1. We have devised a method to fractionate and quantitate the amounts of PLC isozymes in human platelets. With this approach, we showed that compared to normal platelets, platelets from the patient with a mild inherited bleeding disorder contained approximately one-third the amount of PLC-beta2 and 70 to 80 % of PLC-gamma2. PLC-beta4, the least abundant isoform, was increased fourfold. These results suggest that the impaired platelet function in the patient is probably attributable to a deficiency of PLC-beta2. 2. We have cloned cDNA corresponding to a previously unidentified PLC-delta, which we named PLC-delta4. PLC-delta4 exist in at least two alternatively spliced forms of 90- and 93-kDa. Only the 90-kDa form of PLC-delta4 was visible by immunoblot analysis in most rat tissues, whereas testis contains mainly the 93-kDa form. 3. Heat-stable proteins that can activate PLC-gamma1 were purified from bovine brains. These proteins were proved to be members of tau, a microtubule-associated proteins. The tau proteins are rich in proline and contain two consensus sequences for the binding of PLC-gamma1 SH3 domain. The tau-dependent activation required a cofactor arachidonic acid and markedly inhibited by phosphatidylcholine. This suggests that the hydrolysis of PC and concomitant generation of arachidonic acid by PLA2 an activate PLC-gamma1 at a low concentration of calcium without involving tyrosine kinases. 4. PLC-beta isozymes, which are known to be activated by G-protein beta- gamma subunits, contains a pleckstrin homology (PH) domain. To understand the role of PH domain in the regulation of PLC isozymes, 16 point mutants including Trp 131, the most conserved residue in all PH domains, were constructed in the PLC-beta3 PH domain and expressed in Sf9 insect and E.coli. Studies with the W131 mutants suggest that in contrast to suggestions made for other PH-containing proteins, this residue may be involved in the interaction with neither G-beta-gamma nor PIP2.
