Deltorphin II-induced rapid desensitization of delta-opioid receptor requires both phosphorylation and internalization of the receptor

Similar to other G protein-coupled receptors, rapid phosphorylation of the delta-opioid receptor in the presence of agonist has been reported. Hence, agonist-induced desensitization of the delta-opioid receptor has been sugge sted to be via the receptor phosphorylation, arrestin-mediated pathway. How ever, due to the highly efficient coupling between the delta-opioid recepto r and the adenylyl cyclase, the direct correlation between the rates of rec eptor phosphorylation and receptor desensitization as measured by the adeny lyl cyclase activity could not be established. In the current studies, usin g an ecdysone-inducible expression system to control the 6-opioid receptor levels in HEK293 cells, we could demonstrate that the rate of deltorphin II -induced receptor desensitization is dependent oil the receptor level. Only at receptor concentrations less than or equal to 90 fmol/mg of protein wer e rapid desensitizations (t(1/2) <10 min) observed. Apparently, deltorphin II-induced receptor desensitization involves cellular events in addition to receptor phosphorylation. Mutation of Ser(363) in the carboxyl tail of the 6-opioid receptor to Ala completely abolished the deltorphin II-induced re ceptor phosphorylation but not the desensitization response. Although the m agnitude of desensitization was attenuated, the rate of deltorphin II-induc ed receptor desensitization remained the same in the S363A mutant as compar ed with wild type. Also, the S363A mutant could internalize in the presence of deltorphin II. Only when the agonist-induced clathrin-coated pit-mediat ed receptor internalization was blocked by 0.4 M sucrose that the deltorphi n II-induced receptor desensitization was abolished in the S363A mutant. Si milarly, 0.4 M sucrose could partially block the agonist-induced rapid dese nsitization in HEK293 cells expressing the wild type delta-opioid receptor. Taken together, these data supported the hypothesis that rapid desensitiza tion of the delta-opioid receptor involves both the phosphorylation and the internalization of the receptor.