Effects of Large Clostridial Cytotoxins on Activation of RBL 2H3-hm1 Mast Cells Indicate Common and Different Roles of Rac in Fc RI and M1-Receptor Signaling

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 2H3hm1 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 (Van Aelst and D’Souza-Schorey, 1997; Bishop and Hall, 2000). They are critically involved in the regulations of the actin cytoskeleton by extracellular signals (Kaibuchi et al., 1999) but function as switches also in various other signal processes (Nobes and Hall, 1995; Kjoller and Hall, 1999; Bokoch, 2000). Clostridial cytotoxins are established as pharmacological tools to study the function and the role of Rho GTPase proteins in signal transduction pathways. C3 transferases selectively ADP-ribosylate RhoA, RhoB, and RhoC, thereby inhibiting their biological functions (Aktories et al., 1989; Chardin et al., 1989; Paterson et al., 1990). The family of large clostridial cytotoxins inactivates small GTPases by glucosylation (Aktories and Just, 1995). Whereas Clostridium difficile toxins A and B inactivate all Rho GTPases, including Rho, Rac, and Cdc42 (Just et al., 1995), the lethal toxin from Clostridium sordellii inactivates Rac, possibly Cdc42, but not Rho (Just et al., 1996). In addition, Ras subfamily proteins (e.g., Ras, Ral, and Rap) are targets of the