(Received for publication, October 17, 1996)
From the Departments of Biochemistry and Molecular
Biology and ¶ Physiology and Biophysics, Wright State
University School of Medicine, Dayton, Ohio 45435
Pasteurella multocida toxin (PMT) has
been hypothesized to cause activation of a GTP-binding protein
(G-protein)-coupled phosphatidylinositol-specific phospholipase C (PLC) in intact cells. We used voltage-clamped Xenopus oocytes to test for direct PMT-mediated stimulation
of PLC by monitoring the endogenous
Ca2+-dependent Cl current.
Injection of PMT induced an inward, two-component Cl
current, similar to that evoked by injection of IP3 through
intracellular Ca2+ mobilization and Ca2+ influx
through voltage-gated Ca2+ channels. These PMT-induced
currents were blocked by specific inhibitors of Ca2+ and
Cl
channels, removal of extracellular Ca2+,
or chelation of intracellular Ca2+. Specific antibodies
directed against an N-terminal, but not a C-terminal, peptide of PMT
inhibited the toxin-induced currents, implicating that the N terminus
of PMT is important for toxin activity. Injection with specific
antibodies against PLC
1, PLC
2, PLC
3, or PLC
1 identified
PLC
1 as the primary mediator of the PMT-induced Cl
currents. Injection with guanosine
5
-O-(2-(thio)diphosphate), antibodies to the common
GTP-binding region of G-protein
subunits, or antibodies to
different regions of G-protein
subunits established the involvement
of a G-protein
subunit in PMT-activation of PLC
1. Injection with
specific antibodies against the
-subunits of Gq/11,
Gs/olf, Gi/o/t/z, or Gi-1/i-2/i-3
isoforms confirmed the involvement of Gq/11
.
Preinjection of oocytes with pertussis toxin enhanced the PMT response.
Overexpression of Gq
in oocytes could enhance the PMT
response by 30-fold to more than 300-fold, whereas introduction of
antisense Gq
cRNA reduced the response by 7-fold. The
effects of various specific antibodies on the PMT response were
reproduced in oocytes overexpressing Gq
.
Infections of Pasteurella multocida are associated with such severe diseases as pasteurellosis, dermonecrosis resulting from bite wounds, and the irreversible bone atrophy of progressive atrophic rhinitis (1). Purified P. multocida toxin (PMT)1 alone is sufficient to induce experimentally all of the major symptoms of atrophic rhinitis in animals (1, 2, 3, 4, 5). PMT appears to bind to and enter mammalian cells via receptor-mediated endocytosis (6, 7) and acts intracellularly to initiate DNA synthesis (7, 8, 9). Some of the events toward the eventual stimulation of DNA synthesis that occur upon exposure to PMT in cultured fibroblasts and osteoblasts are: enhanced hydrolysis of inositolphospholipids to increase the total intracellular inositol phosphates (9, 10, 11); mobilization of intracellular Ca2+ pools (9, 10, 11, 12); increased production of diacylglycerol (10, 11, 12); decreased ADP-ribosylation of GRP78/BiP (13); and translocation of protein kinase C and increased protein phosphorylation (12). Recently, PMT has also been shown to induce tyrosine phosphorylation of p125Fak and paxillin, as well as actin stress fiber formation and focal adhesion assembly (14).
The reported mitogenic response caused by PMT on intact cells has been
hypothesized to be the result of activation of a cellular phosphatidylinositol-specific phospholipase C (PLC) (10, 11), which
catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-triphosphate (IP3) and
diacylglycerol. Accordingly, PMT-induced release of these second
messengers was presumed to be responsible for initiation of subsequent
signaling events, including stimulation of Ca2+
mobilization and activation of protein kinase C, respectively. There
are a large number of different ligands and receptors that are known to
activate PLC, causing release of IP3 and diacylglycerol from PIP2 (15, 16, 17, 18, 19, 20, 21). Receptor regulation of phosphoinositide
hydrolysis is generally considered to be mediated either through
protein tyrosine phosphorylation of PLC or G-protein activation of
PLC
-isoforms (15, 16, 20, 21, 22). At least two general pathways of
G-protein-regulated PIP2 hydrolysis can be distinguished by
their sensitivity to ADP-ribosylation by pertussis toxin (PT). The
subunits of PT-sensitive Go/i-proteins preferentially stimulate PLC
3 > PLC
2 > PLC
1,
whereas the
subunits of the PT-insensitive Gq family,
including
q and
11, stimulate PLC
1
PLC
3
PLC
2 (16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42). It has been reported that
PMT-induced phosphoinositide hydrolysis could be blocked by the
addition of GDP
S to permeabilized cells, but PMT does not increase
tyrosine phosphorylation of PLC
(10). PMT action may thus involve a
G-protein-dependent PLC activity, such as PLC
1, PLC
2,
or PLC
3.
