The leukemia-associated Rho guanine nucleotide exchange factor (LARG) was recently identified as a fusion partner of the mixed lineage leukemia (MLL) protein in acute myeloid leukemia. LARG is a novel member of the rapidly expanding Dbl family of oncoproteins that promote malignant transformation by activating Ras-related Rho family GTPases. Like other Dbl family proteins, LARG contains a Dbl homology (DH) domain that functions as a guanine nucleotide exchange factor and activator of Rho GTPases. The DH domain is followed by a pleckstrin homology (PH) domain that presumably regulates DH domain function. LARG also contains a regulator of G-protein signaling (RGS) domain, suggesting that it may link G protein-coupled receptor signaling to Rho GTPases. Our preliminary studies determined that LARG is an activator of RhoA and can cause transformation of NIH 3T3 mouse fibroblasts. We have proposed four specific aims to perform detailed structure-function analyses of LARG. Specific aim 1 will determine the roles of the DH and PH domains in mediating LARG activation of RhoA. In particular, whether the PH domain regulates DH domain function in a phosphatidylinositol 3-kinase dependent fashion will be determined. Specific aim 2 will evaluate the role of the RGS domain in linking LARG with G protein coupled receptor signaling. This includes a determination of which heterotrimeric G alpha subunit(s) is regulated by the RGS domain and which G alpha subunit(s) in turn regulates LARG DH domain activation. Specific aim 3 will determine if the tumor-associated MLL-LARG fusion protein is an aberrantly activated form of LARG and can promote growth transformation of epithelial cells and lL-3 independent growth of 32D myeloid cells. Finally, Specific Aim 4 will involve a determination of the crystal structure of the DH/PH domains of LARG complexed with its GTPase target, RhoA, and the determination of the structural basis for DH domain recognition of GTPases. Although the number of Dbl family oncoproteins continue to increase at a rapid pace, to date, LARG is the only functional Dbl protein found to be mutated in human cancer. Our studies will provide a comprehensive, structural, biochemical, and biological analysis of LARG function and assess a role for aberrant LARG activation of RhoA in AML development.