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.