The Xenopus oocyte is a useful model system for mechanistic
studies of signal transduction pathways and has been used widely to
study G-protein coupling to PLC (18, 19, 30, 32, 43, 44, 45, 46, 47, 48, 49, 50).
IP3-induced intracellular Ca2+ mobilization or
Ca2+ influx through a voltage-gated and
IP3-sensitive Ca2+ channel on the cytoplasmic
membrane can be monitored in Xenopus oocytes by observing
the Ca2+-dependent Cl current
using a voltage clamp (18, 19, 43, 44, 45, 46, 47, 48, 49, 50).
We used voltage-clamped Xenopus oocytes to demonstrate
direct PMT-mediated stimulation of PLC activity by monitoring the
endogenous Ca2+-dependent Cl
current evoked upon microinjection with PMT. To identify the intracellular targets involved in the PMT-induced IP3
signaling pathway, we tested the effects of specific antibodies against Gpan
, Gq/11
(C-terminal and N-terminal),
Gi/o/t/z
, Gi-1/i-2/i-3
isoforms,
Gs/olf
, Gpan
(C-terminal, internal, and
N-terminal), PLC
1, PLC
2, PLC
3, PLC
1, an N-terminal peptide
of PMT (toxA28-42), or a C-terminal peptide of PMT
(toxA1239-1253) on the PMT-induced Cl
currents. We also examined the effects of PT on the PMT response. Our
results established the direct involvement of the free, monomeric Gq
protein in PMT activation of PLC
1. The specific
role of Gq
was further confirmed by over- and
underexpression of mouse Gq
in Xenopus
oocytes.
Rabbit polyclonal antisera against two synthetic
peptides, comprising residues 28-42 of PMT (NSDFTVKGKSADEIF) and
residues 1239-1253 of PMT (PVDDWALEIAQRNRA), were obtained from
Bio-Synthesis, Inc., using multiple antigen peptide conjugation
methodology. The anti-peptide IgG antibodies
(anti-toxA28-42 and anti-toxA1239-1253) were
purified using a protein A-agarose column (PURE-1,
Sigma). Rabbit antibodies against the unique C
terminus of the subunit of Gq/11
(anti-Gq/11
, QL) and against the common internal
GTP-binding site of the
subunit of G-proteins
(anti-Gpan
, GA/1) were obtained from DuPont NEN.
Affinity-purified rabbit polyclonal antibodies against the
subunits
of Gs/olf (anti-Gs/olf
, C-terminal, C-18, 377-394), Gq/11 (anti-Gq/11
, N-terminal,
E-17, 13-29), and Gi/o/t/z (anti-Gi/o/t/z
,
C-terminal, C-20, 325-344), the 1-4 G
subunit isoforms
(anti-Gpan
, C-terminal, T-20, 321-340), the 1-4 G
subunit isoforms (anti-Gpan
, N-terminal, M-14, 1-14),
PLC
1 (anti-PLC
1, C-terminal, G-12, 1204-1216), PLC
2
(anti-PLC
2, C-terminal, Q-15, 1170-1181), PLC
3 (anti-PLC
3,
C-terminal, C-20, 1198-1217), and PLC
1 (anti-PLC
1, internal,
530-850) were obtained from Santa Cruz Biotechnology, Inc. Rabbit
polyclonal antibodies against the
subunits of Gi-1
(anti-Gi-1
, internal, 159-168), Gi-1/i-2 (anti-Gi-1/i-2
, C-terminal, 345-354), Gi-3
(anti-Gi-3
, C-terminal, 345-354), and the 1-4 G
subunit isoforms (anti-Gpan
, internal, 127-139) were
obtained from Calbiochem, Inc. Goat anti-rabbit IgG antibodies,
conjugated to alkaline phosphatase, were obtained from Southern
Biotechnology Associates, Inc. Pertussis toxin (PT) catalytic S1
subunit was purchased from List Biological Laboratories, Inc.
IP3 and GDP
S were purchased from Sigma.
Native PMT was purchased from Sigma as a lyophilized
powder with BSA and resuspended in 50 mM Tris-HCl, pH 7.5, containing 10% glycerol, prior to use. Native PMT, purified to
homogeneity, quantified and titered by Vero cell cytotoxicity assays as
described (51), was also obtained as a generous gift from Dr. Clarence
Chrisp. Both toxin samples gave comparable responses in oocyte
experiments, although the highly purified sample was approximately
20-fold more active, and the concentration of the sample was adjusted
to reflect this difference (data not shown).
Anti-toxA28-42 and anti-toxA1239-1253 were
reactive with both toxin samples in Western blots, and
anti-toxA28-42 specifically inhibited the activities of
both toxin samples in oocyte experiments.
