There is compelling evidence that the generation of diacylglycerol and inositol 1,4,5-trisphosphate is intimately associated with stimulus-response coupling in a wide variety of cell systems. Diacylglycerol enhances the phosphorylation of target proteins by the activation of protein kinase C, and inositol 1,4,5-trisphosphate causes the mobilization of calcium from intracellular stores, thereby switching on a host of calcium-regulated functions. The platelet system represents one of the clearest examples of the involvement of phosphatidylinositol catabolism in cell physiology. A number of platelet activating agents trigger the generation of phosphatidylinositol-derived second messengers followed by an increase in the intraplatelet calcium concentration and protein phosphorylation. This process is catalyzed by a receptor- regulated phsophatidylinositol 4,5-bisphosphate (PtdIns-P2) phospholipase C that is modulated by guanine nucleotides and/or stimulatory ligands. At present there is little biochemical information that corroborates this important signal transduction hypothesis. The putative G-protein that interacts with phospholipase C has not been positively identified or purified and there is considerable speculation about its molecular structure. There is also evidence for G-protein-independent mechanisms for phospholipase C regulation. The phospholipase C activity modulated by G-proteins, receptors, or ligands has not been attributed to a purified protein. This proposal focuses on defining the biochemical mechanisms that regulate PtdIns-P2 phospholipase C activity and developing immunological reagents that will be used to test for the involvement of our purified phospholipase C's in ligand-initiated PtdIns-P2 hydrolysis. Our preliminary results indicate that membrane-associated and cytosolic platelet PtdIns-P2 phospholipase C's are allosteric enzymes which are activated by both homotropic (substrate) and heterotropic (phosphatidic acid) agents.
The specific aims are: (1) to purify and characterize platelet phospholipase C's that degrade polyphosphoinositides and determine the relationship between the soluble and membrane-associated forms of the enzyme, and (2) to define the mechanism(s) of substrate, heterotropic activator, receptor and G-protein regulation of platelet PtdIns-P2 phospholipase activity. The results of this work will contribute important new information to our biochemical understanding of phospholipase C regulation in stimulus-response coupling.
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