(Received for publication, November 1, 1995; and in revised form, December 27, 1995)
From the
Treatment of rat basophilic leukemia (RBL) 2H3-hm1 cells with Clostridium difficile toxin B (2 ng/ml), which reportedly
depolymerizes the actin cytoskeleton, blocked
[H]serotonin release induced by
2,4-dinitrophenyl-bovine serum albumin, carbachol, mastoparan, and
reduced ionophore A23187-stimulated degranulation by about
55-60%. In lysates of RBL cells, toxin B
C-glucosylated two major and one minor protein. By using
two-dimensional gel electrophoresis and immunoblotting, RhoA and Cdc42
were identified as protein substrates of toxin B. In contrast to toxin
B, Clostridium botulinum transferase C3 that selectively
inactivates RhoA by ADP-ribosylation did not inhibit degranulation up
to a concentration of 150 µg/ml. Antigen-stimulated tyrosine
phosphorylation of a 110-kDa protein was inhibited by toxin B as well
as by the phosphatidylinositol 3-kinase inhibitor wortmannin.
Depolymerization of the microfilament cytoskeleton of RBL cells by C. botulinum C2 toxin or cytochalasin D resulted in an
increased [
H]serotonin release induced by
antigen, carbachol, mastoparan, or by calcium ionophore A23187, but
without affecting toxin B-induced inhibition of degranulation. The data
indicate that toxin B inhibits activation of RBL cells by glucosylation
of low molecular mass GTP-binding proteins of the Rho subfamily (most
likely Cdc42) by a mechanism not involving the actin cytoskeleton.
Activation of rat basophilic leukemia (RBL) ()2H3-hm1
cells by antigen depends on cross-linking of IgE-receptor complexes and
a cascade of subsequent biochemical reactions that finally result in
secretion of histamine, serotonin, and other inflammatory substances
(for review, see (1) ). So far the precise signal transduction
pathway is not clear. As deduced from the data obtained in several
laboratories, Fc
RI stimulation appears to activate tyrosine
kinases (2, 3, 4, 5, 6) and
phospholipase C
(2, 7, 8, 9) with subsequent
mobilization of Ca
and protein kinase C and may
further involve phospholipase D (10) and/or phospholipase
A
(11, 12) activation. It has been
suggested that GTP-binding proteins are involved in the activation
cascade because GTP
S induces secretion in permeabilized mast
cells(13, 14) . This is particular true for the
activation of peritoneal mast cells by compound 48/80, which is blocked
by pertussis toxin(15, 16) . However, pertussis toxin
has apparently no effect on antigen-stimulated mast cell
activation(16) . Furthermore, some evidence has been presented
for the involvement of a GTP-binding protein in the late step of
secretion (13, 14) .
Low molecular mass GTP-binding
proteins of the Rho subfamily (RhoA, RhoB, RhoC, Rac1, 2, Cdc42Hs) are
involved in the regulation of the actin cytoskeleton(17) . Rho
regulates growth factor-induced formation of stress fibers (18, 19) and formation of cell adhesions(20) .
Rac is involved in membrane ruffling and
lamellipodia(17, 21) , and Cdc42 participates in
receptor-induced formation of microspikes(21) . Furthermore,
Rho subfamily proteins appear to be molecular switches in various
signal transduction pathways. They participate in cell-cell
contact(22) , sensitization of smooth muscle contraction toward
Ca(23) , and are involved in phospholipase D (24) and phosphatidylinositol-4-phosphate-5-kinase (25) regulation. Rho subfamily proteins are suggested to be
involved in signal transduction upstream (26) or downstream (27) from phosphatidylinositol 3-kinase. Moreover, it was shown
very recently that Rho subfamily members are activators of the MAP
kinase cascade(28) . Rho proteins (RhoA, RhoB, RhoC) are the
specific substrates for C3-like
transferases(29, 30, 31, 32, 33, 34) ,
which inactivate the GTP-binding proteins by ADP-ribosylation at
Asn
(35) .
