Recent studies have shown that several hormones, neurotransmitters and growth factors stimulate the breakdown of phosphatidylcholine in addition to phosphoinositides in several tissues. In liver cells, there is evidence that the breakdown occurs in the plasma membrane and involves a G-protein and both phospholipase C and phospholipase D activities. In several tissues there is also evidence for the involvement of protein kinase C. This proposal has two major aims. The first is to define the molecular mechanisms involved in agonist stimulation of phosphatidylcholine breakdown, namely, to identify the receptors and purify the G-protein(s) and phospholipases specifically involved, and to define the role of protein kinase C. The components (receptors, G-proteins, phospholipases) involved in agonist-regulated phosphatidylcholine breakdown will be purified, cloned and compared with those involved in signalling through other G-proteins. A major goal will be to reconstitute agonist-regulated phosphatidylcholine breakdown using purified components. Functional domains in the phospholipases and G-proteins will be determined by chemical and proteolytic modification, and by site- directed mutagenesis of expressed proteins. Their possible phosphorylation and functional modification by protein kinase C will also be examined.
The second aim i s to determine the physiological significance of agonist-induced phosphatidylcholine breakdown in cells i.e. the functions of the diacylglycerol, phosphatidate, choline and phosphocholine produced. This will involve testing for correlations between changes in these compounds and certain physiological responses, e.g., protein kinase C activation and changes in Ca2+ and cAMP. Specific points include determining the extent of diacylglycerol production from phosphatidylcholine and its role in the regulation of protein kinase c and the role of phosphatidate in agonist-induced Ca2+ influx and cAMP decreases. Diacylglycerol is believed to play a major role in the regulation of many cell functions through activation of protein kinase C. The generation of diacylglycerol from phosphatidylcholine may be very important in selective signalling through this enzyme. Several physiological roles have been proposed for phosphatidate and it is possible that choline has other functions besides acetylcholine synthesis. P-choline is elevated in certain human cancer cells and in fibroblasts transformed by the Ha-ras oncogene.
