Site-directed affinity-labeling of delta opioid receptors by SNpys-containing enkephalin and dynorphin analogues

Kaname Isozaki, Hidehiko Fukahori, Takeshi Honda, Naoto Shirasu, Kazushi Okada, Takeru Nose, Kazuyasu Sakaguchi, Yasuyuki Shimohigashi

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6 Citations (Scopus)

Abstract

The S-3-nitro-2-pyridinesulfenyl (SNpys) group in an affinity ligand can bind to a free thiol group of a cysteine residue in a target receptor molecule, forming a disulfide bond via the thiol-disulfide exchange reaction. SNpys-containing Leu-enkephalin analogues of [D-Ala2, Leu 5]-enkephalyl-Cys(Npys)6 and [D-Ala2, Leu(CH2SNpys)5]enkephalin, and dynorphin A analogues of [D-Ala2,Cys(Npys)12]dynorphin A-(1-13) amide and [D-Ala2,Cys(Npys)8]dynorphin A-(1-9) amide have been found to affinity-label all of the δ, μ (rat brain), and κ (guinea pig brain) opioid receptor subtypes. In this study, using these chemically synthesized SNpys-containing analogues, we attempted to identify the analogues that affinity-label the cysteine residue at position 60 of the δ opioid receptor. We first established the assay procedure, principally based on the receptor binding assay to use COS-7 cells expressing the δ opioid receptor. Then, using a mutant δ receptor with the Cys60→Ala substitution, we assayed the SNpys-containing analogues for their specific affinity-labeling. [D-Ala2,Cys(Npys)12]dynorphin A-(1-13) amide was found to have drastically reduced labeling activity for this mutant receptor as compared to its activity for the wild-type δ receptor. Other analogues exhibited almost the same activity for both the wild-type and mutant δ receptors. These results indicate that the δ-Cys60 residue has a free thiol group, which is labeled by [D-Ala2,Cys(Npys) 12]dynorphin A-(1-13) amide.

Original languageEnglish
Pages (from-to)511-522
Number of pages12
JournalLetters in Peptide Science
Volume10
Issue number5-6
DOIs
Publication statusPublished - Nov 2003

All Science Journal Classification (ASJC) codes

  • Biochemistry

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    Isozaki, K., Fukahori, H., Honda, T., Shirasu, N., Okada, K., Nose, T., Sakaguchi, K., & Shimohigashi, Y. (2003). Site-directed affinity-labeling of delta opioid receptors by SNpys-containing enkephalin and dynorphin analogues. Letters in Peptide Science, 10(5-6), 511-522. https://doi.org/10.1007/BF02442583