Abstract

Signaling of G protein-coupled receptors is terminated by phosphorylation of intracellular serine and threonine residues. Resensitization of these receptors requires internalization and subsequent dephosphorylation. We have recently shown that the resensitization rate of the rat μ opioid receptor (MOR) isoforms MOR1 and MOR1B is mainly determined by the amino acid composition of their alternatively spliced C-terminal tails. Upon agonist stimulation, MOR1B passes through an accelerated cycle of receptor endocytosis and reactivation, which in turn promotes a greater resistance to agonist-induced desensitization, as compared with MOR1. Given the fact that MOR1B lacks only one putative phosphorylation site (T394 of MOR1), we replaced this threonine 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-Ala², MePhe⁴, Gly⁵-ol]enkephalin exposure (5 h), the T394A receptor mutant desensitized at a slower rate than MOR1. In contrast, T394A is more rapidly removed from the cell surface than MOR1, as determined by flow cytometry using epitope-tagged receptors. This fast internalization was followed by immediate resensitization of T394A during 20 min of agonist removal while the wild-type MOR1 remained inactive. Similar to MOR1B, rapid internalization 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 residue may influence the time course of μ opioid receptor resensitization.