During the past few years, receptor-regulated phospholipase D has emerged as an important new theme in the area of signal transduction. Its product, phosphatidic acid (and secondarily diacylglycerol) has been implicated in cell growth, differentiation, the inflammatory response and a variety of cell-specific specialized responses. While the occurrence of receptor-activated phospholipase D has been documented in a variety of cell types during recent years, its biochemistry and mechanism of regulation are largely unknown. The neutrophil contains a particularly robust phospholipase D which can be activated by either a G-protein- linked-receptor (the f-Met-Leu-Phe receptor) or by phorbol esters. We have developed a cell-free system in which to investigate this activity, and have documented the existence of a cytosolic factor as well as a membrane-associated GTP-binding protein as components of the phospholipase D system. We have shown that the latter is a small GTPase in the Ras superfamily. The present studies will determine the identify of this GTPase, which we believe is a member of the Rho subfamily. Rho's can be selectively removed from the plasma membrane and from the cytosol using a combination of immunoprecipitation and extraction with recombinant RhoGDI (GDP Dissociation Inhibitor). The extracted material will be analyzed immunochemically and by protein chemistry methods, and candidate recombinant Rho family proteins will be expressed and reconstituted. We will also investigate the relationship between activation of PLD by protein kinase C and by the Rho protein, and will identify the PLD component that is a target of phosphorylation by protein kinase C. The downstream effector of Rho-GTP will be identified using Rho blotting and expression cloning methods, and will be expressed to identify candidate PLD components. The PLD components will also be characterized biochemically and chromatographically to determine the identify and tissue distribution of PLD isoforms and of the 50 kDa cytosolic factor. Techniques in use include lipid biochemistry, recombinant DNA methods, expression cloning, protein expression and protein purification.
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