RALGDS FAMILY MEMBERS INTERACT WITH THE EFFECTOR LOOP OF RAS P21

Using a yeast two-hybrid system, we identified a novel protein which i nteracts with ras p21. This protein shares 69% amino acid homology wit h ral guanine nucleotide dissociation stimulator (ralGDS), a GDP/GTP e xchange protein for ral p24. We designated this protein RGL, for ralGD S-like. Using the yeast two-hybrid system, we found that an effector l oop mutant of ras p21 was defective in interacting with the ras p21-in teracting domain of RGL; suggesting that this domain binds to ras p21 through the effector loop of ras p21. Since ralGDS contained a region highly homologous with the ras p21-interacting domain of RGL, we exami ned whether ralGDS could interact with ras p21. In the yeast two-hybri d system, ralGDS failed to interact with an effector loop mutant of ra s p21. In insect cells, ralGDS made a complex with v-ras p21 but not w ith a dominant negative mutant of ras p21. ralGDS interacted with the GTP-bound form of ras p21 but not with the GDP-bound form in vitro. ra lGDS inhibited both the GTPase-activating activity of the neurofibroma tosis gene product (NF1) for pas p21 and the interaction of Raf with v as p21 in vitro. These results demonstrate that ralGDS specifically in teracts with the active form of ras p21 and that ralGDS can compete wi th NF1 and Raf fbr binding to the effector loop of ras p21. Therefore, ralGDS family members may be effector proteins of ras p21 or play inh ibit interactions between ras p21 and its effecters.