FIG. 8.

RhoH binds GTP only but has no GTPase activity. (a) Nucleotide dissociation assay using GST-RhoH, -Rac, and -RhoA fusion proteins. Similar to RhoA and Rac1, RhoH binds GTP rapidly upon incubation with radiolabeled nucleotide under nucleotide exchange conditions. Note that RhoH is almost completely resistant to GDP dissociation compared to Rac and RhoA under both high- and low-magnesium conditions. (b) GTP hydrolysis assay: Under conditions that allow RhoA and Rac to autohydrolyze greater than 80% of bound GTP, RhoH did not show any hydrolysis of GTP. (c) RhoH is resistant to Rho GAP p50. Addition of Rho GAP p50 enhanced the hydrolysis activity of RhoA but had no effect on RhoH.