Magnesium fluoride-dependent binding of small G proteins to their GTPase-activating proteins.

ABSTRACT : GTPase-activating proteins (GAPs) enhance the intrinsic GTPase activity of small G proteins,such as Ras and Rho, by contributing a catalytic arginine to the active site. An intramolecular arginineplays a similar role in heterotrimeric G proteins. Aluminum fluoride activates the GDP form ofheterotrimeric G proteins, and enhances binding of the GDP form of small G proteins to their GAPs. Theresultant complexes have been interpreted as analogues of the transition state of the hydrolytic reaction.Here, equilibrium binding has been measured using scintillation proximity assays to provide quantitativeinformation on the fluoride-mediated interaction of Ras and Rho proteins with their respective GAPs,neurofibromin (NF1) and RhoGAP. High-affinity fluoride-mediated complex formation between RhoâGDP and RhoGAP occurred in the absence of aluminum; however, under these conditions, magnesiumwas required. Additionally, the novel observation was made of magnesium-dependent, fluoride-mediatedbinding of RasâGDP to NF1 in the absence of aluminum. Aluminum was required for complex formationwhen the concentration of magnesium was low. Thus, either aluminum fluoride or magnesium fluoridecan mediate the high-affinity binding of RhoâGDP or RasâGDP to GAPs. It has been reported thatmagnesium fluoride can activate heterotrimeric G proteins. Thus, magnesium-dependent fluoride effectsmight be a general phenomenon with G proteins. Moreover, these data suggest that some proteinânucleotidecomplexes previously reported to contain aluminum fluoride may in fact contain magnesium fluoride.Small GTPases belonging to the Ras superfamily controla wide range of intracellular signaling events (1, 2). Theycycle between an inactive GDP-bound form and an activeGTP-bound form. The GTP form binds effector molecules,which transmit downstream signals. They are down-regulatedby GTPase-activating proteins (GAPs),