Transduction of Melatonin Receptor Activation in Cultured Cells

这项研究旨在验证褪黑激素 与细胞表面受体结合，该受体通过与 鸟嘌呤核苷酸结合蛋白Gi，减少cAMP产生， 蛋白激酶活性和蛋白磷酸化， 培养的黑素细胞胞浆素重排和黑素体迁移 两栖动物的黑色素细胞 分子机制的表征 褪黑激素在两栖动物黑色素细胞中的作用，一个简单而良好的 对褪黑激素有明显反应的特征性细胞系统 治疗，将提供一个重要的先例，可能导致一个 了解褪黑激素的作用机制， 系统. 拟议的研究是基于PI的发现， 百日咳毒素阻止了两栖类黑色素细胞中的褪黑激素作用， 基于PI开发的体外黑素细胞培养技术。 在拟议的研究中，褪黑激素诱导的重组， 细胞骨架成分的特点，通过研究固定和 用荧光细胞骨架处理的去污剂透化细胞 标记。 褪黑激素诱导的蛋白质磷酸化的变化， 通过使用SDS-PAGE分离掺入的蛋白质来评估 在褪黑激素治疗后使用放射性磷酸盐 蛋白激酶活性的放射酶分析。 本研究 将有助于理解褪黑激素的分子机制 从长远来看，这将表明药理学方法 用于纠正生物节律紊乱或用于同步 生物节律与特定的外部事件相一致。

The proposed research is designed to test the hypothesis that melatonin binds to a cell surface receptor which, through an interaction with the guanine nucleotide binding protein Gi, decreases cAMP production, protein kinase activity, and protein phosophorylation to cause rearrangement of the cytoskelton and melanosome migration in cultured amphibian melanophores. Characterization of the molecular mechanism of melatonin action in the amphibian melanophore, a simple and well characterized cellular system with a demonstrable response to melatonin treatment, will provide an important precedent that may lead to an understanding of melatonin's mechanism of action in more complex systems. The proposed research is based upon the PI's finding that pertussis toxin prevents melatonin action in amphibian melanophores and upon an in vitro melanophore culture technology developed by the PI. In the proposed research, melatonin-induced rearrangements of cytoskeletal components will be characterized, by studying fixed and detergent permeabilized cells treated with fluorescent cytoskeleton markers. Melatonin-induced changes in protein phosphorylation will be evaluated by employing SDS-PAGE to separate proteins incorporating radiophosphate after melatonin treatment and by employing radioenzymatic analysis of protein kinase activities. This research will lead to an understanding of the molecular mechanism of melatonin action which, in the long term, will suggest pharmacological approaches for correcting biological rhythm disorders or for synchronizing biological rhythms to coincide with specific external events.