Abstract

In this study, we investigate the molecular mechanisms by which acute orphanin FQ/nociceptin (OFQ/N), acting through the nociceptin opioid peptide (NOP) receptor, desensitizes the μ-opioid receptor. We described previously the involvement of protein kinase C and G-protein-coupled receptor kinases (GRK) 2 and 3 in OFQ/N-induced μ receptor desensitization. Because phosphorylation of the μ receptor triggers the successive regulatory mechanisms responsible for desensitization, such as receptor uncoupling, internalization, and down-regulation, we investigated the ability of OFQ/N to modulate [d-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO)-induced μ receptor phosphorylation in BE(2)-C human neuroblastoma cells transfected with epitope-tagged μ receptors. OFQ/N treatment (100 nM, 60 min) potentiated DAMGO-induced μ receptor phosphorylation; inhibition of GRK2 or protein kinase C concomitant with OFQ/N treatment blocked the OFQ/N-mediated increase in DAMGO-induced phosphorylation. Inclusion of the NOP antagonist peptide III-BTD during OFQ/N pretreatment blocked the potentiation of DAMGO-induced phosphorylation by OFQ/N, which is consistent with the potentiation being mediated via actions of the NOP receptor. In addition, in cells expressing μ receptors in which the GRK-mediated phosphorylation site Ser³⁷⁵ was mutated to alanine, OFQ/N treatment failed to potentiate DAMGO-induced μ receptor phosphorylation and failed to desensitize the μ receptor. However, DAMGO-induced μ receptor phosphorylation and OFQ/N-induced μ receptor desensitization occurred in cells expressing μ receptors lacking non-GRK phosphorylation sites. These data suggest that OFQ/N binds to NOP receptors and activates protein kinase C, which then increases the ability of GRK2 to phosphorylate the agonist-occupied μ receptor, heterologously regulating homologous μ receptor desensitization.