Compound 48/80 Activates Mast Cell Phospholipase D via Heterotrimeric GTP-Binding Proteins

Abstract

Previous studies have indicated the presence of a cholera toxin-sensitive phospholipase D (PLD) in cultured RBL-2H3 mast cells that is synergistically activated via calcium, protein kinase C, and another unidentified signal. Here we identify a third potential signal for activation transduced by a pertussis toxin-sensitive trimeric GTP-binding protein, most likely via G_i2 or G_i3. Quercetin-treated RBL-2H3 cells in which expression of G_αi2 and G_αi3 is enhanced more than 7-fold respond to the G_i stimulant compound 48/80 with the activation of PLD, a transient activation of phospholipase C, and enhanced membrane GTPase activity. The activation of PLD was blocked in pertussis toxin-treated cells and, as with other stimulants of PLD, was enhanced in cholera toxin-treated cells. The PLD response to compound 48/80 was only partially inhibited by calcium deprivation and inhibition of protein kinase C to indicate a component of the response that was independent of calcium, protein kinase C, and, presumably, phospholipase C. Based on these and other data, we hypothesized that βγ-subunits, released from G_i2 or G_i3 by compound 48/80 or from G_s by cholera toxin, provide an additional signal for the activation of PLD. Consistent with this hypothesis, recombinant G_β2γ2 subunits, but not G_αi-3 subunits, at concentrations of 50 to 300 nM markedly synergized PLD activation by compound 48/80 in permeabilized RBL-2H3 cells.