Activation of phospholipase C by G-protein βγ subunits in DDT 1MF-2 cells

Abstract

Adenosine A 1 receptors directly stimulate inositol phospholipid hydrolysos and Ca 2+ mobilization through a pertussis toxin sensitive mechanism in DDT 1MF-2 cells. In the present study we have investigated whether G protein βγ subunits (Gβγ) are capable of stimulating phospholipase C in DDT 1MF-2 cell membrane preparations using lipid vesicles containing [ 3H]phosphatidylinositol 4,5-bisphosphate. DDT 1MF-2 cell membrane and soluble fractions were found to contain phospholipase C activity which was stimulated by increases in free Ca 2+ ion concentration. Gβγ purified from bovine retinal transducin produced significant increases in phospholipase C activity in DDT 1MF-2 cell membranes. Gβγ-dependent activation of phospholipase C, while virtually absent in the presence of low Ca 2+ ion concentrations, increased markedly with increasing free Ca 2+ ion concentration. These data suggest that membrane bound phospholipase C in DDT 1MF-2 cells is sensitive to Ca 2+, and may be stimulated conditionally by Gβγ subunits i.e. Gβγ subunits activate the enzyme only in the presence of Ca 2+. Gβγ subunits also stimulated soluble phospholipase C in DDT 1MF-2 cells. These findings support the hypothesis that G i βγ subunits are involved in adenosine A 1 receptor stimulated phospholipase C/Ca 2+ signalling in DDT 1MF-2 cells.