Complementary DNA coding for mouse Gq
protein subunit in the pcDNAI cloning vector (5.4 kilobases)
was obtained as a generous gift from Dr. Petra Schnabel. The plasmid
containing the cDNA insert was linearized by digestion with the
restriction enzyme ApaI (Life Technologies, Inc.). Using the
Ampliscribe Transcription System (Epicentre Technologies, Inc.), sense
cRNA was transcribed by the T7 promoter and antisense cRNA by the SP6
promoter, according to the manufacturer's procedure. In
vitro transcriptions of the cRNA were performed in the presence of
methylated cap (m7G[5
]ppp[5
]G) and catalyzed by T7 or
SP6 DNA-dependent RNA polymerase, respectively, at 37 °C
for 2 h. In vitro transcribed cRNA was dissolved in
RNase-free water at a final concentration of 1 ng/nl.
Adult female Xenopus laevis frogs (Xenopus I, Michigan) were anesthetized by immersion in a 0.15% tricaine methanesulfonate (Ayerst) solution for 30 min. A small incision was made on one side of the abdomen to remove several ovarian lobes. The lobes were gently torn apart and immersed in a Ca2+-free OR-2 solution (100 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM Hepes-Tris, pH 7.5). Oocytes were defolliculated by incubation with 2 mg/ml collagenase (Sigma, type 1A) at room temperature (22-24 °C) for 2-3 h. The oocytes were then washed five times with OR-2 solution and five times with a modified Barth's solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.3 mM Ca(NO3)2, 0.4 mM CaCl2, 0.8 mM MgSO4, 15 mM Tris-HCl, pH 7.6, containing 100 µg/ml penicillin and 100 µg/ml streptomycin). Stage 5-6 oocytes were selected and stored at 18 °C in modified Barth's solution.
Two-microelectrode Voltage ClampTwo microelectrodes, made by a horizontal puller (PD-5, Narishige) and filled with 3 M KCl to give a resistance of 1.5-2.0 megaohms, were used for voltage clamping. Voltage clamp experiments were performed in a continuously perfused bath (10 ml/min) at room temperature (22 °C). The bath was connected through an Ag-AgCl-Agar-3 M-KCl bridge to the voltage-recording amplifier (Axoclamp 2A, Axon Instruments). The data were filtered with a four-pole Bessel filter at 500 Hz. Voltage pulse protocols and data acquisition were performed on a 486 IBM computer with pCLAMP software (Axon Instruments), and graphics were obtained using Origin software (Microcal) with a pCLAMP module. Membrane currents were measured in normal Ringer's solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 11.8 mM CaCl2, and 5 mM Hepes-NaOH, pH 7.4), or where indicated, in Ca2+-free Ringer's solution (96 mM NaCl, 2 mM KCl, 12 mM MgCl2, and 5 mM Hepes-NaOH, pH 7.4).
Oocyte MicroinjectionsMicroinjections of toxins,
antibodies, and other reagents were performed under voltage-clamp
conditions at 80 mV holding potential using a pulse-controlled
picoliter injector (Dagan model PMI200). Toxins and other reagents were
prepared in 50 mM Tris-HCl, pH 7.5, or 50 mM
potassium phosphate buffer, pH 7.5, prior to injection. In all oocytes
tested, if a stable membrane potential could be achieved, PMT always
elicited a response. However, rarely, an entire group of oocytes from a
particular donor gave only a very weak response to PMT, upon which the
entire group of oocytes was discarded, and the responses were not
included in the data analysis; oocytes from that donor were not used
again. In all experiments reported here, injection of PMT into control,
untreated oocytes gave detectable responses. In a number of cases, a
group of oocytes from a particular donor elicited robust responses to PMT, of which several oocytes would give extremely large responses (exceeding the detection limits of the instrument), and the responses from these oocytes were also not included in the data analysis. The
number of oocytes used for data analysis is given in parentheses in the
figure legends, and the total number of oocytes tested, including those
giving overwhelming (but not those giving weak) responses, is indicated
in brackets. All results are expressed as the mean ± S.D. or the
mean ± S.E. (as indicated in the figure legends) of the responses
assayed in oocytes from at least two different donors (except where
indicated), with N denoting the number of groups tested and
n denoting the number of oocytes tested in each group. To
evaluate the statistical significance of the results, p
values were determined for each group using the two-ended t
test with unequal variance. Oocytes that were not injected with cRNA
are referred to throughout this communication as normal oocytes.
In the experiments
over- or underexpressing Gq, the oocytes were injected
with in vitro transcribed cRNA (50 ng) by positive displacement using a 10-µl micropipetter 2 days prior to the
electrophysiological experiments. Because of the large response
observed in the oocytes overexpressing Gq
, the amount of
PMT was decreased to 0.1 ng/oocyte. Even at this dose, it was
frequently observed that the peak inward current exceeded the
instrument's recording range (similar to that shown in Fig. 2B,
lower trace). Consequently, only those groups of oocytes showing a
moderate increase (up to ~800 nA, similar to that shown in Fig.
