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

我们已标识为“nocistatin （NST）”。NST是从相同的预剂中衍生出来的，即Noceptin/orphanin FQ （Noc/OFQ），它是一种生物活动，由Noceptin/OFQ引起的Noceptive传输。Although NST并没有绑定到Noc/OFQ受体，它绑定到小鼠大脑和脊柱的膜。为了澄清NST对疼痛转移的抑制机制，我们已经对NST受体的CDNA克隆和信号转移分析以及两个肽的释放机制的分析。1.对肽释放机制的分析： （1） Noc/OFQ检测到人类、大鼠、小鼠和骨骼脑以及人类Cerebrospinal fluids中的所有肽，并展示了抗生素活性。（2）两个NST和Noc/OFQ都位于hypothalamus和免疫活性中，位于spinal dorsal horn、spinal trigeminal tract、paramedian rape Nucleus和ventromedial Nucleus。（3）我们生成的前体 ... 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响应能量转移（FRET）已应用于NST和Noc/OFQ的加工研究;当（CFP）的串联聚变蛋白-（NST·Noc/OFQ）-（YFP）已被转移到Cos 7个细胞中，使NST·Noc/OFQ的清除活性失效，绿色排放增加，指示FRET在fusion蛋白中发生的确切距离设计为FRET在两个端fluorescence蛋白之间的近似距离的定义。当串联融合蛋白被转染到NG 108 -15细胞时，在异常的CFP中，发射峰值从绿色到蓝色的转变表明NST和Noc/OFQ.2 NST受体的cDNA克隆和信号转换： （1） NST受体耦合到大脑和脊柱膜中的cAMP通路。（2） We have been trying to clone a cDNA of NST receptor using increase in cAMP as an indicator. Less（低）

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