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

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 (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 , the lethal toxin from Clostridium sordellii inactivates Rac, possibly Cdc42, but not Rho . In addition, Ras subfamily proteins (e.g., Ras, Ral, and Rap) are targets of the lethal toxin.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 (Prepens et al., 1996). In Fc⑀RI signaling in RBL cells, Rac but not RhoA seems to be involved on various levels of the signal pathway, ultimately resulting in exocytosis. Rac is essential for Fc⑀RI-mediated calcium mobilization (Djouder et al., 2000). Rac participates in phospholipase C␥ activation (Hong-Geller et a...

Bacterial Toxins and the Nervous System: Neurotoxins and Multipotential Toxins Interacting with Neuronal Cells

“…Similarly, LT blocks spontaneous glutamate release in cultured rat cerebellar slices (Kojima and Poulain, unpublished observation). These observations are consistent with reports that Rho-acting toxins block the secretory process in many cell types, including mast cells [263,264,265,266]. In muscles isolated from mice previously injected with LT, nerve-evoked muscle twitch is blocked.…”

“…In these cells, exocytosis is triggered by a rise in intracellular Ca ++ levels induced by stimulation of either the Fc-epsilon receptor or muscarinic- type AChR. The Rac GTPase is the only one involved in Ca ++ mobilization within these cells, and the blockade of exocytosis by ToxB or LT is due to inhibition of capacitative Ca ++ entry exclusively through store-operated Ca ++ channels [263,270]. …”

Modulation of Ca2+ Signaling by Na+/Ca2+ Exchangers in Mast Cells

“…The Ca 2ϩ signals were initiated in RBL cells either by the blockade of sarco(endo)plasmic reticulum Ca 2ϩ -ATPases pumps in the internal Ca 2ϩ stores with thapsigargin or by activation of the stably expressed muscarinic receptors with carbachol (13,19). Fig.…”

“…A single cell imaging system (Till Photonics) was used to measure Ca i as previously described (13). Cells were loaded with fura 2-AM (5 M; Molecular Probes) for 40 min at room temperature.…”

“…RBL cells expressing the human muscarinic receptor (RBL-2H3-hm1) were cultured in ␣-MEM medium (Invitrogen Life Technologies) supplemented with 15% FBS and 1% L-glutamine at 37°C/5% CO 2 , as previously described (13). Isolation, differentiation and FACS analysis of BMMC were essentially performed as described (16).…”