Rapid Kinetics of Regulator of G-protein Signaling (RGS)-mediated Gαi and Gαo Deactivation

Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits speeding deactivation. Gα deactivation kinetics mediated by RGS are too fast to be directly studied using conventional radiochemical methods. We describe a stopped-flow spectroscopic approach to visualize these rapid kinetics by measuring the intrinsic tryptophan fluorescence decrease of Gα accompanying GTP hydrolysis and Gα deactivation on the millisecond time scale. Basal k cat values for Gαo, Gαi1, and Gαi2 at 20 °C were similar (0.025–0.033 s−1). GlutathioneS-transferase fusion proteins containing RGS4 and an RGS7 box domain (amino acids 305–453) enhanced the rate of Gα deactivation in a manner linear with RGS concentration. RGS4-stimulated rates could be measured up to 5 s−1 at 3 μm, giving a catalytic efficiency of 1.7–2.8 × 106 m −1 s−1 for all three Gα subunits. In contrast, RGS7 showed catalytic efficiencies of 0.44, 0.10, and 0.02 × 106 m −1s−1 toward Gαo, Gαi2, and Gαi1, respectively. Thus RGS7 is a weaker GTPase activating protein than RGS4 toward all Gα subunits tested, but it is specific for Gαo over Gαi1 or Gαi2. Furthermore, the specificity of RGS7 for Gαo does not depend on N- or C-terminal extensions or a Gβ5 subunit but resides in the RGS domain itself.

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