Phosphorylation of Ser363, Thr370, and Ser 375 Residues within the Carboxyl Tail Differentially Regulates μ-Opioid Receptor Internalization

Abstract

Prolonged activation of opioid receptors leads to their phosphorylation, desensitization, internalization, and down-regulation. To elucidate the relationship between μ-opioid receptor (MOR) phosphorylation and the regulation of receptor activity, a series of receptor mutants was constructed in which the 12 Ser/Thr residues of the COOH-terminal portion of the receptor were substituted to Ala, either individually or in combination. All these mutant constructs were stably expressed in human embryonic kidney 293 cells and exhibited similar expression levels and ligand binding properties. Among those 12 Ser/Thr residues, Ser363, Thr370, and Ser 375 have been identified as phosphorylation sites. In the absence of the agonist, a basal phosphorylation of Ser363 and Thr370 was observed, whereas [D-Ala2,Me-Phe4,Gly 5-ol]enkephalin (DAMGO)-induced receptor phosphorylation occurs at Thr370 and Ser375 residues. Furthermore, the role of these phosphorylation sites in regulating the internalization of MOR was investigated. The mutation of Ser375 to Ala reduced the rate and extent of receptor internalization, whereas mutation of Ser363 and Thr370 to Ala accelerated MOR internalization kinetics. The present data show that the basal phosphorylation of MOR could play a role in modulating agonist-induced receptor internalization kinetics. Furthermore, even though μ-receptors and δ-opioid receptors have the same motif encompassing agonist-induced phosphorylation sites, the different agonist-induced internalization properties controlled by these sites suggest differential cellular regulation of these two receptor subtypes.