Phosphoinositide binding specificity among phospholipase C isozymes as determined by photo-cross-linking to novel substrate and product analogs

We tested for the presence of high-affinity phosphatidylinositol 4,5-bisphosphate [PI(4,5)P-2] and PI(3,4,5)P-3 binding sites in four phospholipase C (PLC) isozymes (delta(1), beta(1), beta(2), and beta(3)), by probing these proteins with analogs of inositol phosphates, D-Ins(1,4,5)P-3, D-Ins(1,3,4,5)P-4, and InsP(6), and polyphosphoinositides PI(4,5)P-2 and PI(3,4,5)P-3, which contain a photoactivatable benzoyldihydrocinnamide moiety. Only PLC-delta(1) 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 delta(1)-PH domain was also specifically labeled. Equilibrium binding of D-Ins(1,4,5)P-3 to PLC-delta(1) indicated the presence of a single, high-affinity binding site; binding of D-Ins(1,4,5)P-3 to PLC-beta(1), -beta(2), or -beta(3) was not detected. The catalytic activity of PLC-delta(1) was inhibited by the product D-Ins(1,4,5)P-3, whereas no inhibition of PLC-beta(1), -beta(2), or -beta(3) activity was observed. These results demonstrate that the PH domain is the sole high-affinity PI(4,5)P-2 binding site of PLC-delta(1) and that a similar site is not present in PLC-beta(1), -beta(2), or -beta(3) The data are consistent with the idea that the PH domain of PLC-delta(1), but not the beta isozymes, directs the catalytic core to membranes enriched in PI(4,5)P-2 and is subject to product inhibition.