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 -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 receptor desensitization. Because phosphorylation of the 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-Ala 2 ,N-MePhe 4 ,Gly 5 -ol]-enkephalin (DAMGO)-induced receptor phosphorylation in BE(2)-C human neuroblastoma cells transfected with epitope-tagged receptors. OFQ/N treatment (100 nM, 60 min) potentiated DAMGO-induced receptor phosphorylation; inhibition of GRK2 or protein kinase C concomitant with OFQ/N treatment blocked the OFQ/N-mediated increase in DAMGOinduced 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 receptors in which the GRK-mediated phosphorylation site Ser 375 was mutated to alanine, OFQ/N treatment failed to potentiate DAMGOinduced receptor phosphorylation and failed to desensitize the receptor. However, DAMGO-induced receptor phosphorylation and OFQ/N-induced receptor desensitization occurred in cells expressing 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 receptor, heterologously regulating homologous receptor desensitization.Receptor agonists acting through -opioid receptors are widely used analgesics in the treatment of severe pain, despite the fact that long-term treatment with these drugs results in the development of tolerance and dependence. At the molecular level, receptor desensitization or loss of receptor function has been suggested to be the underlying reason for the development of tolerance to receptor agonists (Harrison et al., 1998).Like many GPCRs, the receptor can undergo homologous and heterologous receptor desensitization. Homologous receptor desensitization occurs when a cognate agonist leads to a decrease in the receptor responsiveness through the induction of regulatory events such as phosphorylation, internalization, and down-regulation of the receptor, and it often involves G-protein-coupled receptor kinases (GRKs). Agonist binding to receptor stimulates G proteins, and the ␤␥ subunits of activated G proteins recruit GRKs to the plasma membrane, in which GRKs can phosphorylate agonistoccupied receptors (Lefkowitz et al., 1998). Several GRK2-mediated phosphorylation sites of the receptor have been identified, but Ser 375 seems to be the primary site for DAMGO-induced receptor phosphoryla...

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