5A, lower trace) in the peak current upon injecting the
reduced PMT dose were arbitrarily selected for the antibody studies.
The total number of oocytes tested, including those giving overwhelming
responses (all oocytes gave a response), is denoted in brackets in the
figure legends.
Antibody Injections
Prior to injection, the antibodies were
dialyzed against 50 mM Tris-HCl, pH 7.5, or 50 mM potassium phosphate buffer, pH 7.5, and subsequently
diluted to the original concentrations as supplied by the commercial
source. The optimal preincubation period for oocytes with each antibody
prior to toxin injection was determined to be 2-4 h (data not shown).
Oocytes were injected with 50 nl of undiluted antibody 3 h prior
to injection with PMT. Anti-toxA28-42, diluted to a
concentration that could neutralize the PMT effect in normal oocytes,
as determined by titration (Fig. 1B), were used for
co-injection with PMT. Anti-toxA1239-1253 was used at the
same concentration as anti-toxA28-42.
PMT-induced
Ca2+-dependent Cl currents in
Xenopus oocytes. A, voltage-clamped oocytes were
injected with 10 ng of IP3 (trace shown is representative
of 11 oocytes) and 1 ng of PMT (representative of 74). I1,
the first peak current from the mobilization of intracellular
Ca2+; I2, the second peak current from
Ca2+ influx through voltage-gated Ca2+ channels
on the plasma membrane. No response occurred if PMT was heat-denatured
for 10 min at 95 °C prior to injection (representative of 4).
B, PMT (1.0 µg) was neutralized on ice for 30 min with increasing amounts of anti-toxA28-42 in a total volume of 20 µl prior to injection of 20 nl of the resulting mixture. The relative ratios of the PMT and antibody solutions were: 1:2
(representative of 9); 1:1 (representative of 11); 1:0.5
(representative of 6); and 1:0 (representative of 18). Injection of a
neutralizing amount of anti-toxA28-42 alone failed to elicit a response (representative of 2), whereas
co-injection of a mixture of anti-toxA28-42 and 10 ng of
IP3 evoked the characteristic
Ca2+-dependent Cl
currents
(representative of 2). C, both I1 and
I2 were blocked if the Cl
channel blocker
anthracene-9-carboxylic acid (9-AC; 200 µM)
was added to the extracellular bath solution (representative of 4) or
the intracellular Ca2+ was chelated with EGTA at a final
intracellular concentration of 100 µM (representative of
5). I2 was blocked if calcium from the extracellular bath
solution was removed (representative of 8) or if Cd2+ ions
(1 mM) were added to the bath solution (representative of 9).
SDS-Polyacrylamide Gel Electrophoresis and Western Analysis
Each individual oocyte was solubilized in lysis buffer,
containing 50 mM Hepes, pH 7.4, 1% Triton X-100, 0.5%
Nonidet P-40, 100 mM NaCl, 5 mM
MgCl2, 10 mM benzamidine, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.2 mM phenylmethylsulfonyl
fluoride, followed by the addition of 2 × SDS-polyacrylamide gel
electrophoresis sample buffer. The mixture was heated at 95 °C for
10 min, and the entire contents of each oocyte were separated by 10%
SDS-polyacrylamide gel electrophoresis and transferred onto
nitrocellulose membrane. The membrane was immunoblotted first with
rabbit polyclonal antibodies to Gq, revealing a major
band due to Gq
(data not shown), then with rabbit
polyclonal antibodies to RhoA (Fig. 5C), revealing an
additional band due to RhoA and an unidentified higher molecular weight
band at Mr ~60,000. The blots were developed
using secondary antibodies conjugated to alkaline phosphatase.
Xenopus
oocytes clamped at negative holding potentials (80 mV) produced an
inward current when microinjected with the 146.3-kDa protein toxin from
P. multocida (Fig. 1A).
Examination of the activation of the current showed that it was
characteristic of the IP3-mediated
Ca2+-dependent Cl
conductance,
exhibiting two components: an initial faster peak current
I1 in response to mobilization of intracellular
Ca2+ pools; and a second, slower and larger peak current
I2 caused by Ca2+ influx through voltage-gated
Ca2+ channels on the plasma membrane. Pre-incubation of PMT
with polyclonal antibodies against a 15-amino acid synthetic peptide
from the N-terminal region of PMT (anti-toxA28-42)
prevented the response (Fig. 1B), as did heat inactivation
of the toxin (Fig. 1A), demonstrating the specificity of the
PMT-induced response and the importance of the N terminus of PMT in its
action. Similar preincubation of PMT with polyclonal antibodies against
a 15-amino acid peptide from the C-terminal region of PMT
(anti-toxA1239-1253) had no effect on the PMT response
(statistical analysis included in Fig. 3A).
