MEMBRANE-BINDING PROPERTIES OF PHOSPHOLIPASE C-BETA(1) AND PHOSPHOLIPASE C-BETA-2 - ROLE OF THE C-TERMINUS AND EFFECTS OF POLYPHOSPHOINOSITIDES, G-PROTEINS AND CA2+

We have studied the binding of two G-protein-regulated phospholipase C (PLC) enzymes, PLCs-beta(1) and -beta(2), to membrane surfaces using sucrose-loaded bilayer phospholipid vesicles of varying compositions. Neither enzyme binds appreciably to pure phosphatidylcholine vesicles at lipid concentrations up to 10(-3) M. PLC-beta(1) and PLC-beta(2) bi nd vesicles composed of phosphatidylcholine, phosphatidylserine and ph osphatidylethanolamine (molar ratio 1:1:1) with an approximate K-d of 10(-5) M. Inclusion of 2 % PtdIns(4,5)P-2 in these vesicles had no eff ect on the affinity of this interaction. As reported by others, remova l of the C-terminus of PLC-beta(1) and PLC-beta(2) produces catalytica lly active fragments. The affinity of these truncated proteins for pho spholipid vesicles is dramatically reduced suggesting that this region of the proteins contains residues important for membrane binding. Inc lusion of G-protein alpha- and beta gamma-subunit activators in the ph ospholipid vesicles does not increase the binding of PLC-beta(1) or PL C-beta(2), and the magnitude of G-protein-mediated PLC activation obse rved at low phospholipid concentrations (10(-6) M) is comparable to th at observed at concentrations at which the enzymes are predominantly m embrane-bound (10(-3) M). PLC-beta(1) and -beta(2) contain C2 domains but Ca2+ does not enhance binding to the vesicles. Our results indicat e that binding of these enzymes to membranes involves the C-temini of the proteins and suggest that activation of these enzymes by G-protein s results from a regulated interaction between the membrane-bound prot eins rather than G-protein-dependent recruitment of soluble enzymes to a substrate-containing phospholipid surface.