Phosphatidic acid (PA) is interactive with G alpha(q)-linked agonists to Stimulate GPCR signaling via phospholipase C-beta(1) (PLC-beta(1)). Phorbol 12-myristate 13-acetate (PMA) increases cellular levels of PA and phospholipase D activity (PLD). This study evaluated whether PMA can stimulate PLC-beta(1) activity via PA, independent of GPCR input in transfected COS 7 cells. PMA alone had little effect on PLC activity in cells co-transfected with PLC-beta(1) and G alpha(q). Activated G alpha(q), induced by co-transfecting muscarinic cholinergic receptor (m1R), was necessary for stimulation of PLC-beta(1) activity by PMA. Stimulation by PMA was dependent on the PA-regulatory motif of PLC-beta(1) implicating PA in this mechanism. PLD1 knockdown by antisense decreased responsiveness of PLC-beta(1) to both PMA and carbachol. PA alone thus has little effect on PLC-beta(1) activity, but PA and PLD1 synergize with activated G alpha(q) to stimulate PLC-beta(1) signaling. Coordinate interaction with activated G alpha(q) may serve as an important mechanism to fine tune response to ligands while preventing spurious initiation of PLC-beta signaling by PA in cells. (C) 2009 Elsevier Inc. All rights reserved.