Melatonin is a pineal hormone with potent biological effects. The experiments proposed in this application represent a focused effort, applying contemporary anatomical, biochemical and molecular techniques to bring our level of understanding of melatonin action up to that of other hormones and neurotransmitters. Our long-term goals include defining the structure of a melatonin receptor and understanding the cellular events of melatonin signal transduction. First, using enriched receptor sources, melatonin receptors will be identified by covalent labeling, purified, and more rigorously characterized in terms of receptor-G protein interactions. Second, using the polymerase chain reaction with degenerate cloned. This will allow stable expression of the receptor protein in a cell line, and examination of structure-function relationships and transcriptional regulation of melatonin receptors. Third, melatonin receptor antibodies and in situ hybridization methods will be developed to study experimental issues regarding the cellular and subcellular distribution of melatonin receptor. Fourth, the intracellular effector systems influenced by melatonin will be fully investigated in dispersed ovine pars tuberalis cells and cells stably transfected with a cDNA encoding a melatonin receptor. This will include examination of acute melatonin effects on chemical second messenger systems, ionic responses and protein phosphorylation. Furthermore, the effects of melatonin on secretory outputs from pars tuberalis will be explored. Fifth, experiments are proposed to bridge the gap between acute responses to receptor occupation and the physiological responses to sustained receptor occupation. This will include studying whether the melatonin receptor undergoes homologous desensitization and whether melatonin alters synthesis or phosphorylation of specific proteins in a duration-dependent manner. Sixth, the mechanisms by which melatonin patterns experienced in the past influence the response to subsequent melatonin patterns will be examined. Understanding the mechanisms of action of melatonin has health implications, as melatonin influences circadian rhythmicity in humans and may be useful for treating biological rhythm disorders. Furthermore, elucidating the mechanisms by which melatonin regulates reproduction in animals that breed seasonally may be of value in delineating general mechanisms for the control of reproduction.
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