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      Constitutive activation of the opioid receptor-like (ORL1) receptor by mutation of Asn133 to tryptophan in the third transmembrane region.

      Journal of Neurochemistry
      Amino Acid Substitution, Animals, Binding, Competitive, physiology, COS Cells, Cell Line, Cyclic AMP, antagonists & inhibitors, biosynthesis, GTP-Binding Proteins, metabolism, Humans, Inositol Phosphates, Kidney, cytology, Ligands, Mutagenesis, Site-Directed, Opioid Peptides, Radioligand Assay, Receptors, Opioid, genetics, Signal Transduction, Structure-Activity Relationship, Transfection

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          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.

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