Inhibition of calcium release-activated calcium current by Rac/Cdc42-inactivating clostridial cytotoxins in RBL cells.

Using large clostridial cytotoxins as tools, the role of Rho GTPases in activation of RBL 2H3 hm1 cells was studied. Clostridium difficile toxin B, which glucosylates Rho, Rac, and Cdc42 and Clostridium sordellii lethal toxin, which glucosylates Rac and Cdc42 but not Rho, inhibited the release of hexosaminidase from RBL cells mediated by the high affinity antigen receptor (FcepsilonRI). Additionally, toxin B and lethal toxin inhibited the intracellular Ca(2+) mobilization induced by FcepsilonRI-stimulation and thapsigargin, mainly by reducing the influx of extracellular Ca(2+). In patch clamp recordings, toxin B and lethal toxin inhibited the calcium release-activated calcium current by about 45%. Calcium release-activated calcium current, the receptor-stimulated Ca(2+) influx, and secretion were inhibited neither by the Rho-ADP-ribosylating C3-fusion toxin C2IN-C3 nor by the actin-ADP-ribosylating Clostridium botulinum C2 toxin. The data indicate that Rac and Cdc42 but not Rho are not only involved in late exocytosis events but are also involved in Ca(2+) mobilization most likely by regulating the Ca(2+) influx through calcium release-activated calcium channels activated via FcepsilonRI receptor in RBL cells.

Cross-linking of the high affinity IgE receptor (Fc⑀RI) 1 by antigen-binding induces a cascade of morphological and biochemical reactions, finally resulting in degranulation (1). Several signaling events have been described, which appear to be essential for Fc⑀RI-mediated degranulation, including activation of protein tyrosine kinases and increase of intracellular Ca 2ϩ (Refs. 2 and 3; for review, see Refs. 4 and 5). Activation of Fc⑀RI leads to the stimulation of phospholipase C␥ and an increase of IP 3 levels, which depletes endoplasmic Ca 2ϩ stores.
Subsequently, a transient increase in the intracellular concentration of Ca 2ϩ ([Ca 2ϩ ] i ) is followed by a sustained plateau which reflects the entry of extracellular Ca 2ϩ (6). This Ca 2ϩ entry is suggested to depend on the depletion of intracellular Ca 2ϩ stores and is termed capacitative Ca 2ϩ entry (for review, see Refs. 7 and 8). The inward Ca 2ϩ current in RBL cells that seems to contribute to this entry is designated I CRAC for calcium release-activated calcium current. However, the regulatory mechanisms leading to activation of I CRAC is still unclear. Small GTPases have been suggested to participate in receptormediated Ca 2ϩ influx in RBL cells and mast cells (9).
The small GTPases of the Rho family including Rho, Rac, and Cdc42 play important roles in regulation of the actin cytoskeleton (10). RhoA participates in growth factor-mediated formation of stress fibers and cell adhesions (11), Rac regulates membranes ruffling and lamellipodia (12), and Cdc42 controls the formation of filopodia (13). In addition, Rho GTPases participate as molecular switches in various signaling processes including regulation of phospholipase D (14), phospholipase C␤ 2 (15), phosphoinositide 3-kinase (16,17), and phosphatidylinositol-4-phosphate 5-kinase (18). Furthermore, Rho subfamily proteins are involved in activation of transcription, cell cycle progression, and transformation (for review, see Refs. 10 and 19). The low molecular mass GTP-binding proteins of the Rho family (Rho, Rac, Cdc42) appear to be involved in activation of mast cells and RBL cells (20). Introduction of Rac into permeabilized mast cells cause secretion (21), and expression of dominant inhibitory forms of Cdc42 and Rac1 inhibits antigeninduced degranulation (22).
Here we studied the effects of toxins on degranulation, Ca 2ϩ mobilization, and I CRAC in RBL 2H3 hm1 cells. We report that toxin B and lethal toxin but not the Rho-modifying chimeric toxin C2IN-C3 inhibit secretion and increase of [Ca 2ϩ ] i by the Fc⑀RI receptor in RBL cells. Moreover, the toxins inhibit thapsigargin-induced Ca 2ϩ mobilization and the activation of I CRAC by depletion of intracellular Ca 2ϩ stores, indicating that Rac/ Cdc42 but not Rho participates in regulation of capacitative Ca 2ϩ entry.
