Peptide hormones interact with cells at specific cell-surface receptors. Cellular activation requires transmembrane signalling and possible generation of intracellular second messengers. Hormone-regulated polyphosphoinositide (PPI) metabolism has been identified as a likely transduction mechanism. We will investigate the molecular basis of receptor-stimulated PPI metabolism in clonal pituitary cells (GH3 cells) which are responsive to thyrotropin-releasing hormone (TRH). TRH stimulates protein secretion and previous studies identified rapid hormone-triggered events involving Ca2+-dependent diglyceride-activated protein phosphorylation which may be involved in regulating secretion. TRH-stimulated PPI turnover has been linked to regulation of these pathways. The objective of our research is to establish the means by which TRH receptor occupancy can regulate PPI turnover. Three of the specific aims for this project period will be: 1. to identify the reactions of PPI metabolism subject to regulation by TRH; 2. to characterize and purify enzymes catalyzing these reactions and 3. to develop a cell-free TRH-responsive membrane/cytosol system. PPI-metabolizing enzymes have previously been studied in systems which are incapable of hormone regulation. Recently-developed techniques allow TRH-stimulated PPI turnover to be studied in permeable GH3 cells. This permeable cell system will enable us to identify low molecular weight cofactors involved and to selectively inhibit individual reactions. Once the requirements for hormonal regulation in permeable cells are established and we have identified the critical enzymes involved, a hormone-responsive broken cell system will be developed. The subcellular distributions and regulatory characteristics of PPI-metabolizing enzymes will be determined. We will then reassemble membranes and cytosolic enzymes systematically to reconstitute PPI metabolism in a TRH-responsive manner. Success in this endeavor would enable fuller biochemical elucidation including identification of coupling proteins. The fourth specific aim for this project period will be to develop probes for characterizing and purifying the TRH receptor so that receptor-associated elements can be identified. TRH is representative of a large number of physiological regulators (hormones, neurotransmitters and mitogens) whose actions may be mediated through receptor-activated PPI turnover. Elucidation of the components involved in TRH action will contribute to understanding basic cell regulatory mechanisms.
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