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 s electively activated by the recently discovered peptide nociceptin. To probe the functional homology amongst these receptors, a Chinese hams ter 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 ca used a 2-fold increase in the rate of [S-35]GTP gamma S binding to mem branes derived from CHO cells expressing the ORL1 receptor. Following incubation with nociceptin mitogen-activated protein kinase activity w as increased by 2-fold in cells expressing the ORL1 receptor. In non-t ransfected CHO cells, nociceptin had no effect on cAMP accumulation, t he rate of [S-35]GTP gamma S binding or mitogen-activated protein kina se activity. Human ORL1 receptors expressed in CHO cells selectively b ound [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 bin ding 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 o f opioid receptor-selective agonists and antagonists. However, high mi cromolar levels of opioid receptor-selective agents inhibited the bind ing. 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, respecti vely. These results imply that ORL1 is a G-protein-coupled receptor wi th functional as well as structural homology to opioid receptors. In a ddition, opioid receptor ligands may serve as starting templates for t he development of ORL1, specific ligands. (C) 1997 Elsevier Science B. V.