Agonist-induced Desensitization of the μ Opioid Receptor Is Determined by Threonine 394 Preceded by Acidic Amino Acids in the COOH-terminal Tail

Abstract

To identify the structural determinants necessary for mu opioid receptor desensitization, we serially ablated potential phosphorylation sites in the carboxyl tail of the receptor and examined their effects on [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO)-induced desensitization. First, we replaced Thr394 with alanine (T394A) and stably expressed this mutant receptor in Chinese hamster ovary cells. The T394A receptor did not desensitize after 1 h of treatment with DAMGO, indicating that Thr394 is required for agonist-induced early desensitization. To test whether Thr394 was the only residue necessary, we investigated the importance of 7 potential phosphorylation sites between residues 363 and 383, which were all replaced by alanines with the Thr394 maintained. This mutant (AT) showed partial loss of desensitization (30%), which was attributable to the Ala mutation at Thr383, since complete desensitization was achieved by restoring Thr383 (ATT). These results suggest that Thr394 is the primary recognition site for G protein-coupled receptor kinases, but Thr383 is also required for complete agonist-induced desensitization. The specificity of Thr394 as the primary initiation site appears to be dependent on the preceding acidic amino acid stretch, because in a mutant in which glutamic acid residues at 388, 391, and 393 were replaced by glutamines (EQ), agonist-induced desensitization was completely abolished, identical to the T394A mutant.