Cell Culture-Rat Basophilic Leukemia cells transfected with the human muscarinic receptor (34) (RBL 2H3 hm1, a gift from Dr. G. Schultz (Institut fü r Pharmakologie, Freie Universitä t Berlin, Berlin, Germany) and Dr. P. Jones (University of Vermont, Burlington, VT)) were grown in Eagle's minimum essential medium with Earle's salts supplemented with 15% (v/v) heat-inactivated fetal calf serum, 4 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin in a humidified atmosphere of 5% CO 2 at 37°C. RBL 2H3 m1 cells were detached from culture plates with SK buffer (125 mM NaCl, 1.5 mM EDTA, 5.6 mM glucose, 10 mM HEPES, pH 7.2); no trypsin was used in order to avoid a partial destruction of membrane receptors. Subconfluent cells were preloaded with anti-TNP IgE (0.3 g/ml) 12-24 h prior to antigen stimulation experiments. Thereafter, the medium was changed and the cells were treated with toxins for the indicated times and concentrations.
Treatment with Toxins-RBL cells were treated with C. difficile toxin B (40 ng/ml, 2-4 h), C. sordellii lethal toxin (40 ng/ml, 2-4 h) C. botulinum C2 toxin (100 ng/ml C2I and 200 ng/ml C2II 4 h), or C. limosum C3 exoenzyme (100 ng/ml C2I and 200 ng/ml C2IN-C3, 4 h) for the indicated times and concentrations. After toxin treatment, cells were washed with the appropriate buffer and used for the assays. To compare properly the effects of toxins, paired experiments were carried out on control and toxin-treated cells that were grown under identical conditions. Additionally, the experiments were repeated with cells of at least two independent passages.
Hexosaminidase Release Assay-Cells were seeded in 96-well culture plates and incubated without or with toxins for the indicated times and concentrations. Hexosaminidase release was determined as described (35). Briefly, the medium was removed, and cells were washed two times with Tyrode buffer (130 mM NaCl, 5 mM KCl, 1.4 mM CaCl 2 , 1 mM MgCl 2 , 5.6 mM glucose, 10 mM HEPES, and 0.1% bovine serum albumin, pH 7.4). Incubation at 37°C with stimuli at the indicated concentrations followed for 1 h. Thereafter aliquots (30 l) of cells were incubated with 50 l of 1.3 mg/ml p-nitrophenyl-N-acetyl-␤-D-glucosamide in 0.1 M sodium citrate buffer (pH 4.5) at 37°C for 1 h. At the end of the incubation, 50 l of 0.4 M glycine (pH 10.7) stop buffer was added. The total amount of hexosaminidase release was determined using 2% Triton X-100 in Tyrode buffer. Absorbance was measured at 410 nm, referring to 630 nm. The values were expressed as percentage of total amount of hexosaminidase.
Measurements of [Ca 2ϩ ] i in Cell Suspension-RBL cells were detached from culture plates with SK buffer (125 mM NaCl, 1.5 mM EDTA, 5.6 mM glucose, 10 mM HEPES, pH 7.2). Following centrifugation, cells were washed and resuspended in serum-free minimal essential medium and loaded with fura-2 acetoxymethylester (2.5 M) for 45 min at 37°C. Then, cells were washed three times with HEPES-buffered salt solution (130 mM NaCl, 5.4 mM KCl, 0.9 mM NaH 2 PO 4 , 0.8 mM MgSO 4 , 1.8 mM CaCl 2 , 10 mM glucose, and 20 mM HEPES, pH 7.4), and cell density was adjusted to 10 6 cells/ml. Experiments were carried out at room temperature in HEPES-buffered salt solution using a Perkin Elmer LS 50B spectrofluorimeter. The fluorescence of cells suspension was examined at an emission wavelength of 510 nm and excitation wavelengths of 340 and 380 nm, respectively. Results are presented as changes in fluorescence ratio 340/380 over time.