To confirm that the PMT-induced Cl currents were indeed
Ca2+-dependent, the effects of removing
extracellular Ca2+ and applying specific blockers of
Ca2+ and Cl
channels on the PMT-induced
currents were examined (Fig. 1C). For the IP3
response, it has been shown that removal of extracellular Ca2+ or blockage of the voltage-gated Ca2+
channel by Cd2+ will abolish I2, but not
I1, whereas induction of both I1 and I2 can be blocked by intracellular chelation of
Ca2+ with EGTA or by anion transport inhibitors of
Cl
channels, such as anthracene-9-carboxylic acid (51).
In agreement with these observations, injection of PMT into oocytes
bathed in Ca2+-free solution induced only I1,
whereas I2 was not observed. Likewise, when the
Ca2+-channel blocker Cd2+ was present at 1 mM concentration in the medium, I2 was
diminished. Both I1 and I2 were inhibited if
PMT was co-injected with EGTA or if anthracene-9-carboxylic acid was
perfused in the bath solution.
The overall effect of microinjecting PMT on the
voltage-clamped oocytes was direct and almost immediate, occurring
within 20 s from microinjection. An EC50 value of 0.28 ng/oocyte for PMT (for the Sigma sample) in normal
oocytes was determined from the dose-response curve in Fig.
2A. Contrary to what was observed for
multiple IP3 injections (Fig. 2B, upper trace),
after the first injection with PMT in normal oocytes, additional
injection with PMT gave little or no further response (Fig. 2B,
middle trace), suggesting that the action of PMT is not readily
reversible. For oocytes unable to elicit a response to a second dose of
PMT, additional injection of IP3 in the same oocyte was
still able to evoke both I1 and I2. In
Gq-overexpressing oocytes, even at 20-fold less PMT
doses, the initial injection elicited an overwhelming response (Fig.
2B, lower trace), more than 15 times that observed for
normal oocytes (i.e. >300-fold more potent). After the
first injection, a second dose of PMT elicited a greatly diminished
response (Fig. 2B, lower trace inset). Despite numerous
attempts, it was not technically possible to perform a subsequent
IP3 injection in oocytes showing an overwhelming effect of
PMT.
Specific antibodies against each of
the PLC isoforms, 1,
2,
3, or
1, were microinjected into
normal oocytes 3 h prior to microinjection with PMT. Antibodies
against PLC
1 and PLC
2 did not block (p > 0.4 for
both) the Ca2+-dependent Cl
current induced by PMT; in fact, anti-PLC
2 appeared to cause a 25%
enhancement of the response. On the other hand, antibodies directed
against PLC
1 greatly diminished the PMT-mediated response (p < 0.01), similar to the effect of
anti-toxA28-42, strongly supporting a direct role for
PLC
1 in PMT action. Antibodies against PLC
3 showed a 25%
reduction (p
0.3) in the PMT-induced response, suggesting the possibility that PLC
3 may play a minor role in the
PMT response. The results are summarized in Fig.
3A.
We investigated the ability of specific antibodies
against different G-protein subunits, Gpan,
Gs/olf
, Gi/o/t/z
, Gi-1
, Gi-1/i-2
, Gi-3
, Gq/11
, or
Gpan
to block the PMT-induced currents. Specific
antibodies against the common GTP-binding region of most G-protein
subunits (Gpan
) greatly diminished the PMT-mediated response (p < 0.07). Specific antibodies to the
C-terminal regions of Gs/olf
, Gi/o/t/z
,
or to the N-terminal region of Gq
had no significant
effect (p > 0.4) on the PMT-induced Cl
currents. Antibodies to the C-terminal regions of Gi-1
,
Gi-1/i-2
, or Gi-3
only slightly increased
the PMT response (p < 0.3). On the other hand,
antibodies directed against the unique C-terminal region of
Gq/11
caused a pronounced reduction (p < 0.006) in the PMT response, strongly supporting a direct role for
Gq
-coupled PLC
1 in PMT action. To determine whether
release of the
subunits from the
subunits of the G-proteins
might account for activation of the PLC activity, antibodies to the
common N-terminal, internal, and C-terminal regions of the 1-4
isoforms of G
(anti-Gpan
) were tested. Rather than
blocking the PMT-induced response, results revealed a marked 4-fold
increase in the PMT response (p < 0.0003) for the
C-terminally directed antibodies and a 2-fold increase (p < 0.0001) for the internally directed antibodies.
Antibodies against the N terminus of Gpan
had little
effect on the PMT response (p > 0.1). The results are
summarized in Fig. 3B.
