Structural Biology of Rho-Mediated Signaling

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Rho GTPases mediate diverse pathways in the cardiovascular system, including smooth muscle Ca 2+ sensitization and cell migration phenomena critical for the vasculogenesis. The primary objective of the project is to define the molecular mechanisms that control Ca sensitization pathways upstream of RhoA. These pathways involves activation of the RhoA GTPase by a family of nucleotide exchange factors, including PDZRhoGEF and LARG, which contain domains coupling these proteins to G and receptors of the plexin family, although their direct participation in smooth muscle physiology has not been proven. Structures of the individual domains, as well as of the multi-domain fragments of these GEFs will be determined by a combination of NMR and X-ray crystallography, as well as other complementary biophysical methods. The function and mechanism of the PDZ domain which binds the C-terminus of plexins will be assayed by isothermal titration calorimetry and phage display to determine specificity and affinity of interactions. The strategic aim is to understand how signals transduced membrane receptors ultimately activate the DH-PH domain tandem, which catalyzes the nucleotide exchange on RhoA. The selectivity of the latter reaction will also be probed by site-directed mutagenesis and X, ray crystallography. The functional properties of isolated domains and multi-domain fragments of GEFs, as well as their cellular localization, will be studied in collaboration with Project 1 and 3 within this PPG. The project will also focus on the structural biology of plexins and neuropilins, which function upstream of PDZRhoGEF and LARG in many tissues, and which are of primary importance to vasculogenesis. The central domain of plexin is believed to sequester the active form of Rac through a putative CRIB domain, although the boundaries of structural domains have not been determined. Crystallographic and/or NMR studies will determine the molecular architecture of these proteins and the interaction with Rac. Finally, structures of isolated domains of neuropilins, and the regulatory mechanisms of these receptors will also be probed by a synergistic combination of NMR and X-ray crystallography to determine interdomain communication patterns. This work will provide a model for the PDZRhoGEF activation, and will yield data directly relevant to cardiovascular biology.