Transduction of Melatonin Receptor Activation in Cultured Cells

这项拟议的研究旨在检验一种假设，即褪黑素与细胞表面受体结合，通过与鸟核苷酸结合蛋白Gi的相互作用，降低cAMP产生、蛋白激酶活性和蛋白质磷酸化，从而导致培养的两栖黑素载体中细胞kelton的重排和黑素小体的迁移。褪黑素在两栖黑素载体中作用的分子机制的表征，将提供一个重要的先例，可能有助于理解褪黑素在更复杂系统中的作用机制。这项拟议的研究是基于国际百日咳毒素阻止两栖类黑素载体中褪黑激素作用的发现，以及国际百日咳毒素研究所开发的体外黑素载体培养技术。在拟议的研究中，将通过研究荧光细胞骨架标记物处理的固定和洗涤剂渗透性细胞来表征褪黑素诱导的细胞骨架成分的重排。通过使用SDS-PAGE分离褪黑素处理后含有放射性磷酸盐的蛋白质和通过放射酶分析蛋白激酶活性来评估褪黑素诱导的蛋白质磷酸化的变化。这项研究将有助于理解褪黑素作用的分子机制，从长远来看，这将为纠正生物节律紊乱或使生物节律与特定的外部事件同步提供药理学方法。

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.