Replacement of threonine 394 by alanine facilitates internalization and resensitization of the rat mu opioid receptor

Signaling of G protein-coupled receptors is terminated by phosphorylation o f intracellular serine and threonine residues. Resensitization of these rec eptors requires internalization and subsequent dephosphorylation. We have r ecently shown that the resensitization rate of the rat mu opioid receptor ( MOR) isoforms MOR1 and MOR1 B is mainly determined by the amino acid compos ition of their alternatively spliced C-terminal tails. Upon agonist stimula tion, MOR1B passes through an accelerated cycle of receptor endocytosis and reactivation, which in turn promotes a greater resistance to agonist-induc ed desensitization, as compared with MOR1. Given the fact that MOR1 B lacks only one putative phosphorylation site (T394 of MOR1), we replaced this th reonine by an alanine and stably expressed the wild-type MOR1 and its T394A mutant in mouse neuroblastoma Neuro2a cells. We show that during prolonged [D-Ala(2), MePhe(4), Gly(5)-ol]enkephalin exposure (5 h), the T394A recept or mutant desensitized at a slower rate than MOR1. In contrast, T394A is mo re rapidly removed from the cell surface than MOR1, as determined by flow c ytometry using epitope-tagged receptors. This fast internalization was foll owed by immediate resensitization of T394A during 20 min of agonist removal while the wild-type MOR1 remained inactive. Similar to MOR1B, rapid intern alization and reactivation of T394A may explain its delayed desensitization . These findings suggest that T394 represents a negative regulatory signal for MOR1 internalization. Furthermore, phosphorylation of this threonine re sidue may influence the time course of mu opioid receptor resensitization.