Phosphoinositide Binding Specificity among Phospholipase C Isozymes as Determined by Photo-Cross-Linking to Novel Substrate and Product Analogs

Abstract

We tested for the presence of high-affinity phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] and PI(3,4,5)P₃ binding sites in four phospholipase C (PLC) isozymes (δ₁, β₁, β₂, and β₃), by probing these proteins with analogs of inositol phosphates, d-Ins(1,4,5)P₃, d-Ins(1,3,4,5)P₄, and InsP₆, and polyphosphoinositides PI(4,5)P₂ and PI(3,4,5)P₃, which contain a photoactivatable benzoyldihydrocinnamide moiety. Only PLC-δ₁ was specifically radiolabeled. More than 90% of the label was found in tryptic and chymotryptic fragments which reacted with antisera against the pleckstrin homology (PH) domain, whereas less than 5% was recovered in fragments that encompassed the catalytic core. In separate experiments, the isolated δ₁-PH domain was also specifically labeled. Equilibrium binding of d-Ins(1,4,5)P₃ to PLC-δ₁ indicated the presence of a single, high-affinity binding site; binding of d-Ins(1,4,5)P₃ to PLC-β₁, -β₂, or -β₃ was not detected. The catalytic activity of PLC-δ₁ was inhibited by the product d-Ins(1,4,5)P₃, whereas no inhibition of PLC-β₁, -β₂, or -β₃ activity was observed. These results demonstrate that the PH domain is the sole high-affinity PI(4,5)P₂ binding site of PLC-δ₁ and that a similar site is not present in PLC-β₁, -β₂, or -β₃. The data are consistent with the idea that the PH domain of PLC-δ₁, but not the β isozymes, directs the catalytic core to membranes enriched in PI(4,5)P₂ and is subject to product inhibition.