Measurements of [Ca 2ϩ ] i in Attached
Cells-RBL cells were seeded on coverslips, and the intracellular Ca 2ϩ was measured at room temperature 2 days later using an cell-imaging system (Till Photonics, Planegg, Germany). In the day of experiments, the cells were incubated in medium containing fura-2 acetoxymethylester (4 M) for 1 h at room temperature. Subsequently, the culture medium was replaced by a bath solution with a Ca 2ϩ concentration of Ͻ10 nM (zero Ca o : 115 mM NaCl, 0.5 mM EGTA, 2 mM MgCl 2 , 5 mM KCl, 10 mM HEPES, pH 7.2 (NaOH)). The Ca 2ϩ concentration in bath was increased to 1 mM (1 mM Ca o : 115 mM NaCl, 1 mM CaCl 2 , 2 mM MgCl 2 , 5 mM KCl, 10 mM HEPES, pH 7.2 (NaOH)) during the fluorescence measurements. Images of 10 -25 cells/ coverslip were obtained every 3 s at an emission wavelength of 510 nm and excitation wavelengths of 340 and 380 nm, respectively. The fluorescence ratios were calibrated in vivo as described previously (38). Experiments were paired by alternating Ca 2ϩ measurements in control and toxin-treated coverslips. The data were pooled for statistical analysis and are given as mean Ϯ S.E.
Patch-clamp Techniques-Ionic currents were measured in whole cell mode (39) using an EPC-9 amplifier (HEKA Electronic, Lambrecht, Germany). Whole cell recordings were conducted at room temperature 2-3 days after plating the RBL cells in plastic dishes. For most experiments, depletion of Ca 2ϩ stores was induced by cell dialysis through the patch-clamp pipettes (1-2 megohms) with 20 M IP 3 ,115 mM CsCl, 4 mM MgCl 2 , 10 mM EGTA, and 10 mM HEPES (pH 7.2 (CsOH)). When cells were stimulated with the antigen, the dialysate contained no IP 3 , instead 300 M GTP⅐Na and 2 mM ATP⅐Mg plus 2 mM MgCl 2 were added. The bath solution contained 10 mM CaCl 2 , 115 mM NaCl, 2 mM MgCl 2 , 5 mM KCl, and 10 mM HEPES (pH 7.2 (NaOH)). Liquid junction potentials were corrected a posteriori (40). The membrane potential was clamped at 0 mV throughout the experiments and whole cell currents were scanned with ramps from ϩ80 mV to Ϫ100 mV (0.9 V/s). Since the equilibrium potential for chloride (E Cl ) was 0 mV, a current segment of 15 ms was recorded at the holding potential of 0 mV before each ramp was applied. After breaking into whole cell, 12 ramps were delivered every 2 s to obtain reliable leak currents for subsequent leak subtraction. Thereafter, the development of the whole cell currents was followed with ramps delivered every 4 s. Whole cell currents were sampled at 10 kHz and filtered at 1.5 kHz. Series resistance (R s ) and whole cell membrane capacitance (C m ) were electronically compensated (40 -50%) before each ramp.
The experiments were scheduled to obtain a similar number of whole cell recordings with control and toxin-treated cells in the same day. Typically, the size (C m ) of the RBL 2H3 hm1 cells corresponds to 10 -20 pF and was not changed by the treatment with toxins (controls, 15.3 Ϯ 0.7 pF, n ϭ 19; C2IN-C3, 13.7 Ϯ 1.4 pF, n ϭ 8; toxin B, 15.7 Ϯ 0.8 pF, n ϭ 22; lethal toxin, 16.6 Ϯ 1.2 pF; n ϭ 12). Immediately after breaking into whole cell, an outward current component was usually observed in RBL 2H3 hm1 cells (Fig. 5A) but not in RBL-3 cells (data not shown). This outward current was not apparently modified by the treatment with toxins (Fig. 5B) and disappeared within the first 4 s of recording. Therefore, the currents obtained at 4 -6 s of recording were used for leak subtraction when IP 3 was dialyzed into the cells. In the experi-ments with the antigen, whole cell currents were recorded for 60 s before antigen stimulation and averaged for leak subtraction. Whole cell recordings longer than 60 s showed subthreshold activation of currents probably by depletion of Ca 2ϩ stores passively induced by EGTA present in the dialysate. Cells that were not exposed to anti-TNP IgE (n ϭ 6) did not respond to antigen stimulation. To obtain current densities, the leak-subtracted currents were normalized to cell size using the corresponding C m values, except in Fig. 5, which shows raw data. Current densities at Ϫ80 mV represent the mean value obtained within a window of 5 mV placed around Ϫ80 mV. The holding currents recorded before each ramp were used for calculations of current densities at 0 mV. Only cells with R s below 7 megohms were pooled for statistical analysis. Data are given as mean Ϯ S.E.

