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

Research output: Contribution to journalArticle

2 Citations (Scopus)

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 [ 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
Volume19
Issue number24
DOIs
Publication statusPublished - Dec 15 2011

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Labeling
Ethylmaleimide
Cysteine
Binding Sites
Sulfhydryl Compounds
Ligands
Alkylation
Sulfhydryl Reagents
COS Cells
Opioid Receptors
Cell membranes
Disulfides
Assays
Cell Membrane
Peptides
nociceptin receptor
Population
nociceptin

All Science Journal Classification (ASJC) codes

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

Cite this

Capturing of the free cysteine residue in the ligand-binding site by affinity labeling of the ORL1 nociceptin receptor. / Matsushima, Ayami; Nishimura, Hirokazu; Inamine, Shogo; Uemura, Shiho; Shimohigashi, Yasuyuki.

In: Bioorganic and Medicinal Chemistry, Vol. 19, No. 24, 15.12.2011, p. 7597-7602.

Research output: Contribution to journalArticle

Matsushima, Ayami ; Nishimura, Hirokazu ; Inamine, Shogo ; Uemura, Shiho ; Shimohigashi, Yasuyuki. / Capturing of the free cysteine residue in the ligand-binding site by affinity labeling of the ORL1 nociceptin receptor. In: Bioorganic and Medicinal Chemistry. 2011 ; Vol. 19, No. 24. pp. 7597-7602.
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