Differential regulation of the human kappa opioid receptor by agonists: etorphine and levorphanol reduced dynorphin A- and U50,488H-induced internalization and phosphorylation.

We previously observed that (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide (U50,488H) promoted internalization and phosphorylation of the FLAG-tagged human kappa opioid receptor (FLAG-hkor) stably expressed in Chinese hamster ovary (CHO) cells. In this study, we compared regulation of the FLAG-hkor expressed in CHO cells by U50,488H, dynorphin A, etorphine, and levorphanol, which were potent full agonists as determined by stimulation of guanosine 5'-O-(3-[(35)S]thio)triphosphate binding. Using fluorescence flow cytometry, we found that dynorphin A(1-17), like U50,488H, promoted internalization of the FLAG-hkor in a time- and dose-dependent manner. The antagonists naloxone and norbinaltorphimine, having no effect on FLAG-hkor internalization, effectively blocked dynorphin A(1-17)- and U50,488H-induced internalization. Interestingly, the full agonists etorphine and levorphanol did not cause internalization of the FLAG-hkor but significantly reduced dynorphin A(1-17)- and U50,488H-induced internalization in a dose-dependent manner. Immunofluorescence staining of FLAG-hkor yielded similar results. Dynorphin A(1-17) and U50,488H enhanced phosphorylation of FLAG-hkor to a greater extent than etorphine, but levorphanol did not increase FLAG-hkor phosphorylation. Etorphine or levorphanol decreased dynorphin- or U50,488H-induced phosphorylation. It is likely that conformations of the hkor required for phosphorylation and initiation of internalization are different from those for activation of G proteins. We also examined whether the four agonists had differential effects on superactivation of adenylate cyclase. Pretreatment with U50,488H, dynorphin A(1-17), or etorphine enhanced forskolin-stimulated adenylate cyclase activity to approximately 200 to 250% of the control, whereas levorphanol pretreatment did not result in significant adenylate cyclase superactivation. Thus, the degree of superactivation caused by an agonist is unrelated to its ability to promote internalization of the hkor.