Phospholipase C beta 2 association with phospholipid interfaces assessed by fluorescence resonance energy transfer - G protein beta gamma subunit-mediated translocation is not required for enzyme activation

Phospholipase C beta 2 (PLC beta 2) is activated by G protein beta gamma subunits and calcium. The enzyme is soluble and its substrate, phosphatidylinositol 4,5-bisphosphate (PIP2), is present in phospholipid membranes. A potential mechanism for regulation of this enzyme is through influencing the equilibrium association of the enzyme with membrane surfaces. In this paper we describe a fluorescence resonance energy transfer (FRET) method for measuring the association of PLC beta 2 with phospholipid bilayers. The method allows equilibrium measurements to be made under a variety of conditions, including those that support enzymatic activity and ability to be regulated by G proteins. Using this method it was found that PLC beta 2 bound to vesicles containing anionic lipids and demonstrated a selective and unique interaction with PIP2 containing vesicles. The FRET data were corroborated with a centrifugation based method for estimating the affinity of PLC beta 2 for vesicles. Apparently different modes of association of PLC beta 2 with vesicles of different composition can be distinguished based on alterations in resonance energy transfer efficiency. Association of PLC beta 2 with PLP(2) vesicles requires an intact lipid bilayer, is blocked by neomycin, and is not affected by D-myo-inositol 1,4,5-trisphosphate (D-IP3). G protein beta gamma subunits do not alter the affinity of PLC beta 2 for lipid bilayers and at the PIP2 concentrations used to measure beta gamma-dependent stimulation of PLC activity, the majority of the PLC beta 2 is already associated with the vesicle surface. Furthermore, under conditions where beta gamma subunits strongly activate PLC activity, the extent of association with vesicles is unaffected by beta gamma subunits or calcium. These results indicate that activation of PLC beta 2 by G protein py subunits or Ca2+ in vitro does not involve translocation to the vesicle surface.