Development of a novel photoaffinity probe for labeling nocistatin receptor

Highlights: • Biotin-(AC₅)₂-[Y⁶,azF¹⁴]bNST is a novel nocistatin photoaffinity probe. • Biotin-(AC₅)₂-[Y⁶,azF¹⁴]bNST inhibits the tactile pain allodynia. • Biotin-(AC₅)₂-[Y⁶,azF¹⁴]bNST-binding proteins are observed at 58 and 64 kDa. Nocistatin (NST) is a neuropeptide produced from the same precursor protein of opioid peptide nociceptin/orphanin FQ, and it is involved in a broad range of central functions including pain transmission in the nervous system. However, the composition and structure of the receptor(s) for NST remain unclear. Here, we developed NST photoaffinity probe to identify NST receptor. The NST photoaffinity probe contains an azide moiety for the tagging of the binding protein as well as biotin for protein detection. Intrathecal administration of a NST photoaffinity probe, biotin-(AC₅)₂-[Y⁶,azF¹⁴]bNST, inhibited the nociceptin/orphanin FQ-evoked tactile pain allodynia in a manner similar to that of NST. The biotin-(AC₅)₂-[Y⁶,azF¹⁴]bNST-binding proteins were primarily localized in the gray matter of the spinal cord. After photo-crosslinking of the protein complex with biotin-(AC₅)₂-[Y⁶,azF¹⁴]bNST, two dominant binding protein bands were observed at 58 and 64 kDa. Thus, biotin-(AC₅)₂-[Y⁶,azF¹⁴]bNST has pharmacological activity and is useful for characterizing the NST receptor.