Specific affinity-labeling of the nociceptin ORL1 receptor using a thiol-activated Cys(Npys)-containing peptide ligand

Ayami Matsushima, Hirokazu Nishimura, Yutaka Matsuyama, Xiaohui Liu, Tommaso Costa, Yasuyuki Shimohigashi

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

We previously showed that an antagonist-based peptide ligand, H-Cys(Npys)-Arg-Tyr-Tyr-Arg- Ile-Lys-NH2, captures the free thiol groups in the ligand-binding site of the nociceptin receptor ORL1. However, the exact receptor sites of this thiol-disulfide exchange reaction have not been uncovered, although such identification would help to clarify the ligand recognition site. Since the Cys→Ala substitution prevents the reaction, we performed the so-called Ala scanning for all the Cys residues in the transmembrane (TM) domains of the ORL1 receptor. Seven different mutant receptors were soundly expressed in the COS-7 cells and examined for their specific affinity labeling by a competitive binding assay using nociceptin and [3H]nociceptin. The results of in vitro Ala scanning analyses revealed that the labeled residues were Cys59 in TM1, Cys215 and Cys231 in TM5, and Cys310 in TM7. The present study has provided a novel method of Cys(Npys)-affinity labeling for identification of the ligand-binding sites in the ORL1 receptor.

Original languageEnglish
Pages (from-to)460-469
Number of pages10
JournalBiopolymers
DOIs
Publication statusPublished - Nov 4 2016

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Biomaterials
  • Organic Chemistry

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