Identification of serine 356 and serine 363 as the amino acids involved in etorphine-induced down-regulation of the μ-opioid receptor

Agonist-induced internalization of G protein-coupled receptors is influenced by many structural determinants including the carboxyl tail. To investigate the role of serine and threonine residues within the carboxyl tail, several mutants were constructed by truncating the carboxyl tail of the hemagglutinin-tagged μ-opioid receptor, thereby removing serines and threonines systematically. Neuro(2A) cells stably expressing the truncated receptors did not exhibit a significant alteration in the affinity of [³H]diprenorphine or etorphine for the receptor or the potency of etorphine to inhibit forskolin-stimulated adenylyl cyclase activity. Chronic etorphine treatment resulted in a time-dependent down-regulation of all the truncated receptors, except MOR1TAG355D, thus revealing the importance of the four amino acids between Set³⁵⁵ and Glu³⁵⁹ (STIE). Surprisingly, deletion of the STIE sequence resulted in a receptor that down-regulated the same as the wild-type receptor. The involvement of multiple amino acids within the carboxyl tail was demonstrated by combining alanine substitutions of several putative G-protein-coupled receptor kinase phosphorylation sites. Systematic analysis of these receptors indicated that mutation of Set³⁵⁶ and Ser³⁶³ to alanine attenuated agonist-mediated down-regulation. The magnitude of etorphine-induced phosphorylation of this mutant receptor, however, was similar to that of the wild-type μ-opioid receptor. Thus, phosphorylation of the carboxyl tail of the μ-opioid receptor is not an obligatory event for etorphine-induced down-regulation of the receptor.