Effects of large clostridial cytotoxins on activation of RBL 2H3-hm1 mast cells indicate common and different roles of Rac in FcepsilonRI and M1-receptor signaling. J Pharmacol Exp Ther 304

ABSTRACT Using Rho GTPases-inhibiting clostridial cytotoxins, we showed recently in RBL cells that the GTPase Rac is involved in Fc⑀RI (high-affinity receptor for IgE) signaling and receptormediated calcium mobilization, including influx via calcium release-activated calcium channels. Here, we studied the role of Rho GTPases in muscarinic M1 receptor signaling in RBL 2H3-hm1 cells. Clostridium difficile toxin B, which inactivates Rho, Rac, and Cdc42, and Clostridium sordellii lethal toxin, which inhibits Rac but not Rho, blocked M1-mediated exocytosis, indicating that Rac but not Rho is involved in the regulation of receptor-mediated exocytosis. Although antigen-induced Fc⑀RI stimulation caused tyrosine phosphorylation of the Rac guanine nucleotide exchange factor Vav, M1 stimulation by carbachol activated Rac independently of Vav. The Rac-inactivating toxins blocked M1 receptor-induced membrane translocation of the pleckstrin homology domain of protein kinase B, which is a phosphoinositide 3-kinase effector. The M1-induced calcium release from internal stores was not affected by toxin B; however, the subsequent calcium influx from the extracellular space was inhibited. The data suggest that besides capacitative calcium entry, the M1 signaling pathway activates further calcium entry channels with mechanisms that are not affected by the inhibition of Rac. The low molecular mass GTPases of the Rho family (e.g., Rho, Rac, and Cdc42) are molecular switches in many cellular signaling cascades Using the clostridial toxins as pharmacological tools, it was shown that exocytosis of RBL 2H3-hm1 cells induced by stimulation of the high-affinity antigen receptor Fc⑀RI involves Rho GTPases ABBREVIATIONS: PI3-kinase, phosphoinositide 3-kinase; PKB, protein kinase B; GEF, guanine nucleotide exchange factor; GST, glutathione S-transferase; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; PAK, p21-activated kinase; PH-PKB-GFP, pleckstrin homology domain of protein kinase B tagged with green fluorescent protein; [Ca 2ϩ ] i, cytoplasmic free calcium; TNP-OVA, trinitrophenyl-conjugated ovalbumin; PBS, phosphate-buffered saline; I CRAC , calcium release-activated calcium current; IP 3 , inositol 1,4,5-triphosphate