Activation of human phospholipase C-η2 by Gβγ

Phospholipase C-eta 2 (PLC-eta 2) was recently identified as a novel broadly expressed phosphoinositide-hydrolyzing isozyme [Zhou, Y., et al. (2005) Biochem. J. 391, 667-676; Nakahara, M., et al. (2005) J. Biol. Chem. 280, 29128-29134]. In this study, we investigated the direct regulation of PLC-eta 2 by G beta gamma subunits of heterotrimeric G proteins. Coexpression of PLC-eta 2 with G beta(1)gamma(2), as well as with certain other G beta gamma dimers, in COS-7 cells resulted in increases in inositol phosphate accumulation. G beta(1)gamma(2)-dependent increases in phosphomositide hydrolysis also were observed with a truncation mutant of PLC-eta 2 that lacks the lona alternatively spliced carboxy-terminal domain of the isozyme. To begin to define the enzymatic properties of PLC-eta 2 and its potential direct activation by G beta gamma, a construct of PLC-eta 2 encompassing the canonical domains conserved in all PLCs (PH domain through C2 domain) was purified to homogeneity after expression from a baculovirus in insect cells. Enzyme activity of purified PLC-eta 2 was quantified after reconstitution with Ptdlns(4,5)P-2-containing phospholipid vesicles, and values for K-m (14.4 mu M) and V-max [12.6 mu mol min(-1) (mg of protein)(-1)] were similar to activities previously observed with purified PLC-beta or PLC-epsilon isozymes. Moreover, purified G beta(1)gamma(2) stimulated the activity of purified PLC-eta 2 in a concentration-dependent manner similar to that observed with purified PLC-beta 2. Activation was dependent on the presence of free G beta(1)gamma(2) since its sequestration in the presence of G alpha(i1) or GRK2-ct reversed G beta(1)gamma(2)-promoted activation. The PH domain of PLC-eta 2 is not required for G beta(1)gamma(2)-mediated regulation since a purified fragment encompassing the EF-hand through C2 domains but lacking the PH domain nonetheless was activated by G beta(1)gamma(2). Taken together, these studies illustrate that PLC-eta 2 is a direct downstream effector of G beta gamma and, therefore, of receptor-activated heterotrimeric G proteins.