The enterotoxin A and the cytotoxin B are the major virulence factors of Clostridium difficile, which is the organism causing the antibiotic-associated diarrhea and pseudomembranous colitis(36) . Recently, it was reported that C. difficile toxin A and B monoglucosylate Rho subfamily proteins(37, 38) . Modification of Rho by these toxins prevents subsequent ADP-ribosylation by C3-like transferases and inhibits the biological activity of the low molecular mass GTP-binding proteins(39, 40) . Because C. difficile toxins are able to enter RBL cells readily, we used toxin B as a novel tool for studying the involvement of low molecular mass GTP-binding proteins of the Rho family in antigen-induced signal transduction. Here we report that toxin B inhibits activation of RBL cells induced by various activators in a manner apparently independent of the actin cytoskeleton.
The
lysates from RBL cells (50 µg) were incubated in buffer (3 mM MgCl, 1 mM EDTA, 1 mM dithiothreitol, 50 mM triethanolamine/HCl, pH 7.5)
containing 1 µM [
-
P]NAD (0.5
µCi) and 1 µg/ml ADP-ribosyltransferase C3 for 15 min at 37
°C.
For ADP-ribosylation of actin, cell lysates were incubated
in buffer (2 mM MgCl 1 mM dithiothreitol, 10 mM thymidine, 20 mM triethanolamine/HCl, pH 7.5) containing 5 µM [
-
P]NAD (0.5 µCi) and 1 µg/ml
ADP-ribosyltransferase C2I for 15 min at 37 °C.
[
P]ADP-ribosylated proteins were visualized
after 12.5% SDS-PAGE by autoradiography.
In RBL cells preloaded with
[H]serotonin and sensitized with anti-DNP IgE,
DNP-BSA stimulated serotonin release in a concentration-dependent
manner. A maximal effect was observed at 30 ng/ml. In line with
previous reports, serotonin release was reduced at higher concentration
of DNP-BSA (Fig. 1A). After treatment with C.
difficile toxin B (3 ng/ml), Fc
RI-mediated degranulation of
RBL cells was completely inhibited (Fig. 1A). Toxin B
inhibited serotonin release at concentrations as low as 0.3 ng/ml,
indicating the specificity of the effect. Maximal inhibition (>90%)
was observed at 2 ng/ml after 90 min of incubation (not shown). The
time course of the toxin B effect was characterized by a delay of about
30 min, which was most likely due to binding and transfer of the toxin
into cells (Fig. 1B). RBL cell activation by carbachol
(1 mM) and mastoparan (20 µM), respectively, were
also completely blocked by toxin B (3 ng/ml) (Table 1). In
contrast, toxin B inhibited serotonin secretion stimulated by the
calcium ionophore A23187 maximally by about 50%. Under these
conditions, toxin B caused a half-maximal and maximal inhibition at
about 0.5 and 2.0 ng/ml, respectively (Fig. 2A). The
inhibitory effect of toxin B was largely independent of the
concentration of the ionophore (Fig. 2B).
Figure 1:
A,
effects of C. difficile toxin B on DNP-BSA-induced
[H]serotonin release. RBL cells were incubated
overnight with 2 µCi/ml [
H]serotonin and
primed by DNP-specific IgE. After treatment of cells without (control,
) and with toxin B (3 ng/ml,
) for 2 h, medium was removed
and cells were washed three times. Degranulation was initiated by
addition of DNP-BSA at the indicated concentrations for 25 min at 37
°C. Thereafter, supernatant was removed, and the amount of
[
H]serotonin released was determined by
scintillation counting. Data are means ± S.E. of three separate
experiments and are given as percent of the total amount of serotonin
after correction for spontaneous release (1-3% of total amount). B, time dependence of C. difficile toxin B effects on
DNP-BSA-induced [
H]serotonin release. RBL cells
were incubated overnight with 2 µCi/ml
[
H]serotonin and primed by DNP-specific IgE.
After treatment without (control,
) or with toxin B (3 ng/ml)
(toxin B,
) for the indicated times, medium was removed and cells
were washed three times. Incubation with 30 ng/ml DNP-BSA followed for
25 min at 37 °C. Thereafter, supernatant was removed and the amount
of [
H]serotonin released was determined by
scintillation counting. Data are mean ± S.E. of three separate
experiments and are represented as percent of the total amount of
serotonin after correction for spontaneous release (1-3% of total
amount).
