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-b isphosphate [PI(4,5)P-2] and PI(3,4,5)P-3 binding sites in four phosph olipase 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 benzoyldihyd rocinnamide moiety. Only PLC-delta(1) was specifically radiolabeled. M ore than 90% of the label was found in tryptic and chymotryptic fragme nts which reacted with antisera against the pleckstrin homology (PH) d omain, whereas less than 5% was recovered in fragments that encompasse d the catalytic core. In separate experiments, the isolated delta(1)-P H 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-affi nity 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 o f PLC-beta(1), -beta(2), or -beta(3) activity was observed. These resu lts 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, d irects the catalytic core to membranes enriched in PI(4,5)P-2 and is s ubject to product inhibition.