Abstract
Phospholipase C-beta (PLC-beta) isozymes are key effectors in G protein-coupled signaling pathways. Previously, we showed that PLC-beta1 and PLC-beta3 bound immobilized PIP(3). In this study, PIP(3) was found to potentiate Ca(2+)-stimulated PLC-beta activities using an in vitro reconstitution assay. LY294002, a specific PI 3-kinase inhibitor, significantly inhibited 10 min of agonist-stimulated total IP accumulation. Both LY294002 and wortmannin inhibited 90 sec of agonist-stimulated IP(3) accumulation in intact cells. Moreover, transfected p110CAAX, a constitutively activated PI 3-kinase catalytic subunit, increased 90 sec of oxytocin-stimulated IP(3) accumulation. Receptor-ligand binding assays indicated that LY294002 did not affect G protein-coupled receptors directly, suggesting a physiological role for PIP(3) in directly potentiating PLC-beta activity. When coexpressed with p110CAAX, fluorescence-tagged PLC-beta3 was increasingly localized to the plasma membrane. Additional observations suggest that the PH domain of PLC-beta is not important for p110CAAX-induced membrane association.