It was determined that injection of 1.0 ng
of PMT into normal oocytes elicited a response comparable to that
evoked by 10 ng of IP3. Oocytes preinjected with sense
mouse Gq cRNA two days prior to injection of PMT
resulted in a marked increase in the PMT-induced Cl
current (Fig. 4, A and B). Among
those sense Gq
cRNA-treated oocytes showing moderate
responses (see "Experimental Procedures"), there was a ~3-fold
increase in the PMT-induced response with 10-fold less toxin
(p < 0.001). The PMT response could be blocked by
preinjection of the nonhydrolyzable GDP analog, GDP
S (final intracellular concentration was estimated to be 500 µM),
even at a dose of 1.0 ng/oocyte. Preinjection with antisense
Gq
cRNA into Xenopus oocytes reduced the
response mediated by PMT (p < 0.01) by ~7-fold,
compared to that observed for normal oocytes (Figs 4, A and
B). Western blot analysis of total cell lysates confirmed
that the Gq
protein was indeed being overexpressed in
the oocytes showing overwhelming response to PMT (Fig. 4C) but was not as evident in oocytes showing only a moderate response (data not shown). In the Gq
-underexpressed oocytes, only
a slight reduction in Gq
protein level was noticed. The
effects of various specific antibodies on the PMT-induced response were
also investigated in sense Gq
cRNA-treated oocytes. The
results, summarized in Fig. 5, were consistent with
those found for normal oocytes (Fig. 3).
The effect of injection of sense and
antisense mouse Gq cRNA on the peak inward currents
induced by PMT in Xenopus oocytes. The amount of PMT
injected was 1.0 ng/oocyte for all experiments, except for those with
sense Gq
cRNA, in which case 0.1 ng/oocyte PMT was
injected. A, PMT-induced
Ca2+-dependent Cl
currents in
normal oocytes (upper), oocytes injected with antisense Gq
cRNA (middle), and oocytes injected with
sense Gq
cRNA and showing moderate response
(lower). B, comparison of the
Ca2+-dependent Cl
currents in
normal oocytes injected with 10 ng of IP3 or 1.0 ng of PMT
to that of oocytes over- or underexpressing Gq
protein and injected with 0.1 ng and 1.0 ng of PMT, respectively. Also shown
are results from oocytes overexpressing Gq
but first
injected with GDP
S (final concentration, 500 µM) prior
to injection with 1.0 ng of PMT. The data are given as the mean of the
peak inward Cl
current (nA) induced by injection with PMT
or IP3; bars, S.E. p(t
test) values were determined by comparing data obtained from each of
the N groups to that of normal oocytes from the same donor. For IP3,
N = 4, n = 2-3
(ntotal = 11[20]); normal oocytes,
N = 14, n = 3-8
(ntotal = 74 [104]); oocytes injected with
antisense Gq
cRNA, N = 4, n = 3-4 (ntotal = 14 [14])
(p < 0.01); oocytes injected with sense
Gq
cRNA, N = 11, n = 2-6 (ntotal = 37 [69]) (p < 0.001); oocytes injected with sense Gq
cRNA and GDP
S,
N = 3, n = 3 (ntotal = 9 [9]) (p < 0.01).
C, Western analysis of over- and underexpression of mouse
Gq
protein in Xenopus oocytes. The entire
content of each oocyte lysate was analyzed by 10% SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-Gq
and
anti-RhoA antibodies, as described under "Experimental Procedures."
The results shown are representative of five independent experiments. Lane 1, a single oocyte 48 h after injection with sense
Gq
cRNA and showing overwhelming response to PMT similar
to that shown in Fig. 2B, lower trace; Lane 2, a
single oocyte 48 h after injection with antisense
Gq
cRNA and having diminished response similar to that
shown in Fig. 4A, middle trace; Lane 3, a single
normal oocyte without cRNA treatment and having a response similar to that in Fig. 4A, upper trace.
Effect of Pertussis Toxin on the PMT-induced Response
Normal
oocytes were injected with PT (1 ng/oocyte) at 80 mV to give a
PT-induced conductance (Fig. 6A). When a
stable base line was recovered (after ~2 h), this was followed by
injection of PMT (0.5 ng/oocyte) at various time intervals. After PT
injection, a progressively increased PMT response was observed (Fig.
6B), which was enhanced more than 20-fold after 3.5 h
(Fig. 6A), compared to that of control oocytes not
preinjected with PT (p < 0.0001).
