In addition to their established role in the generation of second messengers by phospholipase C-catalysed hydrolysis, three of the inositol-containing phospholipids, and phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-biphosphate are substrates for a second receptor-regulated enzyme, phosphoinositide 3-kinase. This ATP-dependent enzyme catalyses the phosphorylation of the D-3 OH group of each of the above lipids and is activated by a range of growth factors and hormones that stimulate receptor tyrosine kinases and guanine nucleotide-dependent regulatory protein [G-protein]-linked receptors in a diverse variety of target cells. Accumulation of the products of phosphoinositide 3-kinase, phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5- triphosphate is rapid in stimulated cells. It is probable that these 3- phosphorylated inositol lipids or some metabolite derived from them serves as an important signal in cellular responses to hormones and growth factors. Recent observations implicate phosphoinositide 3-kinase products as regulators of a calcium and phospholipid-dependent protein kinase, and modulators of protein secretion and trafficking. The long- term goal of the proposed research is to understand the cellular functions of receptor-stimulated formation of 3-phosphorylated inositol lipids. Two major questions will be addressed: of receptor-stimulated How do receptors, particularly those coupled to G-proteins, regulate phosphoinositide 3-kinase activity and what are the metabolic fates of the 3-phosphorylated inositol lipids when formed in stimulated cells. The specificity with which G-protein-coupled receptors stimulate the accumulation of 3-phosphorylated inositol lipids will be established. Cell lines expressing pathways for both tyrosine kinase receptor and G- protein-coupled receptor stimulation will be identified. The pathways by which these two classes of receptors regulate phosphoinositide 3- kinase will be compared in intact cells emphasizing the role of role of second messengers, protein kinases and G-proteins in this process. Using cell-free assays, the biochemical mechanisms by which G-protein-coupled receptors activate phosphoinositide 3-Kinase will be determined and the protein components of this system identified. The substrate-specificity of phosphoinositide 3-kinase and the metabolic interrelationships of the products of this enzyme and the other inositol-containing lipids will be examined in both intact and broken cell systems. The 3-phosphorylated inositol lipids will be prepared in radiolabeled and unlabeled forms. The metabolism of these lipids will be examined in cell and tissue homogenates. Successful completion of this program of research will greatly advance our understanding of the regulation and functions of phosphoinositide 3-kinase and its products, both of which promise to be pivotal components of an important signal transduction system that mediates cellular responses to stimulation by hormones and growth factors.
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