IDENTIFICATION OF THE GUANINE-NUCLEOTIDE DISSOCIATION STIMULATOR FOR RAL AS A PUTATIVE EFFECTOR MOLECULE OF R-RAS, H-RAS, K-RAS, AND RAP

To identify proteins that bind to the Ras-related protein R-ras we per formed a yeast two-hybrid cDNA library screen. Several clones were obt ained encoding the C-terminal region of the guanine nucleotide dissoci ation stimulator for Ral (RalGDS). The R-ras-binding domain of RalGDS (RalGDS-RBD) is distinct from the conserved catalytic exchange factor regions. Using the two hybrid system, we show that RalGDS-RBD interact s with H-ras, K-ras, and Rap, and with active but not with inactive po int mutants of these Ras-like GTPases. Moreover, using purified protei ns, we demonstrate the direct GTP-dependent interaction of the Ras-lik e GTPases with RalGDS-RBD and full-length RalGDS in vitro. Furthermore , we show that RalGDS-RBD and the Ras-binding domain of Raf-l compete for binding to the Ras-like GTPases. These data indicate that RalGDS i s a putative effector molecule for R-ras, H-ras, K-ras, and Rap.