STIMULATION OF PHOSPHOLIPASE-C BY GUANINE-NUCLEOTIDE-BINDING PROTEIN-BETA-GAMMA SUBUNITS

We have previously shown that soluble fractions' obtained from human HL-60 granulocytes contain a phospholipase C which is markedly stimulated by the stable GTP analogue guanosine 5'-[3-O-thio]triphosphate (Camps, M., Hou, C., Jakobs, K. H. and Gierschik, P. (1990) Biochem. J. 271, 743 - 748]. To investigate whether this stimulation was due to a soluble alpha-subunit of a heterotrimeric guanine-nucleotide-binding protein or a soluble low-molecular-mass GTP-binding protein, we have examined the effect of purified guanine-nucleotide-binding protein beta-gamma dimers on the phospholipase-C-mediated formation of inositol phosphates by HL-60 cytosol. We found that beta-gamma-subunits, purified from bovine retinal transducin (beta-gamma(t)), markedly stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate by this phospholipase C preparation. The stimulation of phospholipase C by beta-gamma(t), was not secondary to a phospholipase-A2-mediated generation of arachidonic acid, was prevented by the GDP-liganded transducin alpha-subunit and was additive to activation of phospholipase C by guanosine 5'-[3-O-thio]triphosphate. beta-gamma(t), also stimulated soluble phospholipase C from human and bovine peripheral neutrophils, as well as membrane-bound, detergent-solubilized phospholipase C from HL-60 cells. Stimulation of soluble HL-60 phospholipase C was not restricted to beta-gamma(t), but was also observed with highly purified beta-gamma-subunits from bovine brain. Fractionation of HL-60 cytosol by anion-exchange chromatography revealed the existence of at least two distinct forms of phospholipase C in HL-60 granulocytes. Only one of these forms was sensitive to stimulation by beta-gamma(t), demonstrating that stimulation of phospholipase C by beta-gamma-subunits is isozyme specific. Taken together, our results suggest that guanine-nucleotide-binding protein beta-gamma-subunits may play an important and active role in mediating the stimulation of phospholipase C by heterotrimeric guanine-nucleotide-binding proteins.