Orphanin FQ/Nociceptin Potentiates [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-Enkephalin–Induced μ-Opioid Receptor Phosphorylation

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 -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...

GRK2 Protein-mediated Transphosphorylation Contributes to Loss of Function of μ-Opioid Receptors Induced by Neuropeptide FF (NPFF2) Receptors

“…Such indirect regulation has been described in the case of MOP and nociceptin receptors in BE(2)-C neuroblastoma cells, where activation of nociceptin receptors potentiates DAMGOinduced phosphorylation of the MOP receptor by GRK2, by a mechanism that necessitates PKC activation and the presence of the MOP agonist (58). In our case, the fact that inhibitors of several protein kinases (PKC, PKA, Src, or EGF receptor tyrosine kinase), all known to stimulate GRK2 activity (44,45), did not inhibit the 1DMe-induced phosphorylation of the MOP receptor indicates that GRK2 is directly involved in this heterologous regulation.…”

Nociceptin/Orphanin FQ Receptor Structure, Signaling, Ligands, Functions, and Interactions with Opioid Systems

“…In BE(2)-C cells, short treatment with N/OFQ induces translocation of PKCa, GRK2, and GRK3 to the plasma membrane. The increase in GRK2 levels at the plasma membrane resulted in enhanced DAMGO-mediated mu receptor phosphorylation and a resultant increased desensitization (Mandyam et al, 2002;Ozsoy et al, 2005). Prolonged N/OFQ treatment reduced the ability of mu agonists to inhibit cAMP accumulation in BE(2)-C and SH-SY5Y cells (Thakker and Standifer, 2002a), although N/OFQ treatment had no effect on the ability of mu agonists to activate ERK1/2 (Thakker and Standifer, 2002b).…”

Cellular Mechanisms of Nociceptin/Orphanin FQ (N/OFQ) Peptide (NOP) Receptor Regulation and Heterologous Regulation by N/OFQ

“…Because of its ability to modulate morphine analgesia and morphine tolerance, it is important to understand how N/OFQ can regulate the MOR. Indeed, N/OFQ pretreatment produced MOR desensitization (Hawes et al, 1998;Mandyam et al, 2000Mandyam et al, , 2002Thakker and Standifer, 2002a ;Ozsoy et al, 2005), phosphorylation (Ozsoy et al, 2005), internalization (Evans et al, 2010), and downregulation (Mandyam et al, 2002) in cells endogenously expressing and stably transfected with NOP receptor. However, mechanisms of N/OFQ-mediated MOR desensitization also are cell, time, and signal pathway specific.…”

“…However, N/OFQ-induced activation of PKCa does promote the membrane translocation (Mandyam et al, 2000) and phosphorylation (Ozsoy et al, 2005) of GRK2/3, resulting in GRK2/3-mediated desensitization of the NOP receptor (Mandyam et al, 2002). GRKs phosphorylate serine residues 334 and 335 on the C-terminal tail of the rat NOP receptor (corresponding to Ser 337 in the human NOP receptor) (Fig.…”

“…PKC also plays an important role in mediating NOP receptor desensitization. N/OFQ binding to the NOP receptor activates PKC, as demonstrated by translocation of cytosolic PKC to the plasma membrane and increased phosphorylation of proteins by PKC in cell lines that endogenously express or have been transfected to stably express the NOP receptor (see Table 1 for specific names of cell lines, phenotype, level of expression, and use) (Lou et al, 1997;Pei et al, 1997;Pu et al, 1999;Mandyam et al, 2002;Ozsoy et al, 2005). Pretreatment with the phorbol ester, phorbol 12-myristate 13-acetate, a PKC activator, facilitates the acute desensitization of the NOP receptor (Pu et al, 1999).…”