A Point Mutation in G alpha sub(o) and G alpha sub(i1) Blocks Interaction with Regulator of G Protein Signaling Proteins

Regulator of G protein-signaling (RGS) proteins accelerate GTP hydrolysis by G alpha subunits and are thought to be responsible for rapid deactivation of enzymes and ion channels controlled by G proteins. We wanted to identify and characterize G sub(i)-family alpha subunits that were insensitive to RGS action. Based on a glycine to serine mutation in the yeast G alpha subunit Gpa1 super(sst) that prevents deactivation by Sst2, site-directed mutagenesis of alpha sub(o) and alpha sub(i1) was done. G184S alpha sub(o) and G183S alpha sub(i1) show kinetics of GDP release and GTP hydrolysis similar to wild type. In contrast, GTP hydrolysis by the G arrow right S mutant proteins is not stimulated by RGS4 or by a truncated RGS7. Quantitative flow cytometry binding studies show IC sub(50) values of 30 and 96 nM, respectively, for aluminum fluoride-activated wild type alpha sub(o) and alpha sub(i1) to compete with fluorescein isothiocyanate- alpha sub(o) binding to glutathione S-transferase-RGS4. The G arrow right S mutant proteins showed a greater than 30-100-fold lower affinity for RGS4. Thus, we have defined the mechanism of a point mutation in alpha sub(o) and alpha sub(i1) that prevents RGS binding and GTPase activating activity. These mutant subunits should be useful in biochemical or expression studies to evaluate the role of endogenous RGS proteins in G sub(i) function.