Identification of serine 356 and serine 363 as the amino acids involved inetorphine-induced down-regulation of the mu-opioid receptor

Agonist-induced internalization of G protein-coupled receptors is influence d by many structural determinants including the carboxyl tail. To investiga te the role of serine and threonine residues within the carboxyl tail, seve ral mutants were constructed by truncating the carboxyl tail of the hemaggl utinin-tagged mu-opioid receptor, thereby removing serines and threonines s ystematically. Neuro(2A) cells stably expressing the truncated receptors di d not exhibit a significant alteration in the affinity of [H-3]diprenorphin e or etorphine for the receptor or the potency of etorphine to inhibit fors kolin-stimulated adenylyl cyclase activity. Chronic etorphine treatment res ulted in a Lime-dependent down-regulation of all the truncated receptors, e xcept MOR1TAG355D, thus revealing the importance of the four amino acids be tween Ser(355) and Glu(359) (STIE). Surprisingly, deletion of the STIE sequ ence resulted in a receptor that down-regulated the same as the wild-type r eceptor. The involvement of multiple amino acids within the carboxyl tail w as demonstrated by combining alanine substitutions of several putative G-pr otein-coupled receptor kinase phosphorylation sites. Systematic analysis of these receptors indicated that mutation of Ser(356) and Ser(363) to alanin e attenuated agonist-mediated down-regulation. The magnitude of etorphine-i nduced phosphorylation of this mutant receptor, however, was similar to tha t of the wild-type mu-opioid receptor. Thus, phosphorylation of the carboxy l tail of the mu-opioid receptor is not an obligatory event for etorphine-i nduced down-regulation of the receptor.