Phosphorylation of Ser(363), Thr(370), and Ser(375) residues within the carboxyl tail differentially regulates mu-opioid receptor internalization

Prolonged activation of opioid receptors leads to their phosphorylation, de sensitization, internalization, and down-regulation. To elucidate the relat ionship between mu -opioid receptor (MOR) phosphorylation and the regulatio n of receptor activity, a series of receptor mutants was constructed in whi ch the 12 Ser/Thr residues of the COOH-terminal portion of the receptor wer e substituted to Ale, either individually or in combination, All these muta nt constructs were stably expressed in human embryonic kidney 293 cells and exhibited similar expression levels and ligand binding properties, Among t hose 12 Ser/Thr residues, Ser(363), Thr(370), and Ser(375) have been identi fied as phosphorylation sites, In the absence of the agonist, a basal phosp horylation of Ser(363) and Thr(370) was observed, whereas [D-Ala(2), Me-Phe (4), Gly(5)-ol] enkephalin (DAMGO)-induced receptor phosphorylation occurs at Thr(370) and Ser(375) residues, Furthermore, the role of these phosphory lation sites in regulating the internalization of MOR was investigated, The mutation of Ser(375) to Ala reduced the rate and extent of receptor intern alization, whereas mutation of Ser(363) and Thr(370) to Ala accelerated MOR internalization kinetics, The present data show that the basal phosphoryla tion of MOR could play a role in modulating agonist-induced receptor intern alization kinetics, Furthermore, even though mu -receptors and delta -opioi d receptors have the same motif encompassing agonist-induced phosphorylatio n sites, the different agonist-induced internalization properties controlle d by these sites suggest differential cellular regulation of these two rece ptor subtypes,