RESULTS
To obtain information about the involvement of Rho GT-Pases in RBL cell activation, we applied the glucosylating C. difficile toxin B and C. sordellii lethal toxin, which differ in substrate specificity. Treatment of RBL cells with toxin B (40 ng/ml) or with lethal toxin (40 ng/ml) inhibited the release of hexosaminidase induced by stimulation of the Fc⑀RI (Fig. 1A). Using UDP-[ 14 C]glucose and Western blotting, we confirmed that in RBL cells, lethal toxin glucosylates the Rho GTPases Rac and possibly Cdc42 but not Rho. In addition, Ral and Ras (detected by immunoprecipitation, see Fig. 2B) were substrates for lethal toxin (Fig. 2, A and B). As reported recently (36), toxin B modified Rho, Rac, and Cdc42 but not Ras proteins in RBL cell lysates. In contrast to inhibition of secretion by toxin B and lethal toxin, the C3 fusion toxin C2IN-C3 (27), that selectively and completely ADP-ribosylated Rho (Fig. 2C) but not Rac and Cdc42 had no effect on secretion induced by TNP-OVA (Fig. 1B). Thus, these findings confirmed that Rac/Cdc42 but not Rho are essential for secretion in RBL cells.
Since the increase in [Ca 2ϩ ] i is a prerequisite for secretion in RBL cells, we studied the effects of the lethal toxin and toxin B on the Ca 2ϩ mobilization induced by TNP-OVA in RBL cells.
Because treatment of RBL cells with clostridial toxins affect the cytoskeleton possibly interfering with the adherence of cells, we first tested the effects of the toxins in suspension. Under these conditions and in the presence of extracellular Ca 2ϩ , toxin B and lethal toxin blocked the Ca 2ϩ mobilization (Fig. 3A). Next, we tested the effects of C3 exoenzyme, which selectively modifies RhoA, -B, and -C but not Rac or Cdc42. Because cell accessibility of C3 exoenzyme is rather poor, we used the fusion toxin C2IN-C3, which is able to enter cells readily and shows the same substrate specificity as C3 (27). As shown in Fig. 3B, C2IN-C3 did not affect TNP-OVA-induced Ca 2ϩ mobilization. The glucosylating cytotoxins affect the actin cytoskeleton in many cell types including RBL cells (23); therefore, we studied the role of the redistribution of the actin cytoskeleton on the Ca 2ϩ response using C. botulinum C2 toxin, which ADP-ribosylates actin and induces depolymerization of actin filaments (33). C2 toxin did not inhibit but rather increased the late phase of the Ca 2ϩ transients induced by TNP-OVA (Fig. 3C).
The effects of the toxin B and lethal toxin shown in Fig. 3 can be explained by inhibition of one or several steps in the intracellular signaling cascade initiated by stimulation of the Fc⑀RI receptor and leading to mobilization of intracellular Ca 2ϩ . To test a possible effect of toxin B and lethal toxin on the signaling cascade between release of Ca 2ϩ from intracellular stores and activation of capacitative Ca 2ϩ influx, we used thapsigargin, a potent inhibitor of Ca 2ϩ ATPases involved in Ca 2ϩ storage, that is frequently used to induce opening of store-regulated Ca 2ϩ channels in a receptor-independent manner (41). Moreover, thapsigargin and antigen reportedly activate the same store-regulated Ca 2ϩ influx in RBL cells (42). As shown in Fig.  4A, toxin B and lethal toxin inhibited the thapsigargin-induced Ca 2ϩ mobilization. Again, thapsigargin-induced Ca 2ϩ mobilization was not inhibited by C2 toxin (Fig. 4B). This finding supported the view that toxin B and lethal toxin likely affect the capacitative Ca 2ϩ influx underlying the Ca 2ϩ mobilization induced by Fc⑀RI receptor stimulation. Therefore, we studied the effects of the toxins in patch clamp experiments to detect a possible inhibition of I CRAC . The depletion of Ca 2ϩ stores was induced in a receptor-independent manner by cell dialysis with IP 3 (Fig. 5). Typically, the currentvoltage relationships changed very rapidly reflecting the increase of inward currents both in control and toxin-treated cells and steady-state current levels were attained after 60 s after beginning of the dialysis of IP 3 (Fig. 5A). No difference in the time course of current activation was observed between control and toxin treated cells, except that the steady-state currents were consistently smaller in cells treated with toxin B (Fig. 5B). To analyze this difference, the current amplitudes were normalized to cell size (Fig. 6). In control and toxin-treated