Elise Camacho, Benjamin Aguila, Nicolas Elie, Jana Sopkova, Caroline Marte, Audrey Davis, Cédric Lecoutey and Stéphane Allouche
Study background: We previously revealed that the arrestin 2 was differentially involved in the regulation of the human delta opioid receptor (hDOR) by peptidic and alkaloid agonists. In the present study, we examined whether i) hDOR regulation by two related alkaloid agonists (etorphine and morphine) involved the same arrestins ii) similar arrestin-dependent mechanisms occur upon short- and long-term agonist exposure. Methods: The human neuroblastoma SK-N-BE cells, endogenously expressing hDOR, were transfected with wild-type or mutant arrestins. Using shRNA, we also generated a clonal cell line depleted in arrestin 2, the only arrestin isoform expressed in this cell line. Cells were then exposed or not to either morphine ou etorphine for short- (1 h) or long-term (18 h), then receptor desensitization was examined on the cAMP pathway and internalization was visualized by confocal microscopy. Results: In arrestin 2-depleted cells, we observed a strong reduction of desensitization after 18 h of morphine exposure but not with etorphine. Over-expression of wild-type or mutant arrestins produced an opposite modulation of receptor desensitization induced by morphine and etorphine both upon short- and long-term exposure. Confocal fluorescence microscopy experiments did not reveal any strong impact on receptor internalization when different arrestins were over-expressed. Conclusion: Our results showed that i) two related opioid alkaloid agonists produce hDOR desensitization by different mechanisms ii) the involvement of arrestins in hDOR desensitization depends on the duration of agonist exposure.