Transient elevation of free Ca2+ concentration in the
cytosol is one of the first events observed in various cell types on
stimulation with hormones and growth factors (13, 14, 15, 16, 17, 18, 19, 44, 45, 46, 47, 48, 49). This
elevation is due both to Ca2+ release from intracellular
stores and to Ca2+ influx through Ca2+ channels
on the plasma membrane (15, 18, 19, 44, 45, 46, 47, 48, 49). It has been reported that
treatment of cultured cells with PMT causes an increase in
intracellular inositol phosphate and Ca2+ levels within
3-4 h after exposure to toxin (9, 10, 11, 12). The lag period, before
detectable intracellular responses are observed, has been attributed to
the requirement for PMT to first bind to cell surface receptors and be
internalized and presumably processed through endocytic vesicles (6,
7). With the objective of deciphering early events in the action of the
toxin, we bypassed this lengthy internalization process by directly
injecting toxin into Xenopus oocytes. Upon microinjection of
PMT into oocytes, we observed an immediate IP3-like
response (Fig. 1A) within 20 s. Control experiments
confirmed that this IP3-like response was indeed a
Ca2+-dependent Cl current (Fig.
1C).
The observed response was PMT-dependent, as demonstrated by the lack of response from heat-denatured toxin (Fig. 1A) and by the ability of specific antibodies against an N-terminal peptide of PMT, anti-toxA28-42, to block the activity (Fig. 1B). Unlike anti-toxA28-42, specific antibodies to a C-terminal peptide of PMT, anti-toxA1239-1253, did not block the activity (Fig. 3A), strongly implicating that the N terminus of PMT is crucial for its activity. This PMT-induced response was dose-dependent (Fig. 2A). The action of PMT was not readily reversible, for although repeated IP3 injections produced repeated responses, a second injection of PMT did not (Fig. 2B). PMT did not impair the IP3 response, because oocytes first injected with PMT and showing no response to a second dose of PMT were still able to show an IP3-dependent response (Fig. 2B, middle trace); therefore, PMT action must occur upstream to IP3 release.
It has been proposed that PMT might facilitate G-protein coupling to
PLC, causing the observed increased inositol phosphates and increased
intracellular Ca2+ concentrations (10). However, other than
the similarity of its eventual intracellular effects to that caused by
different neuropeptides, there was no direct evidence linking PMT to
G-protein coupled PLC activity. We have now established a link between
PMT action and IP3 release and subsequent Ca2+
mobilization in Xenopus oocytes. To determine which of the
several known phospholipases (15, 16, 20, 21) is responsible for the
IP3 release due to PMT action, we first investigated the
effects of specific antibodies to PLC1, PLC
2, PLC
3, or PLC
1
on the PMT-induced response. Each of the anti-PLC
antibodies were
directed against the C-terminal regions of the corresponding proteins
known to be required for interaction with G-proteins (20, 22, 26-28, 41). The anti-PLC
1 antibodies were directed against the region containing SH2 and SH3 domains, which is known to be involved in
interaction with tyrosine-phosphorylated growth factor receptors, and
contains the tyrosine residues required for PLC
1 activation via
phosphorylation (20). As summarized in Fig. 3A, only
antibodies to PLC
1 were able to block the PMT-induced response in a
manner similar to anti-toxA28-42. Anti-PLC
1 had no
effect on the PMT-induced response, consistent with reported results
showing no tyrosine phosphorylation-dependent activation of
PLC
in PMT-treated cultured fibroblasts (10). Anti-PLC
2 actually
showed a 25% increased response, whereas anti-PLC
3 showed a 25%
decrease. From these results, it is clear that PLC
1 is the primary
mediator of the PMT-induced IP3 release; however, PLC
3
might also have a minor role (see discussion below for overexpressed
Gq
experiments).
Activation of PLC1 has been shown to be primarily mediated through
subunits of the Gq family (16, 19, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41), whereas co-transfection of Goa
, Gob
,
Gt
, or Gz
with PLC
1 in COS-7 cells
failed to increase IP3 formation (39). PLC
3 can also be
activated by
subunits of the Gq family to an equal or
lesser extent than PLC
1 (40). Overexpression of Gq
,
G11
, Goa
, or Gob
in
Xenopus oocytes has been shown to enhance the
receptor-initiated Ca2+-dependent
Cl
currents, but overexpression of Gs
,
Golf
, or Gt
did not (19). Another
mechanism for activation of PLC
2 and PLC
3 isoforms is through the
subunits of the PT-sensitive Gi- or
Go-proteins (23, 24, 25, 26, 27, 30, 42). Specific antibodies against
Gpan
or Gpan
were used to distinguish
between the two possible mechanisms of G-protein activation of PLC
isoforms. Anti-Gpan
antibodies were directed against the
common GTP-binding region of G
proteins. Anti-Gpan
antibodies were against the common C-terminal, internal, or N-terminal
regions of the G
1-4 isoforms. As summarized in Fig. 4,
anti-Gpan
nearly abolished the PMT-induced response in a
manner similar to anti-toxA28-42, implicating a
G
-dependent pathway. Anti-Gpan
did not
block, but instead enhanced, the response by as much as 4-fold (see
discussion below).
