The Guanine-Nucleotide-Binding Protein Subunit Galphai2 is Involved in Calcium Activation of Phospholipase A2. Effects of the Dominant Negative Galphai2 Mutant, [G203T]Galphai2, on Activation of Phospholipase A2 in Chinese Hamster Ovary Cells

Abstract

The mechanisms of activation of cytoplasmic phospholipase A2 (cPLA2) are complex and incompletely defined. In Chinese hamster ovary (CHO) cells, receptor stimulation of cPLA2 is due to the interaction of pathways involving the α subunits of at least two guanine-nucleotide-binding (G) proteins, Gαi2 and Gαq. Activation of cPLA2 is inhibited by pertussis toxin and Gαi2 mutants. In addition, activation of phospholipase C via Gαq results in increased intracellular calcium ([Ca2+]i) and activation of protein kinase C., both of which interact with and activate cPLA2. The present study was undertaken to analyze the mechanism of interaction of Gαi2 with the phospholipase-C-stimulated pathway in the activation of cPLA2. We addressed this question using a dominant negative Gαi2 mutant, [G203T]Gαi2, in which Gly203 is mutated to Thr. [G203T]Gαi2 inhibits ATP receptor activation of cPLA2. The effect of [G203T]Gαi2 was specific to Gαi2-activated pathways, as shown by its lack of effect on other purinergic receptor stimulated pathways: ATP stimulation of [Ca2+]i; or mitogen-activated protein kinase phosphorylation is unaltered by [G203T]Gαi2. We addressed the possibility that the activation of cPLA2 by Ca2+ and/or protein kinase C is dependent on Gαi2. Activation of cPLA2 by the Ca2+ ionophore, ionomycin, was inhibited by 61±9% (n=5) in [G203T]Gαi2-expressing cells; however the ionomycin-induced [Ca2+]i rise was unaffected by [G203T]Gαi2. Thus, [G203T]Gαi2 specifically inhibits Ca2+ activation of cPLA2. In contrast, activation of cPLA2 via protein kinase C by phorbol 12-myristate 13-acetate was unaffected by [G203T]Gαi2. Our results demonstrate that Ca2+ but not phorbol ester activation of cPLA2 in CHO cells is Gαi2-dependent. The possibility is discussed that Gαi2 is downstream of Ca2+ but upstream of protein kinase C activation of cPLA2.