SIGNALING THROUGH RHO GTP/GDP EXCHANGE FACTORS

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DESCRIPTION (Verbatim from the Applicant's Abstract): Intracellular signaling pathways depend upon appropriate and unique subcellular locations of their constituent proteins. Frequently, key intracellular signaling proteins move from one subcellular location to another (e.g., from cytoplasm to plasma membranes or from cytoplasm to nucleus) in response to extracellular stimuli. Incorrectly localized proteins can prevent completion of a particular signaling pathway or can cause unregulated signaling, such as uncontrolled cell growth. Understanding how cellular proteins come together at specific times and subcellular sites will lead to insight into ways to block inappropriate signaling in disease states such as cancer. This research grant will address this issue in the context of subcellular localization of and interactions between p11 5rhoGEF/PDZrhoGEF and Ga12/13. The heterotrimeric (abg) G proteins act as molecular switches to relay information from activated cell-surface receptors to appropriate intracellular effectors. One family of G protein a subunits, consisting of a12 and a13, can activate the rho family of small GTPases. Rho, in turn, activates signaling pathways that stimulate cell growth and morphological changes. Recently, two large, multi-domain proteins, p115rhoGEF and PDZrhoGEF, have been described that may function as direct links between a 12/13 and rho. a12 and a13 are typically found tightly associated with plasma membranes. On the other hand, we have recently demonstrated that, in response to activation of a13, p11 5rhoGEF translocates from the cytoplasm to plasma membranes, and at least one rho family member has also been shown to redistribute from cytoplasm to plasma membranes. The main objectives of this research proposal are to elucidate the underlying mechanisms of regulated plasma membrane targeting of p11 5rhoGEF and PDZrhoGEF, understand the importance of proper localization for proper cell signaling by p11 5rhoGEF and PDZrhoGEF, and define critical and specific interactions between a12/13 and p11 5rhoGEF/PDZrhoGEF. These objectives will be addressed through the following specific aims: (1) Define subcellular localization of p115rhoGEF and PDZrhoGEF, and determine changes in their localization in response to activated a12, a13 and GPCRs. (2) Define mechanisms of plasma membrane localization of p1l5rhoGEF and PDZrhoGEF. (3) Investigate relationship between subcellular localization and signaling ability for p115rhoGEF and PDZrhoGEF. (4) Investigate interactions between a12/13 and p115rhoGEF and PDZrhoGEF. To address these specific aims, this research grant application proposes experiments focusing on model mammalian cell culture systems. A combination of assays for subcellular localization of p115rhoGEF and PDZrhoGEF, assays for protein-protein interactions, and assays of rho-dependent signal transduction willbe employed.