Phospholipase C-beta1 directly accelerates GTP hydrolysis by Galphaq and acceleration is inhibited by Gbeta gamma subunits.
Phospholipase C-beta, the principal effector protein regulated by Galphaq, has been shown to increase the agonist-stimulated, steady-state GTPase activity of Gq in proteoliposomes that contain both heterotrimeric Gq and m1 muscarinic receptor. We now use a moderately stable complex of R183C Galphaq bound to GTP to show that PLC-beta1 acts directly as a GTPase-activating protein (GAP) for isolated Galphaq in a membrane-free system. PLC-beta1 accelerated the hydrolysis of GalphaqR183C.GTP up to 20-fold. The Km was 1.5 nM, which is similar both to the EC50 with which R183C and wild type Galphaq activate PLC-beta1 and to the EC50 with which PLC-beta1 acts as a Gq GAP in the vesicle-based assay. The Galphaq GAP activity of RGS4 can also be quantitated by this assay; it accelerated hydrolysis of bound GTP about 100-fold. The Gq GAP activities of both PLC-beta1 and RGS4 are blocked by Gbeta gamma subunits, probably by a competitive mechanism. These data suggest either that the Gbeta gamma subunits are not continuously required for receptor-catalyzed GDP/GTP exchange during steady-state GTP hydrolysis or that GAPs, either PLC-beta or RGS proteins, can substitute for Gbeta gamma in this set of reactions.
Chidiac
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Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041, USA. pchidiac@julian.uwo.ca
Ross
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D000818:Animals; D001667:Binding, Competitive; D004791:Enzyme Inhibitors / Q000494:pharmacology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D009154:Mutation; D054799:Phospholipase C beta; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D020710:RGS Proteins; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism*
D004791:Enzyme Inhibitors; D020690:GTPase-Activating Proteins; D007527:Isoenzymes; D011506:Proteins; D020710:RGS Proteins; C098799:RGS4 protein; D006160:Guanosine Triphosphate; D010738:Type C Phospholipases; D054799:Phospholipase C beta; C518034:Plcb1 protein, mouse; D019204:GTP-Binding Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11