Phosphorylation of the δ-opioid receptor regulates its β-arrestins selectivity and subsequent receptor internalization and adenylyl cyclase desensitization

In the current study, we investigated the role of receptor phosphorylation and β-arrestins in δ-opioid receptor (DOR) signaling and trafficking by using a DOR mutant in which all Ser/Thr residues in the C terminus were mutated to Ala (DTS). We demonstrated that the DOR agonist D-[Pen ²,Pen⁵]enkephalin could induce receptor internalization and adenylyl cyclase (AC) desensitization of DTS, but with comparatively slower kinetics than those observed with wild type DOR. Blockade of the internalization of DTS by the dominant-negative mutant dynamin, dynamin K44E, did not affect AC desensitization. However, depletion of β-arrestins almost totally blocked both internalization and AC desensitization of DTS. A BRET assay suggested that DOR phosphorylation promotes receptor selectivity for β-arrestin 2 over β-arrestin 1. Furthermore, in mouse embryonic fibroblast (MEF) cells lacking either β-arrestin 1 (βarr1^-/-) or β-arrestin 2 (βarr2^-/-), agonist-induced DTS desensitization and internalization were similar to that observed in wild type MEFs. In contrast, although DOR internalization decreased in both βarr1^-/- MEFs and βarr2^-/- MEFs, DPDPE-induced DOR desensitization was significantly reduced in βarr2^-/- MEFs, but not in βarr1^-/- MEFs. Additionally, the BRET assay suggested that depletion of phosphorylation did not influence the stability of the receptor-β-arrestin complex. Consistent with this observation, DTS did not recycle after internalization, which is like wild type DOR. Taken together, these results indicate that receptor phosphorylation confers DOR selectivity for β-arrestin 2 without affecting the stability of the receptor-β- arrestin complex and the fate of the internalized receptor.