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Site-directed affinity-labeling of delta opioid receptors by

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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 [-Ala2, Leu5]-enkephalyl-Cys(Npys)6 and [-Ala2,Leu(CH2SNpys)5]enkephalin, and dynorphin A analogues of [-Ala2,Cys(Npys)12]dynorphin A-(1-13) amide and [-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. [-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 [-Ala2,Cys(Npys)12]dynorphin A-(1-13) amide.

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Authors and Affiliations

  1. Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Sciences, Kyushu University, Fukuoka, 812-8581, Japan

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

  2. Division of Chemistry, Graduate School of Science, Hokkaido university, Sapporo, 060-0810, Japan

    Kazuyasu Sakaguchi

Authors
  1. Kaname Isozaki
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  2. Hidehiko Fukahori
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  3. Takeshi Honda
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  4. Naoto Shirasu
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  5. Kazushi Okada
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  6. Takeru Nose
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  7. Kazuyasu Sakaguchi
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  8. Yasuyuki Shimohigashi
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Isozaki, K., Fukahori, H., Honda, T. et al. Site-directed affinity-labeling of delta opioid receptors by. Int J Pept Res Ther 10, 511–522 (2003). https://doi.org/10.1007/s10989-004-2414-7

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  • DOI: https://doi.org/10.1007/s10989-004-2414-7

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