AbstractsBiology & Animal Science

Phospholipase C-β (PLC-β) isozymes are key effectors in G protein-coupled signaling pathways. Prior research suggested that some isoforms of PLC-β may exist and function as dimers, but little is known about dimerization of PLC-β. Data from coimmunoprecipitation assays of differentially-tagged PLC-β constructs and sizeexclusion chromatography of native PLC-β support homodimerization of PLC-β3 and PLC-β1 isozymes, but not heterodimerization of these isozymes. Size-exclusion chromatography data also suggest that PLC-β3 and PLC-β1 form higher affinity homodimers than PLC-β2. Evidence supportive of limited PLC-β monomerhomodimer equilibrium appears at 100 nM and lower. Further assessment of homodimerization status by co-immunoprecipitation assays with differentially-tagged PLC-β3 fragments demonstrated that at least two subdomains of PLC-β3 are involved in dimer formation, one in the catalytic X and Y domains, and the other in the G protein-regulated carboxy-terminal domain. Additionally, microscopic fluorescence resonance energy transfer assays provide evidence consistent with the existence of PLC-β homodimers in a whole cell context. Phosphatidylinositol 3,4,5-trisphosphate (PIP₃) has been proposed as a second messenger that affects a variety of cellular responses. Previously, we had shown that PLC-β1 and PLC-β3 bound immobilized PIP₃. In this study, PIP₃ was found to potentiate Ca²⁺-stimulated PLC-β activities using an in vitro reconstitution assay. LY294002, a specific PI 3-kinase inhibitor, significantly inhibited 10 minutes agoniststimulated 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-β activity. When co-expressed with p110CAAX, fluorescence-tagged PLC-β3 was increasingly localized to the plasma membrane. Conversely, a greater proportion of PLC-β3 associated with cytosolic fraction following H9c2 cells treatment with LY294002. Additional observations suggest that the C-tail domain of PLC-β1 and β3, not the PH or catalytic XY domain, is important for membrane association.