cells (Fig. 6A), the whole cell currents showed an inward rectification and reversed at strong positive potentials (data not shown), as described for I CRAC in various cell systems (43). When the whole cell currents of cells treated with toxin B and lethal toxin were scaled by a factor of 1.4 -1.7, the scaled currents superimposed on whole cell currents measured in paired control cells (Fig. 6A, inset). By contrast, I CRAC was not much changed by treatment with C2IN-C3 (Fig. 6A). When paired experiments were compared, the current densities of cells treated with toxin B and lethal toxin were 46% and 40% smaller than control current densities at Ϫ80 mV, respectively (Fig. 6B). At Ϫ80 mV, the current densities of cells treated with C2IN-C3 were not significantly smaller than controls but significantly larger than current densities of cells treated with toxin B and lethal toxin (p Ͻ 0.01). Since the equilibrium potential for chloride was 0 mV in the present experimental conditions, we compared also current densities at 0 mV in order to rule out a possible contamination of Cl Ϫ currents. Although the amplitude of steady-state currents at 0 mV is small in RBL cells (5-10 pA), the current densities of cells treated with toxin B and lethal toxin but not with C21N-C3 were consistently smaller than current densities of control cells (control, Ϫ0.41 Ϯ 0.23 pA/pF, n ϭ 17; C2IN-C3, Ϫ0.35 Ϯ 0.21 pA/pF, n ϭ 8; toxin B, Ϫ0.25 Ϯ 0.17 pA/pF, n ϭ 18; lethal toxin, Ϫ0.21 Ϯ 0.14 pA/pF, n ϭ 9). Same inhibition by the glucosylating toxins was observed when I CRAC was activated by stimulation of the Fc⑀RI receptor with TNP-OVA in cells primed with anti-TNP-IgE (Fig. 7) instead of depletion of Ca 2ϩ stores induced by dialysis of IP 3 (Figs. 5 and 6). These results strongly indicate both toxin B and lethal toxin but not C2IN-C3 inhibited I CRAC in RBL cells, probably by interfering at steps of the signaling cascade between depletion of Ca 2ϩ stores and activation of CRAC channels in the plasma membrane of RBL cells. Furthermore, since in contrast to the effects of the clostridial cytotoxins, the fusion toxin C2IN-C3 did not reduce whole cell currents, it is likely that Rac and Cdc42 but not Rho are involved in the activation of I CRAC following stimulation of the Fc⑀RI receptor.  The Ca 2ϩ signals of RBL cells are normally characterized by a biphasic response. The initial transient phase due largely to a Ca 2ϩ release from IP 3 is usually followed by a sustained plateau due to capacitative Ca 2ϩ entry through CRAC channels (43,44). Since the experiments shown in Fig. 5-7 indicated that toxin B and lethal toxin inhibited the activation of I CRAC , we tested whether the capacitative Ca 2ϩ entry is modified by toxin B. In the absence of extracellular Ca 2ϩ , stimulation of the Fc⑀RI receptor of adherent RBL cells by TNP-OVA caused a small and transient increase in [Ca 2ϩ ] i due to release of Ca 2ϩ from intracellular stores (Fig. 8A). After increasing the extracellular Ca 2ϩ concentration to 1 mM, the capacitative Ca 2ϩ entry was observed in control and toxin-treated cells (Fig. 8A). Treatment of RBL cells with toxin B did not effect the release of Ca 2ϩ from intracellular stores observed in the absence of extracellular Ca 2ϩ but inhibited the capacitative Ca 2ϩ entry in the presence of 1 mM [Ca 2ϩ ] o by about 52% (Fig. 8B). These results support the view that toxin B and lethal toxin reduce the Ca 2ϩ mobilization induced by activation of the Fc⑀RI receptor mainly by inhibiting I CRAC and consequently the capacitative Ca 2ϩ entry in RBL cells. DISCUSSION Several studies have demonstrated that Rho GTPases are involved in Fc⑀RI-mediated activation of RBL cells and mast cells (22,45,46). Secretion from permeabilized mast cells were increased by dominant active Rac and Rho proteins (20). Gomperts and co-workers suggested Rac and Cdc42 as candidates for "G E ," a GTP-binding protein, mediating exocytosis in cells of hematopoietic origin (21). A role of small GTPases of the Rho family in RBL and mast cell activation was supported by recent findings that toxin B, which glucosylates and inactivates Rho GTPases, completely blocks secretion in these cells (36). However, the precise role of Rho GTPases in activation of RBL cells is still a matter of debate. Here we studied the effects of clostridial cytotoxins that inactivate