Capturing of the free cysteine residue in the ligand-binding site by affinity labeling of the ORL1 nociceptin receptor

Ayami Matsushima, Hirokazu Nishimura, Shogo Inamine, Shiho Uemura, Yasuyuki Shimohigashi

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2 Citations (Scopus)


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 [ 3H] 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.

Original languageEnglish
Pages (from-to)7597-7602
Number of pages6
JournalBioorganic and Medicinal Chemistry
Issue number24
Publication statusPublished - Dec 15 2011

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmaceutical Science
  • Drug Discovery
  • Clinical Biochemistry
  • Organic Chemistry


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