The Rho family GTPases RhoA, Rac1, and Cdc42 are critical signaling components regulating cell actin cytoskeletal organization, adhesion, transcriptional activity, and cell cycle progression. Aberrant signaling through these proteins may lead to certain human diseases such as cancer and inflammation. To date, three classes of regulatory proteins that control their GTPase cycle in cells, i.e. the guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPS), and the guanine nucleotide dissociation inhibitors (GDIs), have been identified. In addition, a large array of effector targets that serve to mediate their physiological responses has also been discovered. Our long term goals are to dissect the unique as well as the commonly shared structure-function properties of individual members of the Rho family and to understand the molecular mechanisms underlying the specific regulatory and targeting interactions of Rho GTPases. We propose three specific aims in the current studies.
In aim 1, we will examine the mechanism of interaction of Rho GTPases with a few regulatory GEF, GAP, and GDI proteins, including FGD1, Dbl, Ost, BcrGAP, and RhoGDIalpha, by identifying the critical amino acid residues of RhoA, Rac1, and/or Cdc42 involved in the specific couplings.
In aim 2, we will dissect the mechanism of interaction of RhoA, Rac1, and Cdc42 with a number of effector targets, which include N-WASP, IQGAP2, Por1, PAK2, p35, PKN, and ROK, by determining the structural elements of the Rho proteins involved in specifying the respective interactions and by quantifying the binding affinity of the Rho GTPases to the p21-binding domain of the effectors. Finally, in aim 3, we will reconstitute a few signaling ternary complexes of Rho GTPases consisting of Rho GTPases, the regulators, and/or the effectors, and determine the effect of the molecular interplay of the GTPase complex on the GTP-binding/effector activation/GTP-hydrolysis functions of the Rho proteins. Throughout the proposed studies special emphasis will be put on the specificity issue of the protein-protein interactions involved and on the direct comparison of the structure-function relationships of RhoA, Rac1, and Cdc42. The knowledge gained from these studies may be explored to formulate rational approaches for the development of novel pharmaceutical agents that interfere with specific functions of Rho GTPases contributing to human diseases.
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