RHOGDI-3 IS A NEW GDP DISSOCIATION INHIBITOR (GDI) - IDENTIFICATION OF A NON-CYTOSOLIC GDI PROTEIN INTERACTING WITH THE SMALL GTP-BINDING PROTEINS RHOB AND RHOG

RhoB is a small GTP-binding protein highly homologous to the RhoA prot ein. While RhoA is known to regulate the assembly of focal adhesions a nd stress fibers in response to growth factors, the function of RhoB r e mains unknown. We have reported that the transient expression of the endogenous RhoB protein is regulated during the cell cycle, contrasti ng with the permanent RhoA protein expression (1). Using the yeast two -hybrid system to characterize proteins interacting with RhoB, we iden tified a new mouse Rho GDP dissociation inhibitor, referenced as RhoGD I-3. The NH2-terminal alpha helix of RhoGDI-3 is strongly amphipatic a nd differs thus from that found in previously described bovine, human, and yeast RhoGDI proteins and mouse and human D4/Ly-GDIs. Contrary to the cytosolic localization of all known GDI proteins, acting on Rab o r Rho, RhoGDI-3 is associated to a Triton X-100-insoluble membranous o r cytoskeletal subcellular fraction. In the two-hybrid system, RhoGDI- 3 interacts specifically with GDP- and GTP-bound forms of post-transla tionally processed RhoB and RhoG proteins, both of which show a growth -regulated expression in mammalian cells. No interaction is found with RhoA, RhoC, or Rac1 proteins. We show that GDI-3 is able to inhibit G DP/GTP exchange of RhoB and to release GDP-bound but not GTP-bound Rho B from cell membranes.