Purification and Characterization from Bovine Brain Cytosol of a Novel Regulatory Protein Inhibiting the Dissociation of GDP from and the Subsequent Binding of GTP to rhoB ~20, a ras p214ike GTP- binding Protein*

A novel regulatory protein for the rho proteins (rhoA p2 1 and rhoB p20), belonging to a ras p2 llras p21like small molecular weight (M,) GTP-binding protein (G protein) superfamily, was purified to near homogeneity from bovine brain cytosol and characterized. This regulatory protein, designated here as GDP dissociation inhibitor (GDI) for the rho proteins (rho GDI), inhibited the dissociation of GDP from rhoB p20 and the binding of guanosine 5’-(3-0-thio)triphosphate (GTP-yS) to the GDP-bound form of rhoB p20 but not of that to the guanine nucleotide-free form. The M, value of rho GDI was estimated to be about 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and from the S value, indicating that rho GDI is composed of a single polypeptide without a subunit structure. The isoelectric point was about pH 5.7. rho GDI made a complex with the GDP-bound form of rhoB p20 with a molar ratio of 1:l but not with the GTP+bound or guanine nucleotide-free form. rho GDI did not stimulate the GTPase activity of rhoB p20 and by itself showed neither GTPyS-binding nor GTPase activity. rho GDI was equally active for rhoA p21 and rhoB p20 but was inactive for other ras p2llraa p21like G proteins including c-Ha-r~21, smgp25A, and smg ~21. rho GDI activity was detected in the cytosol fraction of various rat tissues. These results indicate that, in mammalian tissues, there is a novel type of regulatory protein specific for the rho proteins that interacts with the GDP-bound form of the rho proteins and thereby regulates the GDP/GTP exchange reaction of the rho proteins by inhibiting the dissociation of GDP from and the subsequent binding of GTP to them. Since there is a GTPase-activating protein for the rho proteins stimulating the GTPase activity of the rho proteins in mammalian tissues, the rho proteins appear to be regulated at least by GTPase-activating protein and GDI in a dual manner.