Elucidation of receptor and releasing mechanism of a novel peptide nocistatin that regulates pain transmission

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We identified a novel neuropeptide and named it "nocistatin (NST)". NST is derived from the same precursor as nociceptin/orphanin FQ (Noc/OFQ) and it has the biological activity which blocks Noc/OFQ-evoked nociceptive transmission. Although NST did not bind to the Noc/OFQ receptor, it bound to the membrane of mouse brain and spinal cord. To clarify the inhibitory mechanism of pain transmission by NST, we carried out cDNA cloning and signal transduction analysis of NST receptor and analysis of releasing mechanism of the two peptides.1.Analysis of peptide-releasing mechanism : (1) Noc/OFQ was detected in human, rat, mouse and bovine brain and human cerebrospinal fluids, and all peptides showed the antinociceptive activity. (2) Both NST and Noc/OFQ were abundant in the hypothalamus and the immunoreactivity was located in the superficial laminae of spinal dorsal horn, the spinal trigeminal tract, paramedian raphe nucleus and ventromedial nucleus of hypothalamus. (3) We generated precursor … More proteins by fusing the carboxyl terminus of NST to the GFP mutant YFP and that of Noc/OFQ to the other GFP mutant CFP.Both YFP-tagged NST and CFP-tagged Noc/OFQ proteins were detected in neurites and the cytoplasm of NG108-15 cells which express the precursor mRNA.(4) Fluorescence response energy transfer (FRET) was applied to the study of processing of NST and Noc/OFQ.When the tandem fusion protein of (CFP)-(NST・Noc/OFQ)-(YFP) was transfected into Cos7 cells devoid of cleavage activity of NST・Noc/OFQ, green emission increased, indicating that FRET occurred in the fusion protein with the proximate distance designed for induction of FRET between the two end fluorescent protein. When the tandem fusion protein was transfected into NG108-15 cells, the shift of emission peak from green to blue in the excited CFP indicates the processing of NST and Noc/OFQ.2.cDNA cloning and signal transduction of NST receptor : (1) NST receptor was coupled to the cAMP pathway in brain and spinal membranes. (2) We have been trying to clone a cDNA of NST receptor using increase in cAMP as an indicator. Less