Abstract
Phospholipase Cbeta (PLCbeta) enzymes are activated by Galpha q and Gbetagamma subunits and catalyze the hydrolysis of the minor membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Activation of PLCbeta2 by Gbetagamma subunits has been shown to be conferred through its N-terminal pleckstrin homology (PH) domain, although the underlying mechanism is unclear. Also unclear are observations that the extent of Gbetagamma activation differs on different membrane surfaces. In this study, we have identified a unique region of the PH domain of the PLCbeta2 domain (residues 71-88) which, when added to the enzyme as a peptide, causes enzyme activation similar to that with Gbetagamma subunits. This PH domain segment interacts strongly with membranes composed of lipid mixtures but not those containing lipids with electrically neutral zwitterionic headgroups. Also, addition of this segment perturbs interaction of the catalytic domain, but not the PH domain, with membrane surfaces. We monitored the orientation of the PH and catalytic domains of PLC by intermolecular fluorescence resonance energy transfer (FRET) using the Gbetagamma activatable mutant, PLCbeta2/delta1(C193S). We find an increase in the level of FRET with binding to membranes with mixed lipids but not to those containing only lipids with electrically neutral headgroups. These results suggest that enzymatic activation can be conferred through optimal association of the PHbeta71-88 region to specific membrane surfaces. These studies allow us to understand the basis of variations of Gbetagamma activation on different membrane surfaces.