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

研究成果: ジャーナルへの寄稿記事

4 引用 (Scopus)

抄録

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.

元の言語英語
ページ(範囲)460-469
ページ数10
ジャーナルBiopolymers
DOI
出版物ステータス出版済み - 11 4 2016

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Sulfhydryl Compounds
Labeling
Peptides
Ligands
Binding sites
Binding Sites
Scanning
Competitive Binding
COS Cells
Disulfides
Assays
Substitution reactions
nociceptin
nociceptin receptor
3-nitro-2-pyridinesulfenyl

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Biomaterials
  • Organic Chemistry

これを引用

Specific affinity-labeling of the nociceptin ORL1 receptor using a thiol-activated Cys(Npys)-containing peptide ligand. / Matsushima, Ayami; Nishimura, Hirokazu; Matsuyama, Yutaka; Liu, Xiaohui; Costa, Tommaso; Shimohigashi, Yasuyuki.

:: Biopolymers, 04.11.2016, p. 460-469.

研究成果: ジャーナルへの寄稿記事

Matsushima, Ayami ; Nishimura, Hirokazu ; Matsuyama, Yutaka ; Liu, Xiaohui ; Costa, Tommaso ; Shimohigashi, Yasuyuki. / Specific affinity-labeling of the nociceptin ORL1 receptor using a thiol-activated Cys(Npys)-containing peptide ligand. :: Biopolymers. 2016 ; pp. 460-469.
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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.",
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AU - Liu, Xiaohui

AU - Costa, Tommaso

AU - Shimohigashi, Yasuyuki

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