A point mutation in Gα(o) and Gα(i1) blocks interaction with regulator of G protein signaling proteins

Abstract Regulator of G protein-signaling (RGS) proteins accelerate GTP hydrolysis by Gα 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 Gi-family α subunits that were insensitive to RGS action. Based on a glycine to serine mutation in the yeast Gα subunit Gpa1sst that prevents deactivation by Sst2 (DiBello, P. R., Garrison, T. R., Apanovitch, D. M., Hoffman, G., Shuey, D. J., Mason, K., Cockett, M. I., and Dohlman, H. G. (1998) J. Biol. Chem. 273, 5780–5784), site-directed mutagenesis of αo and αi1 was done. G184S αo and G183S αi1 show kinetics of GDP release and GTP hydrolysis similar to wild type. In contrast, GTP hydrolysis by the G → S mutant proteins is not stimulated by RGS4 or by a truncated RGS7. Quantitative flow cytometry binding studies show IC50 values of 30 and 96 nm, respectively, for aluminum fluoride-activated wild type αo and αi1 to compete with fluorescein isothiocyanate-αo binding to glutathioneS-transferase-RGS4. The G → 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 αo and αi1 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 Gi function.