Nociceptin (ORL‐1) and μ‐Opioid Receptors Mediate Mitogen‐Activated Protein Kinase Activation in CHO Cells Through a G_i‐Coupled Signaling Pathway: Evidence for Distinct Mechanisms of Agonist‐Mediated Desensitization

Abstract

Abstract:The recently identified 17-amino acid peptide nociceptin (orphanin FQ) is the endogenous ligand for the opioid receptor-like-i (ORL-i) receptor. A physiologic role for nociceptin (OFQ) activation of the ORL-1 receptor (OFQR) may be to modulate opioid-induced analgesia. The molecular mechanism by which nociceptin (OFQ) and ORL-1 (OFQR) modify oploid-stimulated effects, however, is unclear. Both ORL-1 (OFQR) and opioid receptors mediate pertussis toxin (PTX)-sensitive signal transduction, indicating these receptors are capable of coupling to GIG0 proteins. This study determines that nociceptin stimulates an intracellular signaling pathway, leading to activation of mitogen-activated protein (MAP) kinase in CHO cells expressing ORL-1 receptor (OFQR). Nociceptin (OFQ)-stimulated MAP kinase activation was inhibited by PTX or by expression of the carboxyl terminus of /3-adrenergic receptor kinase (/3ARKct), which specifically blocks G/3y-mediated signaling. Expression of the proline-rich domain of SOS (SOS-PRO), which inhibits SOS interaction with p21 ras also attenuated nociceptin (OFQ)-stimulated MAP kinase activation. The phosphatidylinositol 3-kinase (P1-3K) inhibitors wortmannm and LY294002 reduced nociceptin (OFQ)-stimulated MAP kinase activation, whereas inhibition of protein kinase C (PKC) activity by bisindolylmaleimide I or cellular depletion of PKC had no effect. In a similar manner, in cells expressing~a-opioid receptor, [D-A1a 2,N-MePhe4 ,Gly-ol] -enkephalin (DAMGO; a p-opioid receptorselective agonist) stimulated PTX-sensitive MAP kinase activation that was inhibited by wortmannin, LY294002, /3ARKct expression, or SOS-PRO expression but not affected by inhibition of PKC activity. These results indicate that both ORL-1 (OFQR) and 1i-opioid receptors mediate MAP kinase activation via a signaling pathway using the /3y-subunit of G, a P1-3K, and SOS, independent of PKC activity. In cells expressing both ORL-i (OFQR) and popioid receptors, pretreatment with nociceptin decreased subsequent nociceptin (OFQ)-or DAMGO-stimulated MAP kinase activation. In contrast, pretreatment of cells with DAMGO decreased subsequent DAMGO-stimulated MAP kinase but had no effect on subsequent nociceptin (OFQ)-stimulated MAP kinase activation. These results demonstrate that nociceptin (OFQ) activation of ORL-i (OFQR) can modulate~a-opioid receptor signaling in a cellular system. Key Words: Nociceptin-p-Opioid receptors-Mitogen-activated protein kinase -GIG0 protein-Pertussis toxin. J. Neurochem. 71, 1024Neurochem. 71, -1033Neurochem. 71, (1998.Nociceptin (orphanin FQ, OFQ) has recently been identified as the endogenous ligand for the orphan opioid receptor-like-I (ORL-l) receptor (Meunier et al., 1995;Reinscheid et al., 1995). The amino acid sequence of the ORL-l receptor (OFQR) is 47% identical to the opioid receptors (~t-, K-, and h-receptors) overall, and is 64% identical in the transmembrane domains (Bunzow et al.,

μ-Opioid-Induced Desensitization of Opioid Receptor-Like 1 and μ-Opioid Receptors: Differential Intracellular Signaling Determines Receptor Sensitivity

“…Previous reports of apparent failures of -agonist pretreatment to alter OFQ/N activity were based upon the unchanging activity of a single, maximal concentration of OFQ/N (Connor et al, 1996;Hao et al, 1997;Hawes et al, 1998). It is quite possible in those studies that pretreatment with the -agonist reduced the potency of OFQ/N without altering its efficacy, as we similarly noted .…”

“…The ability of -agonist pretreatment to alter ORL1 responsiveness varies between cell lines (Hawes et al, 1998;Mandyam et al, 2000) and brain regions (Connor et al, 1996;Hao et al, 1997;Jhamandas et al, 1998). Similarly, -receptor regulation differs between cell populations (Zhang et al, 1996;Kramer and Simon, 1999;Li and Wang, 2001) and supraspinal and spinal sites (Bohn et al, 2002).…”

“…Acute OFQ/N, but not DAMGO, pretreatment decreased OFQ/N-and DAMGO-mediated stimulation of extracellular signal-regulated kinase (ERK)1/2 activity in Chinese hamster ovary (CHO) cells expressing recombinant -and ORL1 (Hawes et al, 1998). DAMGO pretreatment desensitized only -mediated stimulation of ERK1/2.…”

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

“…Although the same treatment of SH-SY5Y cells was ineffective in reducing N/OFQ signaling (Mandyam et al, 2000(Mandyam et al, , 2003. Likewise, in CHO or HEK 293 overexpressing recombinant NOP and mu receptors, mu receptor activation had no effect on NOP receptor-mediated stimulation of ERK1/2 (Hawes et al, 1998) or inhibition of cAMP . This is probably due to differences in signal transduction components native to these cell lines.…”

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

“…OFQ/N binds to NOP receptors and activates protein kinase C, which plays a role in homologous NOP receptor desensitization (Lou et al, 1997;Pei et al, 1997;Pu et al, 1999;Mandyam et al, 2002). Besides homologous NOP receptor desensitization, activation of NOP receptors by OFQ/N heterologously regulates the receptor response to DAMGO (Hawes et al, 1998;Mandyam et al, 2000Mandyam et al, , 2003Thakker and Standifer, 2002).…”