Membrane binding of phospholipases C-β1 and C-β2 is independent of phosphatidylinositol 4,5-bisphosphate and the αand βγ subunits of G proteins

We have measured the membrane binding affinities of purified phosphatidylinositol-specific phospholipases C-β1 and 1-β2 to membranes of varying lipid composition using fluorescence methods. Our studies show that these proteins bind with affinities of I0^-5-10-^-4 M, with a small dependence on lipid type. Binding was relatively insensitive to the presence of phosphatidylinositol-specific phospholipases C-βs' major physiological substrate, phosphatidylinositiol 4,5-bisphosphate, as well as the presence of Ca²⁺, which is required for activity. The presence of purified GTPγS-activated α₁₁ subunits of heterotrimeric guanine nucleotide binding proteins (G proteins) did not alter the membrane binding affinity of phosphatidylinositol-specific phospholipases C-γ1, even though an is a potent activator of this protein. Similarly, the presence of purified βγ subunits of G proteins did not alter the membrane association of phosphatidylinositol-specific phospholipases C-β2 even though these subunits strongly activate this isoform. These results argue against a recruitment model for PLC-β activation by G proteins, negatively charged lipids, Ca²⁺, or substrate, and suggest that activation occurs through association of the membrane-bound species.