A major signaling paradigm for modulation of hormonal and other extracellular stimuli is the use of heterotrimeric G proteins by cell surface receptors. Besides direct regulation of a variety of enzymes that produces intracellular second messengers, these receptor/Gprotein pathways influence the action of several members of the Ras superfamily of monomeric GTP-dependent proteins. Members of this family, such as Ras and the Rho proteins, regulate cellular growth, differentiation, shape and adhesion. Recently, p115 RhoGEF was shown to directly connect the two families of G proteins by stimulating exchanges of guanine nucleotides on Rho when activated by the heterotrimeric G13 protein. Since Rho can regulate phospholipase D (PLD), this suggests the following signaling pathway for regulation of this enzyme by some hormones. Hormone-->Receptor-->G13-->p115RhoGEF-->Rho-->PLD The proposed studies examine regulatory mechanisms among components of this pathway and will explore the hypothesis that p115 RhoGEF exemplifies a general mechanism for communication between heterotrimeric and monomeric G proteins. A variety of affinity approaches for isolation of new molecules that would act in a analogous fashion will test this hypothesis. The functional domains of p115 RhoGEF will be defined by expression of truncated molecules. Reconstitution of p115 RhoGEF with upstream (receptor/G13) and downstream (Rho effectors) components will define mechanisms of regulation. Mutants in function and inhibitory domains will be used to explore in vivo function. Continuing studies with PLD attempt to define regulatory mechanisms for the molecule and its role in regulation. Special interest is focused on the roles of phosphatidylinositol 4,5-bisphosphate and phosphatidic acid, the product of PLD activity, in regulation of this enzyme and protein trafficking. Progress will increase our understanding of these key pathways for regulation and provide a better understanding of how hormones liked to heterotrimeric G proteins impart regulatory control on the Ras superfamily of GTPases and their sequelae.
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