INTERACTION BETWEEN CDC42HS AND RHOGDI IS MEDIATED THROUGH THE RHO-INSERT REGION

Members of the Rho subfamily of GTP binding proteins contain a region of amino acid sequence (residues 122-134) that is absent from other Ra s-like proteins and is termed the Rho insert region, To address the fu nctional role of this domain, we have constructed a Cdc42Hs/Ras chimer a in which loop 8 from Ha-Ras was substituted for the region in Cdc42H s that contains the 13-amino acid insert region, Our data indicate tha t the insert region of Cdc42Hs is not essential for its interactions w ith various target/effector molecules or for interactions with the gua nine nucleotide exchange factor, Dbl, or the Cdc42 GTPase-activating p rotein (GAP), However, the regulation of GDP dissociation and GTP hydr olysis on Cdc42Hs by the Rho GDP-dissociation inhibitor (GDI) is extre mely sensitive to changes in the insert region, such that a Cdc42Hs/Ha -Ras chimera that lacks this insert is no longer susceptible to a GDI- induced inhibition of GDP dissociation and GTP hydrolysis, The insensi tivity to GDI activity is not due to the inability of the GDI molecule to bind to the Cdc42Hs/Ha-Ras chimera, and in fact, the GDI is fully capable of stimulating the release of this chimera from membranes.