Figure 2:
A,
concentration dependence of C. difficile toxin B effects on
A23187-induced [H]serotonin release. RBL cells
were incubated overnight with 2 µCi/ml
[
H]serotonin. After treatment without (first
point of curve) or with increasing concentrations of toxin B (
)
medium was removed and cells were washed three times. Degranulation was
initiated by addition of 300 nM A23187 for 25 min at 37
°C. Thereafter, supernatant was removed, and the amount of
[
H]serotonin released was determined by
scintillation counting. Data are means ± S.E. of three separate
experiments and are represented as percent of the total amount of
serotonin after correction for spontaneous release (1-3% of total
amount). B, effects of C. difficile toxin B on
A23187-induced [
H]serotonin release. RBL cells
were incubated overnight with 2 µCi/ml
[
H]serotonin. After treatment without (control,
) or with toxin B (3 ng/ml) (toxin B,
) for 2 h, medium was
removed, and cells were washed three times. Incubation with A23187 at
the indicated concentrations followed for 25 min at 37 °C.
Thereafter, supernatant was removed, and the amount of
[
H]serotonin released was determined by
scintillation counting. Data are means ± S.E. of three separate
experiments and are represented as percent of the total amount of
serotonin after correction for spontaneous release (1-3% of total
amount).
We have
shown, recently, that toxin A and B act on the cytoskeleton by
inactivation of Rho proteins(39, 40) . Furthermore, we
reported that toxin B glucosylates low molecular mass GTP-binding
proteins of the Rho subtype family by using UDP-glucose as
cosubstrate(40) . Therefore, we studied whether Rho proteins
are modified by toxin B also in RBL cells. To this end, lysates from
RBL cells, which were pretreated with toxin B (up to 2 h) until the
typical morphological changes occurred, were C-glucosylated by toxin B. As shown in Fig. 3A, toxin B induced the incorporation of
[
C]glucose into at least two proteins with M
of about 22,000. Pretreatment of intact RBL
cells with toxin B reduced subsequent incorporation of
[
C]glucose in the cell lysates, indicating
previous modification of proteins in intact cells. We attempted to
identify the glucosylated proteins by applying two-dimensional PAGE and
immunoblotting. Using anti-RhoA, the upper glucosylated band, which
showed a slightly more acidic isoelectric point than the major lower
band, was identified as RhoA, whereas the major lower band
cross-reacted with anti-Cdc42 antibody (Fig. 3B). A
third but minor glucosylated protein with a more acidic isoelectric
point than RhoA and an M
lower than that of Cdc42
was observed after long exposure. Additionally, RhoA was identified by
specific C3-induced ADP-ribosylation. It has been described that
glucosylation of Rho at threonine 37 blocks subsequent ADP-ribosylation
by C3 at asparagine 41(40) . As shown in Fig. 3C, this was also true for RBL cells. After
treatment of intact cells with toxin B, subsequent C3-catalyzed
ADP-ribosylation of Rho in cell lysates was inhibited in a toxin B
concentration-dependent manner. Thus, Rho was one of the protein
substrates of toxin B in intact RBL cells.
Figure 3:
A,
time dependence of toxin B-induced glucosylation of Rho subfamily
proteins in intact RBL cells. RBL cells were treated with toxin B (3
ng/ml) for the indicated times. Thereafter, the medium was removed,
cells were washed and scraped off in the presence of lysis buffer.
Lysates from control and toxin B-treated cells were incubated with 1
µg/ml toxin B and 30 µM UDP-[C]glucose for 1 h at 37 °C.
Thereafter, labeled proteins were analyzed by 15% SDS-PAGE.
PhosphorImager data from SDS-PAGE are shown. B, identification
of toxin B substrates by two-dimensional PAGE and immunoblotting.
Lysates from RBL cells were either glucosylated by toxin B or
ADP-ribosylated by C. botulinum transferase C3. The lysates
were incubated with 1 µg/ml toxin B and 30 µM UDP-[
C]glucose for 1 h at 37 °C.
ADP-ribosylation of the lysates was performed with 1 µg/ml C3 and
0.3 µM [
P]NAD (0.5 µCi) for 15
min at 37 °C. After incubation the proteins were separated on
two-dimensional gels and electroblotted on nitrocellulose. RhoA was
detected with anti-RhoA antibody (b) and Cdc42 with anti-Cdc42
antibody (c). Phosphorimaging of toxin B-catalyzed
[
C]glucosylation is shown in a and
C3-catalyzed ADP-ribosylation of Rho in d. Arrows in a indicate three labeled proteins: arrow 1, RhoA; arrow 2, Cdc42, short arrow, not identified. Arrows in b and d indicate RhoA. The arrow in c indicates Cdc42. The asterisk in c indicates a staining artifact that was not observed in
repeats. C, time dependence of the effects of C. difficile toxin B in intact RBL cells on subsequent C3-catalyzed
ADP-ribosylation of Rho in cell lysates. RBL cells were treated with
toxin B (3 ng/ml) for the indicated times. Thereafter, cell lysates
were [
P]ADP-ribosylated with C3 (1 µg/ml)
and 1 µM [
P]NAD (0.5 µCi).
