title: Capturing of the free cysteine residue in the ligand-binding site by affinity labeling of the ORL1 nociceptin receptor.
abstract: All of the δ, μ, and κ opioid receptors have a free thiol group of the Cys residue in the ligand-binding site, although its functional role is not yet known. In order to examine whether or not a similar Cys is also present in the ORL1 nociceptin receptor, we attempted to identify it by affinity labeling using a specific antagonist peptide. We first treated ORL1-expressing COS-7 cell membrane preparations with the thiol-alkylation reagent N-ethylmaleimide (NEM) to perform a binding assay using [(3)H]nociceptin as a tracer and nociceptin, an ORL1 agonist, or Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2), a nociceptin/ORL1 antagonist, as a competitor. It was suggested that ORL1 has a free Cys in its ligand-binding site, since the NEM treatment reduced the population of ligand-binding sites. This was further confirmed by affinity labeling using Cys(Npys)-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) with the SNpys group that can react with a free thiol group, resulting in the formation of a disulfide bond. This affinity labeling was approximately 23 times more specific than NEM alkylation. The results revealed that the ORL1 nociceptin receptor does contain a free Cys residue in the ligand-binding site.
authors: Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Sciences, and Risk Science Research Center, Kyushu University, Fukuoka 812-8581, Japan.
authors: Ayami
authors: A
authors: Matsushima
authors: Hirokazu
authors: H
authors: Nishimura
authors: Shogo
authors: S
authors: Inamine
authors: Shiho
authors: S
authors: Uemura
authors: Yasuyuki
authors: Y
authors: Shimohigashi
mesh_terms: D000345:Affinity Labels / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000032:analysis*; D005033:Ethylmaleimide / Q000737:chemistry*; D008024:Ligands; D009292:Narcotic Antagonists; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D011957:Receptors, Opioid / Q000819:agonists / Q000737:chemistry* / Q000378:metabolism; D000094942:Nociceptin Receptor
chemical_list: D000345:Affinity Labels; D008024:Ligands; D009292:Narcotic Antagonists; D010455:Peptides; D011957:Receptors, Opioid; D003545:Cysteine; D005033:Ethylmaleimide; D000094942:Nociceptin Receptor