Hierarchical phosphorylation of delta-opioid receptor regulates agonist-induced receptor desensitization and internalization

Treatment of HEK293 cells expressing the delta -opioid re ceptor with agoni st [D-Pen(2,5)]enkephalin (DPDPE) resulted in the rapid phosphorylation of the receptor, We constructed several mutants of the potential phosphorylati on sites (Ser/Thr) at the carboxyl tail of the receptor in order to delinea te the receptor phosphorylation sites and the agonist-induced desensitizati on and internalization. The Ser and Thr were substituted to alanine, and th e corresponding mutants were transiently and stably expressed in HEK293 cel ls. We found that only two residues, i.e. Thr(358) and Ser(363), were phosp horylated, with Ser(363) being critical for the DPDPE-induced phosphorylati on of the receptor. Furthermore, using alanine and aspartic acid substituti ons, we found that the phosphorylation of the receptor is hierarchical, wit h Ser(363) as the primary phosphorylation site. Here, we demonstrated that DPDPE-induced rapid receptor desensitization, as measured by adenylyl cycla se activity, and receptor internalization are intimately related to phospho rylation of Thr(358) and Ser(363), with Thr(358) being involved in the rece ptor internalization.