Fawzi A B, Zhang H, Weig B, Hawes B, Graziano M P
Department of CNS and Cardiovascular Research, Schering-Plough Research Institute, Kenilworth, NJ 07033, USA.
Eur J Pharmacol. 1997 Oct 8;336(2-3):233-42. doi: 10.1016/s0014-2999(97)01227-2.
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 [35S]GTPgammaS 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 [35S]GTPgammaS binding or mitogen-activated protein kinase activity. Human ORL1 receptors expressed in CHO cells selectively bound [125I][Tyr14]nociceptin with a Kd of 2.1 pM and a Bmax of 2.6 pmol/mg protein. Similar to opioid receptors, nociceptin binding to the ORL1 receptor was altered by Na+, GTPgammaS and dithiothreitol. Na+ increased the Kd of nociceptin binding to the ORL1 receptor. GTPgammaS decreased the apparent Bmax of [125I][Tyr14]nociceptin binding but had no effect on the Kd 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 Ki values of 36, 24, 0.4, 8 and 28 microM, 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.
阿片样物质受体样1（ORL1）受体是G蛋白偶联受体超家族的成员，与μ、δ和κ阿片样物质受体具有显著的一级序列同源性。ORL1受体被最近发现的肽痛敏肽选择性激活。为了探究这些受体之间的功能同源性，对表达人ORL1受体的中国仓鼠卵巢（CHO）细胞系进行了特性分析。痛敏肽抑制福斯高林刺激引起的细胞内cAMP增加，IC50为70 pM。痛敏肽刺激使[35S]GTPγS与表达ORL1受体的CHO细胞膜结合速率增加2倍。与痛敏肽孵育后，表达ORL1受体的细胞中丝裂原活化蛋白激酶活性增加2倍。在未转染的CHO细胞中，痛敏肽对cAMP积累、[35S]GTPγS结合速率或丝裂原活化蛋白激酶活性无影响。在CHO细胞中表达的人ORL1受体选择性结合[125I][Tyr14]痛敏肽，Kd为2.1 pM，Bmax为2.6 pmol/mg蛋白。与阿片样物质受体类似，痛敏肽与ORL1受体的结合受Na+、GTPγS和二硫苏糖醇的影响。Na+增加痛敏肽与ORL1受体结合的Kd。GTPγS降低[125I][Tyr14]痛敏肽结合的表观Bmax，但对其余位点的Kd无影响。用二硫苏糖醇预处理可抑制痛敏肽与ORL1受体的结合。痛敏肽结合对低纳摩尔浓度的阿片样物质受体选择性激动剂和拮抗剂不敏感。然而，高微摩尔水平的阿片样物质受体选择性药物可抑制结合。吗啡、纳洛酮、纳曲吲哚、去甲二丙诺啡和CTAP（D-苯丙氨酸-半胱氨酸-酪氨酸-D-色氨酸-精氨酸-苏氨酸-青霉胺-苏氨酸-NH2）抑制痛敏肽与ORL1受体结合，Ki值分别为36、24、0.4、8和28 μM。这些结果表明ORL1是一种与阿片样物质受体具有功能和结构同源性的G蛋白偶联受体。此外，阿片样物质受体配体可能作为开发ORL1特异性配体的起始模板。