Rho GTPases on the Ca 2ϩ mobilization in RBL 2H3 hm1 cells. Our findings indicate that Rho GTPases play an essential role in the Ca 2ϩ response of high affinity IgE receptors. Toxin B as well as lethal toxin inhibited not only the TNP-OVA-induced secretion but also the increase in [Ca 2ϩ ] i . The effects of the toxins were much stronger in the presence of extracellular Ca 2ϩ suggesting an action on Ca 2ϩ entry rather than on release from internal Ca 2ϩ stores. Toxin B modifies all Rho GTPases known including Rho, Rac, and Cdc42 subtypes (29); lethal toxin inactivates Rac and, to a minor extent, Cdc42 but not Rho. In addition, Ras subfamily proteins like Ras and Ral are modified by lethal toxin (30). Thus, our data indicate that Rac (or Cdc42) but not Rho is involved in the toxins' effects on Ca 2ϩ mobilization. This notion is supported by the findings that the fusion toxin C2IN-C3, which selectively inactivates RhoA, -B, and -C, was without effect on secretion and Ca 2ϩ mobilization (27,47). Rho GTPases are master regulators of the actin cytoskeleton. Therefore, it was tested whether redistribution of the actin cytoskeleton plays a major role in toxin-caused inhibition of the Ca 2ϩ re-sponse by Fc⑀RI activation. Because C2 toxin, which causes depolymerization of actin filaments (33), showed no inhibition but rather an increase in Ca 2ϩ mobilization, we conclude that the role of the Rho GTPases in the Ca 2ϩ response is largely independent of the actin cytoskeleton.
Importantly, we observed that the clostridial cytotoxins also inhibited thapsigargin-induced increase in [Ca 2ϩ ] i . Thapsigargin blocks sarco-endoplasmic reticulum Ca 2ϩ -ATPases (41) and thereby promote influx of Ca 2ϩ in RBL cells by passive depletion of IP 3 -sensitive Ca 2ϩ stores (42,48). Thus, our data suggested that the toxin-sensitive step, e.g. the site of action of Rac/Cdc42 in regulation of Ca 2ϩ mobilization, may be located downstream of IP 3 production. To further substantiate this hypothesis, we performed whole cell patch-clamp experiments to characterize the CRAC channel suggested to be involved in ligand-regulated Ca 2ϩ entry in RBL cells. In line with the findings on the Ca 2ϩ mobilization determined with the Fura method, we observed inhibition of the TNP-OVA-induced increase in I CRAC by the clostridial cytotoxins. Moreover, IP 3induced activation of the CRAC channel was inhibited by the Rac/Cdc42-modifying toxins, suggesting a role of the GTPases in this process.
So far the precise regulatory functions of Rac/Cdc42 in Ca 2ϩ responses are unclear. At least three models have been proposed for signaling capacitative Ca 2ϩ entry (7,49). First, it was suggested that a diffusible signaling factor (calcium influx factor) is generated and released from the endoplasmic reticulum (50). Second, conformational coupling model was proposed in which the endoplasmic reticulum IP 3 receptor directly interacts with the Ca 2ϩ channel in the plasma membrane (51). Finally, recent studies suggest a secretion-like coupling leading to fusion of vesicles containing Ca 2ϩ channels with the plasma membrane, thereby allowing Ca 2ϩ entry (52,53).
Rho proteins including Rho, Rac, and Cdc42 have been reported to regulate endocytic and/or exocytic events (54 -56). Moreover, these GTPases have been suggested to be involved in Ca 2ϩ signaling by various receptors. Recent studies on the Fc␥ receptor signaling indicate that Rho GTPases participate in the Ca 2ϩ response in J774 macrophages (57). In these cells, Rho itself appears to be essential, because microinjection of C3 exoenzyme blocked the Fc␥ receptor-mediated Ca 2ϩ response. As reported in the present article, however, in RBL cells, ADPribosylation of Rho by the fusion toxin C2IN-C3 neither effected Ca 2ϩ signaling nor secretion. In HeLa cells, transfection of dominant negative N17Rac blocked Ca 2ϩ influx stimulated by epidermal growth factor, suggesting an essential role of Rac in Ca 2ϩ response mediated by the receptor tyrosine kinase (58).
Thus, it appears that the involvement of Rho GTPases in Ca 2ϩ mobilization largely depends on the cell type studied.
In summary, we show that Rac/Cdc42 but not Rho are not only essential for late secretory events of RBL cell activation but also control specifically the Ca 2ϩ response induced by the high affinity IgE receptor (Fc⑀RI).