Labeled proteins were analyzed by SDS-PAGE. PhosphorImager data from
SDS-PAGE are shown.
In order to clarify the
role of Rho subtype proteins in antigen-induced mast cell activation,
we incubated RBL cells with C3 at high concentrations (150 µg/ml)
for 36 h and tested the efficacy of the treatment by subsequent
[P]ADP-ribosylation of the cell lysate. Although
C3 treatment of intact cells caused significant reduction in labeling
of Rho in the cell lysates (Fig. 4), antigen-induced mast cell
activation was not affected by C3 (Table 1).
Figure 4:
ADP-ribosylation of Rho in intact RBL
cells. RBL cells were treated for 36 h with the indicated
concentrations of C3. Thereafter, ADP-ribosylation of the lysates was
performed with 1 µg/ml C3 and 1 µM [P]NAD (0.5 µCi) for 15 min at 37
°C, and labeled proteins were analyzed by SDS-PAGE. PhosphorImager
data from SDS-PAGE are shown.
It has been
suggested that Rho subfamily proteins are involved in the regulation of
tyrosine phosphorylation(44) . Therefore, we studied the
effects of toxin B on FcRI-mediated tyrosine phosphorylation by
detecting protein tyrosine phosphorylation with anti-phosphotyrosine
antibody. Antigen-receptor activation by DNP-BSA largely increased
tyrosine phosphorylation of various proteins (not shown). Fig. 5A shows that toxin B selectively blocked tyrosine
phosphorylation of a protein of about 110 kDa. Wortmannin, an inhibitor
of phosphatidylinositol 3-kinase, which was shown to block
antigen-induced mast cell activation (45) (Table 2),
similarly inhibited protein tyrosine phosphorylation of the 110-kDa
protein (Fig. 5B). However, in A23187-activated RBL cells,
tyrosine phosphorylation of several proteins were inhibited by toxin B,
but not by wortmannin. Moreover, toxin B and wortmannin inhibited
degranulation of RBL cells in an additive manner (Table 2),
indicating that both agents differ in their modes of action.
Figure 5: A, effects of C. difficile toxin B on tyrosine phosphorylation in RBL cells. Cells were primed overnight with anti-DNP IgE. After incubation without(-) or with toxin B [3 ng/ml] (+) for 2 h, cells were washed and stimulated for 3 min at 37 °C with DNP-BSA (500 ng/ml) or A23187 (0.5 µM). Thereafter, cells were lysed and proteins analyzed by SDS-PAGE. Immunoblotting was performed with anti-phosphotyrosine antibody and detected with the ECL Western blotting detection system (Amersham). Chemiluminogram is shown. Arrows indicate tyrosine phosphorylation of proteins which is inhibited after incubation with toxin B. B, effects of wortmannin (wort.) on tyrosine phosphorylation in RBL cells. Cells were incubated overnight with anti-DNP IgE. After incubation without (control (con.), 2 h) and with toxin B [3 ng/ml] (toxin B, 2 h) or wortmannin [100 nM, 15 min] cells were washed and stimulated for 3 min at 37 °C with DNP-BSA [500 ng/ml] or A23187 [0.5 µM]. Thereafter cells were lysed and proteins analyzed by SDS-PAGE. Immunoblotting was performed with anti-phosphotyrosine antibody and detection with the ECL Western blotting detection system (Amersham). Chemiluminogram of DNP-BSA-stimulated protein tyrosine phosphorylation is shown in I and of A23187-induced protein tyrosine phosphorylation in II. Arrows indicate inhibition of tyrosine phosphorylation of proteins by toxin B.
C.
difficile toxin B is a well known cytotoxin(46) . Its
cytotoxic effects are characterized by rounding up of cells and
disassembly of the actin cytoskeleton(47) . Because
cytoskeleton rearrangement is thought to be involved in FcRI
signal transduction (48, 49) , it was of interest to
clarify whether redistribution of the actin cytoskeleton is the
mechanism by which toxin B inhibits antigen-induced serotonin release.
