Phospholipase C-β, the principal effector protein regulated by Gα(q), has been shown to increase the agonist-stimulated, steady-state GTPase activity of G(q) in proteoliposomes that contain both heterotrimeric G(q) and ml muscarinic receptor. We now use a moderately stable complex of R183C Gα(q) bound to GTP to show that PLC-β1 acts directly as a GTPase-activating protein (GAP) for isolated Gα(q) in a membrane-free system. PLC-β1 accelerated the hydrolysis of Gα(qR183C·GTP up to 20-fold. The K(m) was 1.5 nM, which is similar both to the EC50 with which R183C and wild type Gα(q) activate PLC-β1 and to the EC50 with which PLC-β1 acts as a G(q) GAP in the vesicle-based assay. The Gα(q) GAP activity of RGS4 can also be quantitated by this assay; it accelerated hydrolysis of bound GTP about 100- fold. The G(q) GAP activities of both PLC-β1 and RGS4 are blocked by Gβγ subunits, probably by a competitive mechanism. These data suggest either that the Gβγ subunits are not continuously required for receptor-catalyzed GDP/GTP exchange during steady-state GTP hydrolysis or that GAPs, either PLC- β or RGS proteins, can substitute for Gβγ in this set of reactions.
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