INHIBITION OF REGULATOR OF G-PROTEIN SIGNALING FUNCTION BY 2 MUTANT RGS4 PROTEINS

Regulators of G protein signaling (RGS) proteins limit the lifetime of activated (GTP-bound) heterotrimeric G protein alpha subunits by acti ng as GTPase-activating proteins (GAPs), Mutation of two residues in R GS4, which, based on the crystal structure of RGS4 complexed with G(i alpha 1)-GDP-AlF4-, directly contact G(i alpha 1) (N88 and L159), esse ntially abolished RGS4 binding and GAP activity, Mutation of another c ontact residue (S164) partially inhibited both binding and GAP activit y, Two other mutations, one of a contact residue (R167M/A) and the oth er an adjacent residue (F168A), also significantly reduced RGS4 bindin g to G(i alpha 1)-GDP-AlF4-, but in addition redirected RGS4 binding t oward the GTP gamma S-bound form, These two mutant proteins had severe ly impaired GAP activity, but in contrast to the others behaved as RGS antagonists in GAP and in vivo signaling assays, Overall, these resul ts are consistent with the hypothesis that the predominant role of RGS proteins is to stabilize the transition state for GTP hydrolysis. In addition, mutant RGS proteins can be created with an altered binding p reference for the G(i alpha)-GTP conformation, suggesting that efficie nt RGS antagonists can be developed.