Activation of Human PLC‐eta2 by Gbetagamma

The phospholipase C‐η subfamily of PLC isozymes was recently identified, and PLC‐η2 was shown to be activated by Gβ 1 γ 2 after transient expression in COS‐7 cells. We show here that activation of expressed PLC‐η2 also occur in the presence of other Gβγ dimers in addition to Gβ 1 γ 2 , and illustrate that their activation occur with a truncation mutant that lacks the long carboxyl terminus. To define the enzymatic properties of PLC‐η2 and its potential direct activation by Gβγ, a construct of PLC‐η2 encompassing PH through C2 domain was purified to homogeneity after expression from a baculovirus in insect cells and assayed after reconstitution with phospholipid vesicles. Purified PLC‐η2 hydrolyzed PI(4,5)P 2 with an apparent K m of 9.4 μM and V max of 5 μmol/min/mg, similar to activities previously observed with purified PLC‐β or ‐ε. Moreover, Gβ 1 γ 2 stimulated the lipase activity of PLC‐η2 in a concentration dependent manner similar to that observed with purified PLC‐β2. Activation was dependent on the presence of free Gβ 1 γ 2 since its sequestration in the presence of Gα i1 or GRK2‐ct reversed Gβ 1 γ 2 ‐promoted activation. The PH domain of PLC‐η2 is not required for Gβ 1 γ 2 ‐mediated regulation since a purified fragment lacking the PH domain nonetheless was activated by Gβ 1 γ 2 . Taken together these studies illustrate that PLC‐η2 is a direct downstream effector of Gβγ and therefore, of receptor‐activated heterotrimeric G proteins.