Therefore, the influence of C2 toxin on Fc
RI-mediated RBL cell
activation was tested. C2 toxin ADP-ribosylates actin thereby
inhibiting actin polymerization and causing depolymerization of
F-actin(50, 51, 52, 53) . As shown
in Fig. 6A, treatment of RBL cells with C2 toxin, which
similarly induced rounding up of cells (not shown), substantially
increased the stimulated release of serotonin from RBL cells. This
effect was observed with antigen as well as with ionophore-stimulated
exocytosis, but was not caused by alteration of the basal serotonin
secretion. C2 toxin-induced [
P]ADP-ribosylation
of actin was largely reduced in lysates from RBL cells pretreated for 2
h with C2 toxin, indicating that cellular actin was ADP-ribosylated in
intact cells (Fig. 6B). A comparable increase in
serotonin release occurred in the presence of cytochalasin D (5
µM; not shown). However, even in the presence of C2 toxin, C. difficile toxin B inhibited antigen- and ionophore-mediated
serotonin release (Table 1). This effect was also observed when
RBL cells were pretreated with C2 toxin for 1 h (not shown). Thus, all
these findings suggest that microfilament depolymerization is not
causally involved in toxin B-induced inhibition of antigen-stimulated
serotonin release.
Figure 6:
A, effects of C. botulinum C2
toxin on DNP-BSA-induced [H]serotonin release.
Cultures were incubated overnight with 2 µCi/ml
[
H]serotonin and primed by anti-DNP-IgE. After
treatment without (control,
) or with C2 toxin (100 ng/ml C2I
+ 200 ng/ml C2II) (C2 toxin,
) for 2 h, medium was removed,
and cells were washed three times. Incubation with DNP-BSA at the
indicated concentrations followed for 25 min at 37 °C. Thereafter,
supernatant was removed, and the amount of
[
H]serotonin released was determined by
scintillation counting. Data are means ± S.E. of three separate
experiments and are represented as percent of the total amount of
serotonin after correction for spontaneous release (1-3% of total
amount). B, time dependence of ADP-ribosylation of actin by C. botulinum C2 toxin. RBL cells were incubated without or
with C2 toxin (100 ng/ml C2I + 200 ng/ml C2II) for the indicated
times. Thereafter, the medium was removed, and cells were scraped off
in the presence of lysis buffer. Lysates from control and C2
toxin-treated cells were incubated with 1 µg/ml C2I and 5
µM [
P]NAD for 15 min at 30 °C.
After incubation the proteins were separated by 12.5% SDS-PAGE.
PhosphorImager data from SDS-PAGE are shown. Incubation with enzyme
component (C2I) or binding component (C2II) alone was performed as
controls.
Here we report that the cytotoxin C. difficile toxin
B inhibits antigen-stimulated serotonin release from RBL 2H3-hm1 cells.
Because C. difficile toxins A and B have been shown to act on
Rho subfamily proteins(37, 38) , we conclude that
inhibition of IgE-stimulated serotonin release is caused by the action
of the toxin B on this family of GTP-binding proteins and suggest that
Rho subfamily proteins are involved in the FcRI signal pathway.
Involvement of Rho proteins in the action of C. difficile toxins was first deduced from the finding that the toxins inhibit
C3-induced ADP-ribosylation of Rho proteins(39, 40) .
Recently, the modification of Rho subfamily proteins by C.
difficile toxin was identified as monoglucosylation at threonine
37 of Rho. Here we show that C. difficile toxin B catalyzes
the glucosylation of Rho proteins in intact RBL cells. As deduced from
the inhibitory effect of toxin B on C3-induced ADP-ribosylation, which
is selective for RhoA, RhoB, RhoC, and from immunoblotting with
anti-RhoA antibody, we conclude that RhoA is one protein substrate for C. difficile toxin in RBL cells. In addition, glucosylation
and immunoblot studies indicate that also Cdc42 is modified by toxin B.
Additionally, a minor glucosylated protein was detected after extended
phosphorimaging of two-dimensional PAGE. So far, the nature of this
protein is not clear. It was shown that recombinant Rac is also
substrate for glucosylation by toxin B from C.
difficile(38) . However, applying two-dimensional PAGE and
immunoblotting with anti-Rac antibody, we did not detect glucosylated
Rac in RBL cell lysate.
