CLONING, SEQUENCING, PURIFICATION, AND GQ-DEPENDENT ACTIVATION OF PHOSPHOLIPASE-C-BETA-3

Abstract

Six mammalian phospholipase C isozymes (PLC-beta1, PLC-beta2, PLC-gamma1, PLC-gamma2, PLC-delta1, and PLC-delta2) have been identified at both protein and DNA levels. Here, cDNAs corresponding to a previously unidentified PLC isozyme were isolated from a rat thyroid cell FRTL cDNA library. Comparison of the predicted amino acid sequence of this new PLC with other known PLC isozymes revealed a high degree of overall similarity with PLC-beta1 and PLC-beta2. Thus, the new PLC was named PLC-beta3. Comparison with PLC-beta1 and PLC-Beta2 also revealed that the deduced amino-terminal sequence of PLC-beta3 was incomplete by 10-20 amino acids. With the use of antibodies raised against synthetic peptides corresponding to PLC-beta3-specific amino acid sequences, we purified PLC-beta3 from a rat brain particulate fraction. The purified enzyme exhibited an apparent molecular mass of 152 kDa on SDS-polyacrylamide gels, as compared with 150 and 140 kDa for PLC-beta1 and PLC-beta2, respectively. Studies of the activation of PLC-beta isozymes by three alpha subunits of G(q) class G proteins, alpha(q), all, and alpha1 in the presence of guanosine 5-O-(3-thiotriphosphate) (GTPgammaS) revealed that the extent of activation decreased in the order of PLC-beta1 greater-than-or-equal-to PLC-beta3 >> PLC-beta2 for all three alpha subunits, suggesting a certain degree of specificity in the interaction of G(q)alpha subunits with different PLC-beta isozymes.