The overall goal of this research proposal is to understand the molecular mechanisms underlying regulation of hormone synthesis and secretion by anterior pituitary cells. The proposed studies focus on how the hypothalamic tripeptide thyrotropin-releasing hormone (TRH) interacts with its receptor and transmits a signal. The acute secretory responses of pituitary tumor cells (GH-cells) and normal pituitary cells will be determined in a perifusion system. The importance of calcium ion, cyclic AMP and protein kinase C in the biphasic secretory response will be measured, and the extent of desensitization will be determined. The TRH receptor interacts with a guanyl nucleotide binding protein in the plasma membrane. The importance of this interaction in eliciting hormonal response will be determined in transiently permeabilized cells, and the GTP binding protein will be identified. In order to characterize the TRH receptor, anti-idiotypic antibodies will be prepared, and the receptor will be photoaffinity labeled and identified. The TRH receptor will be localized on pituitary cells under different conditions. To obtain a permanent cell line secreting thyrotropin, GH-cells will be fused with normal pituitary cells and hybrids will be selected. A cell line secreting thyrotropin will be used to determine the mechanism of thyroid hormone inhibition of basal and TRH stimulated secretion. These experiments should provide basic information about the mechanisms of hormonal regulation.
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