Nociceptin activation of the human ORL1 receptor expressed in Chinese hamster ovary cells: Functional homology with opioid receptors

Opioid receptor-like 1 (ORL1) receptor, a member of the superfamily of G-protein-coupled receptors has significant primary sequence homology to the mu-, delta- and kappa-opioid receptors. The ORL1 receptor is selectively activated by the recently discovered peptide nociceptin. To probe the functional homology amongst these receptors, a Chinese hamster ovary (CHO) cell line expressing the human ORL1 receptor has been characterized. Nociceptin inhibited forskolin stimulated increases in intracellular cAMP with an IC50 of 70 pM. Stimulation by nociceptin caused a 2-fold increase in the rate of [S-35]GTP gamma S binding to membranes derived from CHO cells expressing the ORL1 receptor. Following incubation with nociceptin mitogen-activated protein kinase activity was increased by 2-fold in cells expressing the ORL1 receptor. In non-transfected CHO cells, nociceptin had no effect on cAMP accumulation, the rate of [S-35]GTP gamma S binding or mitogen-activated protein kinase activity. Human ORL1 receptors expressed in CHO cells selectively bound [I-125][Tyr(14)]nociceptin with a K-d of 2.1 pM and a B-max of 2.6 pmol/mg protein. Similar to opioid receptors, nociceptin binding to the ORL1 receptor was altered by Na+, GTP gamma S and dithiothreitol. Na+ increased the K-d of nociceptin binding to the ORL1 receptor. GTP gamma S decreased the apparent B-max of [I-125][Tyr(14)]nociceptin binding but had no effect on the K-d of the remaining sites. Pretreatment with dithiothreitol inhibited nociceptin binding to the ORL1 receptor. Nociceptin binding was insensitive to low nanomolar concentrations of opioid receptor-selective agonists and antagonists. However, high micromolar levels of opioid receptor-selective agents inhibited the binding. Morphine, naloxone, naltrindole, nor-Binaltorphimine and CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2) inhibited nociceptin binding to ORL1 receptor with K-1 values of 36, 24, 0.4, 8 and 28 mu M, respectively. These results imply that ORL1 is a G-protein-coupled receptor with functional as well as structural homology to opioid receptors. In addition, opioid receptor ligands may serve as starting templates for the development of ORL1, specific ligands. (C) 1997 Elsevier Science B.V.