Characterization and purification from bovine neutrophils of a soluble guanine-nucleotide-binding protein that mediates isozyme-specific stimulation of phospholipase C β2

Abstract

Members of the β isozyme subfamily of phosphatidylinositol-specific phospholipase C (PLC) are stimulated by a subunits and βγ dimers of heterotrimeric guanine-nucleotide-binding proteins (G proteins). Myeloid differentiated human HL-60 granulocytes and bovine neutrophils contain a soluble phospholipase C, which is stimulated by the metabolically stable GTP analogue guanosine (5'→O)-3-thiotriphosphate (GTP[S]). To identify the component(s) involved in mediating this stimulation, the relevant polypeptide(s) was resolved from endogenous phospholipase C and purified from bovine neutrophil cytosol by measuring its ability to confer GTP[S] stimulation to exogenous recombinant PLCβ2. The resolved factor, which behaved as 48-kDa protein upon gel filtration, stimulated PLCβ2 but not PLCβ1, or PLCδ1. Activation of phosphatidylinositol 4-phosphate 5-kinase was not involved in this stimulation. The purified stimulatory factor consisted of two polypeptides of molecular masses of approximately 23 kDa and 26 kDa. The protein stimulated a deletion mutant of PLCβ2, that lacked a carboxyl-terminal region necessary for stimulation by members of the α(q) subfamily of the G-protein α subunits. The results of this study suggest that a GTP-binding protein distinct from α(q) subunits, probably a low-molecular-mass GTP-binding protein associated with a regulatory protein, is involved in isozyme-specific activation of PLCβ2.