In this application we propose to study the molecular basis of selected aspects of RhoA-mediated signal transduction in smooth muscle. Specifically, we will study the molecular mechanisms involved in Ca2+ sensitization and migration of vascular smooth muscle cells. These phenomena are key to the contractility in smooth muscle and onset of atherosclerosis. RhoA is a 21 kDa cytosolic GTPase of the Ras-related Rho family of signaling proteins. It shuttles from the biologically active, GTP-bound state in the cell under acute control of numerous pathways. RhoA regulates, among others, cell adhesion and motility, contractile responses, cytokinesis, vascular transport, etc., through the reorganization of the actin skeleton and signaling to myosin. Further, in smooth muscles it is responsible for the effect of Ca2+ sensitization. RhoA plays a critical role in the function of cardiac and vascular smooth muscle. The proposed study will provide a much better understanding of the specific mechanisms by which the signaling pathways function. We have already determined the crystal structure of the RhoA.GDP complex at 2.1 A resolution, and crystals of RhoA with GMPPNP, a non-hydrolyzable GP analogue, have been prepared. Coupled to site-directed mutagenesis and functional studies to be conducted in Project 1, the structural characterization will address the question of what epitopes in RhoA are responsible for downstream signaling to Rho-kinase, and/or effectors, in smooth muscle. Further, we will solve and characterize the structure of a unique GAP, a GTPase activating protein, which is functionally associated with the focal adhesions and shows preference for RhoA and Cdc42Hs as substrates. Crystals for this purpose have been prepared. The structure of the native GAP, as well as the planned characterization of the complexes of GAP with RhoA.GMPPNP and RnoA.GDP.AIF4-, will provide data regarding the activation mechanism and the molecular basis of substrate preference. Finally, we will study the structure-function properties in RhoGDI, a protein that solubilizes RhoA in the cytosol and inhibits the exchanges of the nucleotide. High resolution X-ray crystallography will be used to characterize the details of the geranylgeranyl-binding site of GDI, specifically with respect to solvent structure. Different constructs for crystallization have been prepared and shown to yield crystalline proteins. The ultimate goal is to crystalize the RhoDI-RhoA complex and describe the complete mechanism by which RhoA inhibits nucleotide exchange. To this purpose, we have demonstrated the feasibility of preparing such a complex using recombinant generated in E. coli and yeast.
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