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

The recently identified 17-amino acid peptide nociceptin (orphanin FQ) is the endogenous ligand for the opioid receptor-like-1 (ORL-1) 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 opioid-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 G(i)/G(c) 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 β-adrenergic receptor kinase (βARKct), which specifically blocks Gβγ-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 (PI-3K) inhibitors wortmannin 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 μ-opioid receptor, [D-Ala2,N- MePhe4,Gly-ol]-enkephalin (DAMGO; a μ-opioid receptor-selective agonist) stimulated PTX-sensitive MAP kinase activation that was inhibited by wortmannin, LY294002, βARKct expression, or SOS-PRO expression but not affected by inhibition of PKC activity. These results indicate that both ORL- 1 (OFQR) and μ-opioid receptors mediate MAP kinase activation via a signaling pathway using the βγ-subunit of G(i), a PI-3K, and SOS, independent of PKC activity. In cells expressing both ORL-1 (OFQR) and μ- opioid 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-1 (OFQR) can modulate μ-opioid receptor signaling in a cellular system.