SELECTIVE ACTIVATION OF PHOSPHOLIPASE-C BY RECOMBINANT G-PROTEIN ALPHA-SUBUNIT AND BETA-GAMMA-SUBUNITS

Receptor activation of phospholipase C (PLC) via G-proteins occurs by pertussis toxin-sensitive and toxin-insensitive signaling pathways. The alpha-subunits of the G(q) family are presumed to mediate the toxin-insensitive pathway, but the nature of the G-proteins mediating the toxin-sensitive pathway is not established. Recently, PLC-beta has been shown to be activated by G-protein beta subunits of mixed or undefined composition. The relative activities of G-protein subunits that might activate PLC-beta were examined using defined recombinant alpha- and betagamma-subunits obtained from the baculovirus expression system by reconstituting the purified subunits with purified bovine brain PLC-beta1 or turkey erythrocyte PLC-beta in unilamellar phospholipid vesicles. Turkey erythrocyte Galpha11 and recombinant Galpha11 and Galpha(q) obtained after expression in Sf9 cells activated both bovine brain PLC-beta1 and turkey erythrocyte PLC-beta. In contrast, under the same assay conditions, recombinant Galpha(i1), Galpha(i2), Galpha(i3), and Galpha(o) were without effect on either type of PLC. All types of betagamma-subunits tested (rbeta1gamma2, rbeta1gamma3, rbeta2gamma2, rbeta2gamma3, bovine brain betagamma or turkey erythrocyte betagamma) inhibited Galpha11-mediated activation of PLC, presumably by promotion of formation of inactive heterotrimeric G-protein. All types of betagamma-subunits also markedly stimulated the activity of turkey erythrocyte PLC-beta but did not activate bovine brain PLC-beta1. Of the four different betagamma complexes of defined composition, three stimulated PLC with similar activities whereas beta2gamma3 was less effective. The data suggest that pertussis toxin-sensitive activation of PLC is mediated by the betagamma-subunits of G-proteins acting on specific phospholipase C isoenzymes.