Rac2 regulation of phospholipase C-β₂ activity and mode of membrane interactions in intact cells

Phospholipase C-β (PLCβ) isozymes play important roles in transmembrane signaling. Their activity is regulated by heterotrimeric G proteins. The PLCβ₂ isozyme is unique in being stimulated also by Rho GTPases (Rac and Cdc42). However, the mechanism(s) of this stimulation are still unclear. Here, we employed fluorescence recovery after photobleaching to investigate the interaction of green fluorescent protein (GFP)-PLCβ₂ with the plasma membrane. For either GFP-PLCβ₂ or GFP-PLCβ₂Δ, a C-terminal deletion mutant lacking the region required for stimulation by Gα_q, these interactions were characterized by a mixture of exchange with a cytoplasmic pool and lateral diffusion. Constitutively active Rac2(12V) stimulated the activity of both GFP-PLCβ₂ and GFP-PLCβ₂Δ in live cells, and enhanced their membrane association as evidenced by the marked reduction in their fluorescence recovery rates. Both effects required the putative N-terminal pleckstrin homology (PH) domain of PLCβ₂. Importantly, Rac2(12V) dramatically increased the contribution of exchange to the fluorescence recovery of GFP-PLCβ₂, but had the opposite effect on GFP-PLCβ₂Δ, where lateral diffusion became dominant. Our results demonstrate for the first time the regulation of membrane association of a PLCβ isozyme by a GTP-binding protein and assign a novel function to the PLCβ₂ C-terminal region, regulating its exchange between membrane-bound and cytosolic states.