in mediating these processes, five truncated GIP-Rs (T395, T399, T420, T431, T455) were created to delete consecutive serines from the carboxyl end. All mutants except T395 exhibit an identical ligand-binding affinity to the WT receptor. The T395 mutant, which had the entire carboxyl tail removed, does not bind to ligand. Down-regulation and desensitization was assessed by measuring the receptor number and the ability of agonist-induced cAMP or [Ca2+] generation after pre-exposure to 10(-7) M GIP for 24 h. The wild-type (WT) and T421, T431, T455 mutant GIP-Rs are maximally down-regulated by GIP preincubation, whereas T399 mutant does not, indicating that the sequence between amino acids 399 and 420 is critical for this process. Mutation analysis of this area by alanine scanning mutagenesis reveals two critical residues: serine 406 and cysteine 411. Replacement of serine 406 with arginine (S406R) or alanine (S406A) partly attenuates agonist-induced down-regulation and desensitization. In contrast, mutation of the cysteine 411 to glycine (C411G) or alanine (C411A) markedly attenuates both processes. Mutant SCRG, in which both serine 406 and cysteine 411 are mutated, behaves similar to C411G or C4111A. The data suggest that chronic desensitization and down-regulation of the GIP-R may be mediated by similar mechanisms, and that the cysteine in the carboxyl terminus plays an essential role in regulating both processes.