Rapid kinetics of regulator of G-protein signaling (RGS)-mediated Galphai and Galphao deactivation. Galpha specificity of RGS4 AND RGS7.
Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits speeding deactivation. Galpha 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 Galpha accompanying GTP hydrolysis and Galpha deactivation on the millisecond time scale. Basal k(cat) values for Galpha(o), Galpha(i1), and Galpha(i2) at 20 degrees C were similar (0.025-0.033 s(-1)). Glutathione S-transferase fusion proteins containing RGS4 and an RGS7 box domain (amino acids 305-453) enhanced the rate of Galpha deactivation in a manner linear with RGS concentration. RGS4-stimulated rates could be measured up to 5 s(-1) at 3 microm, giving a catalytic efficiency of 1.7-2.8 x 10(6) m(-1) s(-1) for all three Galpha subunits. In contrast, RGS7 showed catalytic efficiencies of 0.44, 0.10, and 0.02 x 10(6) m(-1) s(-1) toward Galpha(o), Galpha(i2), and Galpha(i1), respectively. Thus RGS7 is a weaker GTPase activating protein than RGS4 toward all Galpha subunits tested, but it is specific for Galpha(o) over Galpha(i1) or Galpha(i2). Furthermore, the specificity of RGS7 for Galpha(o) does not depend on N- or C-terminal extensions or a Gbeta(5) subunit but resides in the RGS domain itself.
Lan
K L
KL
Departments of Pharmacology and Internal Medicine/Hypertension, The University of Michigan, Ann Arbor, Michigan 48109-0632, USA.
Zhong
H
H
Nanamori
M
M
Neubig
R R
RR
D005453:Fluorescence; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D021122:Protein Subunits; D020710:RGS Proteins / Q000502:physiology*
D021122:Protein Subunits; D020710:RGS Proteins; C115771:Rgs7 protein, rat; C098799:RGS4 protein; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins