Role of rho family GTP-binding proteins in IGE receptor-mediated phospholipase D activation in mast cells

To investigate the role of Rho family proteins in antigen-mediated phospholipase D (PLD) activation in cultured rat basophilic leukemia (RBL-2H3) mucosal mast cells, we used two toxins, Clostridium difficile toxin B (toxin B) and Clostridium botulinum C3 toxin (C3 toxin), which inhibit Rho family proteins by monoglucosylation and ADP-ribosylation, respectively. Pretreatment with toxin B caused rounding-out of RBL-2H3 cells, strong inhibition of antigen-induced PLD activation, and also a complete blockage of serotonin secretion. By contrast, C3 toxin was without effect on both PLD activation and serotonin secretion, although RhoA was markedly ADP-ribosylated. Recombinant ADP-ribosylation factor (Arf) stimulated the PLD activity in isolated membranes in a dose-dependent manner, and 4 beta-phorbol 12-myristate 13-acetate (PMA) pretreatment of cells potentiated this recombinant Arf effect. The recombinant Arf-and PMA-stimulated PLD activities were partially inhibited by pretreatment with toxin B but not by C3 toxin. Stimulation of RBL cells with antigen induced translocation to membranes of factors involving PLD activation, e.g. protein kinase C (PKC) (alpha, beta 2, delta, epsilon) isozymes, Cdc42 and Arf, but not RhoA. These results suggest that the membrane-translocation of Cdc42 plays a major role in antigen-induced PLD activation in RBL cells and also that the translocated Arf and PKCs exert a co-operative effect for PLD activation.