Abstract
Phospholipase C‐[beta](PLC‐[beta]) isozymes are key effectors in G protein‐coupled signaling pathways. Previously, we had shown that PLC‐[beta]1 and PLC‐[beta]3 bound immobilized PIP3. In this study, PIP3 was found to potentiate Ca2+‐stimulated PLC‐[beta] activities using an in vitro reconstitution assay. LY294002, a specific PI 3‐kinase inhibitor, significantly inhibited 10 minutes agonist‐stimulated total IP accumulation. Both LY294002 and wortmannin inhibited 90 seconds agonist‐stimulated IP3 accumulation in intact cells. Moreover, transfected p110CAAX, a constitutively activated PI 3‐Kinase catalytic subunit, increased 90 seconds oxytocin‐stimulated IP3 accumulation. Receptor‐ligand binding assays indicated that LY294002 did not affect G protein‐coupled receptors directly, suggesting a physiological role for PIP3 in directly potentiating PLC‐[beta] activity. When co‐expressed with p110CAAX, fluorescence‐tagged PLC‐[beta]3 was increasingly localized to the plasma membrane. Conversely, a greater proportion of PLC‐[beta]3 associated with cytosolic fraction following H9c2 cells treatment with LY294002. Additional observations suggest that the PH domain of PLC‐[beta]1 and ‐[beta]3 is not important for p110CAAX‐induced membrane association.This work was supported by a grant from the NIH R01‐GM61244 (to T.M.F.).
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