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 intracellular enzymes that produce intracellular second messengers, these receptor/G protein pathways influence the action of several members of the Ras superfamily of monomeric GTPases. Members of this family, such as the Ras and Rho proteins, regulate cellular growth, differentiation, shape and adhesion. P115 RhoGEF can directly connect the two families of G proteins by stimulating exchange of GTP on Rho when activated by the heterotrimeric G13 protein. Hormone --> Receptor --> ? G13 --> p115RhoGEF --> Rho. This proposal continues examination of the regulatory mechanisms among components of this pathway in vitro and attempts to clearly define the physiological role of proteins in cellular regulation. Proposed studies include attempts to determine structures of regulatory complexes, mutational analysis and fluorescent approaches to discern mechanisms, affinity approaches to identify other components of the pathway, and down-regulation of the endogenous protein to determine functional specificity. Initiatives to develop a chimeric G13alpha protein and define receptor independent pathways for activating the G protein will aid mechanistic studies as well as open new perspectives to determine the role of G13 in the modulation of numerous cellular pathways. Mutations in p115 RhoGEF and its homologues are known to be oncogenic. G13 is required for development. Progress will increase our understanding of these key pathways in regulating growth, differentiation and cell motility. This will help to better understand the regulation imparted by a variety of hormones and the contribution of these proteins to cellular dysfunction.
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