Various GTP-binding proteins have been
postulated to be involved in exocytic processes from mast cells. A
pertussis toxin-sensitive G-protein is involved in mast cell activation
by compound 48/80 but not in FcRI-mediated signal
transduction(15, 16) . Studies with permeabilized mast
cells led to the hypothesis that a GTP-binding protein, G
,
which is part of the membrane fusion mechanism, is involved in
exocytosis(13, 14, 54) . However, whether
G
is actually a heterotrimeric G-protein or not remains to
be clarified. Furthermore, recent studies indicated that activation of
mast cells by compound 48/80 is paralleled by redistribution of the
actin filaments, a phenomenon which may involve Rho and Rac proteins
besides a heterotrimeric G-protein(55) . Moreover, Price et
al.(56) reported that the GTP-binding proteins Rac and
Rho are involved in GTP
S-induced secretion from permeabilized mast
cells. Our study with C. difficile toxins also suggests that
Rho subfamily proteins participate in RBL cell activation. However,
treatment of RBL cells with C3 at high concentrations, which resulted
in ADP-ribosylation of at least 90% of Rho in intact cells, did not
inhibit antigen-induced secretion from RBL cells. Therefore, we
conclude that glucosylation of Cdc42 rather than Rho is responsible for
inhibition of antigen-stimulated secretion in these cells. Because Rho
subfamily proteins (including Cdc42) are involved in regulation of the
actin cytoskeleton(17) , the question arises whether the
perturbation of the actin cytoskeleton by toxin B is responsible for
inhibition of antigen-stimulated secretion. We addressed this question
by using C. botulinum C2 toxin and cytochalasin D, which
directly act on actin. C2 toxin ADP-ribosylates G-actin, thereby
inhibiting actin polymerization and causing depolymerization of
microfilaments(50, 51) . Cytochalasin D acts like a
capping molecule on F-actin to inhibit actin
polymerization(57) . Because depolymerization of actin by C2
toxin or cytochalasin increased rather than decreased antigen- and
ionophore-stimulated secretion, the regulatory functions of Rho
subfamily proteins on the actin cytoskeleton are not causal for the C. difficile toxin-induced inhibition of RBL cell activation.
On the contrary, the data indicate that these small GTP-binding
proteins play a central role in early signal transduction processes, i.e. upstream of regulation of the actin cytoskeleton or
vesicle fusion processes. This is supported by the findings that
antigen-, carbachol-, and mastoparan-induced serotonin release were
completely inhibited by toxin B, whereas ionophore-induced release was
only partially affected. Rho subfamily proteins regulate various enzyme
systems involved in signal transduction for example
phosphatidylinositol-4-phosphate-5-kinase(25) , phospholipase
D(24) , or phosphatidylinositol 3-kinase(26) . Cdc42,
which appears to be the major substrate for toxin B-induced
glucosylation in RBL cells, was shown to interact with p85, the
regulatory component of phosphatidylinositol 3-kinase thereby
increasing kinase activity(58) . Therefore, we compared the
action of toxin B with wortmannin, which inhibits the catalytic subunit
of phosphatidylinositol 3-kinase (p110) (45) . As shown
earlier(45) , wortmannin potently blocked the
antigen-stimulated RBL cell secretion. However, at least two findings
indicate that toxin B and wortmannin might act on different signal
pathways. First, tyrosine phosphorylation pattern of
ionophore-activated RBL cells were different with both agents. Second,
toxin B and wortmannin inhibited activation of RBL cells in an additive
manner. Thus, the target GTPases of toxin B are not only involved in
phosphorylation reactions within the signal pathway of
phosphatidylinositol 3-kinase but may also regulate additional signal
pathways. However, at present it is not clear whether the decrease in
tyrosine phosphorylation observed after toxin B treatment is due to a
direct or indirect effect.
Taken together, we employed C.
difficile toxin B as a novel tool for studying the involvement of
low molecular mass GTP-binding proteins of the Rho subfamily in
FcRI-mediated cell activation. Complete inhibition of
antigen-induced activation indicates an essential role for these
regulatory GTPases in RBL cell activation. As Cdc42 and Rho are the
major targets for glucosylation by toxin B and Rho inactivation by C3
was without effect on secretion, we suggest that Cdc42 is basically
involved in signal transduction via the Fc
RI receptor in RBL
cells.