Gbetagamma inhibits Galpha GTPase-activating proteins by inhibition of Galpha-GTP binding during stimulation by receptor.
Gbetagamma subunits modulate several distinct molecular events involved with G protein signaling. In addition to regulating several effector proteins, Gbetagamma subunits help anchor Galpha subunits to the plasma membrane, promote interaction of Galpha with receptors, stabilize the binding of GDP to Galpha to suppress spurious activation, and provide membrane contact points for G protein-coupled receptor kinases. Gbetagamma subunits have also been shown to inhibit the activities of GTPase-activating proteins (GAPs), both phospholipase C (PLC)-betas and RGS proteins, when assayed in solution under single turnover conditions. We show here that Gbetagamma subunits inhibit G protein GAP activity during receptor-stimulated, steady-state GTPase turnover. GDP/GTP exchange catalyzed by receptor requires Gbetagamma in amounts approximately equimolar to Galpha, but GAP inhibition was observed with superstoichiometric Gbetagamma. The potency of inhibition varied with the GAP and the Galpha subunit, but half-maximal inhibition of the GAP activity of PLC-beta1 was observed with 5-10 nM Gbetagamma, which is at or below the concentrations of Gbetagamma needed for regulation of physiologically relevant effector proteins. The kinetics of GAP inhibition of both receptor-stimulated GTPase activity and single turnover, solution-based GAP assays suggested a competitive mechanism in which Gbetagamma competes with GAPs for binding to the activated, GTP-bound Galpha subunit. An N-terminal truncation mutant of PLC-beta1 that cannot be directly regulated by Gbetagamma remained sensitive to inhibition of its GAP activity, suggesting that the Gbetagamma binding site relevant for GAP inhibition is on the Galpha subunit rather than on the GAP. Using fluorescence resonance energy transfer between cyan or yellow fluorescent protein-labeled G protein subunits and Alexa532-labeled RGS4, we found that Gbetagamma directly competes with RGS4 for high-affinity binding to Galpha(i)-GDP-AlF4.
Tang
Wei
W
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041, USA.
Tu
Yaping
Y
Nayak
Surendra K
SK
Woodson
Jimmy
J
Jehl
Markus
M
Ross
Elliott M
EM
D017607:Aluminum Compounds / Q000737:chemistry; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D004926:Escherichia coli / Q000378:metabolism; D005136:Eye Proteins / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000494:pharmacology; D005459:Fluorides / Q000737:chemistry; D020558:GTP Phosphohydrolases / Q000737:chemistry*; D020555:GTP-Binding Protein Regulators; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006153:Guanosine Diphosphate / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D020128:Inhibitory Concentration 50; D007313:Insecta; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry; D009154:Mutation; D010750:Phosphoproteins / Q000494:pharmacology; D011485:Protein Binding; D020710:RGS Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D010738:Type C Phospholipases / Q000378:metabolism
D017607:Aluminum Compounds; D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D005136:Eye Proteins; D005456:Fluorescent Dyes; C118981:G-protein Beta gamma; D020555:GTP-Binding Protein Regulators; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D020690:GTPase-Activating Proteins; D008164:Luminescent Proteins; D010750:Phosphoproteins; D020710:RGS Proteins; C065631:phosducin; C054562:yellow fluorescent protein, Bacteria; D006153:Guanosine Diphosphate; D049452:Green Fluorescent Proteins; C098799:RGS4 protein; D006160:Guanosine Triphosphate; D010738:Type C Phospholipases; D020558:GTP Phosphohydrolases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D005459:Fluorides; C032311:aluminum fluoride