title: Constitutive activation of the opioid receptor-like (ORL1) receptor by mutation of Asn133 to tryptophan in the third transmembrane region.
abstract: We have introduced a series of point mutations into the human opioid receptor-like (ORL1) receptor and characterized them for their ability to constitutively activate G protein-coupled receptor signalling pathways. Among the 12 mutants generated, mutation at Asn133 (N133W) gave increased basal signalling through three separate pathways. N133W increased the basal activity of G14- and G16-dependent pathways by two- to three-fold. The constitutive activity of the mutant was confirmed by the finding that the enhanced activity is dependent on the level of receptor expression. In HEK-293 cells stably expressing N133W, signalling through Gi/o-dependent pathways was also observed. Radioligand binding studies revealed that the affinity for nociceptin of the wild-type ORL1 receptor and the N133W mutant do not differ significantly, suggesting that the ligand binding and signalling functions of constitutively active mutants of G protein-coupled receptors are not necessarily intrinsically linked. In conclusion, our results demonstrate that a mutation in the third transmembrane domain is able to increase the basal signalling activity of the human ORL1 receptor.
authors.affiliation: Department of Biochemistry, Molecular Neuroscience Center, and the Biotechnology Research Institute, Hong Kong University of Science and Technology, Clearwater Bay, Hong Kong, China.
authors.forename: Kenneth W L
authors.initials: KW
authors.lastname: Kam
authors.forename: David C
authors.initials: DC
authors.lastname: New
authors.forename: Yung H
authors.initials: YH
authors.lastname: Wong
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001667:Binding, Competitive / Q000502:physiology; D019556:COS Cells; D002460:Cell Line; D000242:Cyclic AMP / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D018847:Opioid Peptides / Q000378:metabolism; D011869:Radioligand Assay; D011957:Receptors, Opioid / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013329:Structure-Activity Relationship; D014162:Transfection; D000094942:Nociceptin Receptor; D000097629:Nociceptin
chemical_list: D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D007295:Inositol Phosphates; D008024:Ligands; D018847:Opioid Peptides; D011957:Receptors, Opioid; D000094942:Nociceptin Receptor; D000097629:Nociceptin; C000722283:OPRL1 protein, human