To determine which of the G families is involved in activating the
PLC
isoforms leading to IP3 release, the effects of
specific antibodies against Gs/olf
,
Gi/o/t/z
, Gi-1
, Gi-1/i-2
,
Gi-3
, or Gq/11
on PMT action were
examined. Anti-Gq/11
completely blocked the PMT-induced
response, similar to that observed for anti-Gpan
,
anti-PLC
1, or anti-toxA28-42.
AntiGs/olf
, anti-Gi-1
,
anti-Gi-1/i-2
, anti-Gi-3
, and
anti-Gi/o/t/z
did not block the PMT-induced response,
consistent with the known non-PLC effector targets of the respective
G
proteins (52). Based on these results using specific antibodies to
identify the key mediators of PMT action, the observed PMT-induced
Cl
current involves a signal transduction pathway
composed of Gq/11
-dependent activation of
PLC
1, subsequent hydrolysis of PIP2 to release IP3, Ca2+ mobilization, and eventual activation
of the Ca2+-dependent Cl
channels.
The N-terminal half of the cytotoxic necrotizing factors type 1 and 2 (CNF1 and CNF2) from enteropathogenic Escherichia coli show
24-27% homology to the first ~600 amino acids of PMT (53, 54). The
Rho family of Ras-related, small GTP-binding proteins, involved in
regulating the assembly of focal adhesion and stress fibers in
eukaryotic cells, have recently been implicated as possible intracellular targets of the cytotoxic necrotizing factors (54). Ca2+ and Rho signaling pathways cooperate to regulate
reorganization of actin filaments (55, 56), and Rho regulation of
cytoskeletal function appears to be activated by PLC via a
PKC/diacylglycerol/phorbol ester-sensitive factor (56). We have
observed no inhibitory effect on the PMT-induced Cl
currents using antibodies specific for the GTP-binding region unique to
RhoA or RhoB, as well as antibodies to the conserved GTP-binding region
of Ras-related proteins.2 Our results do
not preclude Rho- or Ras-related proteins as additional PMT targets or
as potential signaling proteins important in the mitogenic effect
stimulated by PMT (14). However, these proteins do not appear to be
required for the PMT-triggered IP3 release in
Xenopus oocytes. The cellular mechanisms that lead to
PMT-induced mitogenesis remain unclear, and there is as yet no direct
evidence linking the effect of PMT on the Gq-coupled PLC
pathway to cell proliferation.
The cloned amino acid sequence of the Xenopus
Gq shares 96% identity to that from mouse (43, 57) and
retains all of the characteristics that distinguish Gq
from other G-protein
subunits (43, 57). To confirm that
Gq
is involved in PMT action, we overexpressed the mouse
Gq
subunit in Xenopus oocytes. Overexpressing Gq
in oocytes could increase the
PMT-dependent Cl
current 30- to more than
300-fold (Fig. 2B), whereas oocytes treated with antisense
Gq
cRNA showed a 7-fold decreased response (Fig. 4,
A and B). Western blot analysis confirmed an
enhancement of Gq
production in the Gq
cRNA-treated oocytes (Fig. 4C). GDP
S, a known inhibitor
of Gq
- and other G
-mediated signaling pathways (10,
17, 34, 58), blocked the PMT-induced response in Gq
-overexpressing oocytes, even at much higher PMT doses
(Fig. 4B). As observed for normal oocytes, a second dose of
PMT elicited a dramatically decreased response in oocytes
overexpressing Gq
(Fig. 2B, lower trace).
These findings further support the direct involvement of
Gq
in PMT-mediated signaling pathways.
Furthermore, the effects of specific antibodies, anti-PLC1,
anti-Gpan
, anti-Gq/11
, or
anti-toxA28-42 on the PMT-induced Cl
currents in normal oocytes were reproduced in
Gq
-overexpressing oocytes (Fig. 5). In the
Gq
-overexpressing oocytes, anti-PLC
2 did not enhance
the PMT response, as observed in normal oocytes (compare Fig. 5 with
Fig. 3A). On the other hand, the 25% decrease in PMT
response observed in normal oocytes for anti-PLC
3 was reproduced in
the Gq
-overexpressing oocytes (Figs. 3A and
5). This effect of anti-PLC
3 suggested the possibility of a minor role for PLC
3 in a partial Gq
- or G
-mediated
pathway (40, 42). However,
anti-Gpan
(C-terminal) was not able to block
PMT action, and in fact, a 2.5-fold enhancement (4-fold for normal oocytes) of the response was observed (Figs. 3B and 5).
Although it is conceivable that anti-Gpan
may not be
effective in blocking the activation of PLC
1 or PLC
3 mediated by
the G
subunits, the anti-Gpan
used in this study
was directed against the C-terminal 20 amino acids of G
1-4. This
region has been shown to be important for G
association with G
(52, 59, 60, 61, 62), as well as dimerization of G
, which is important
for G
-dependent activation of PLC
2 and PLC
3
(23, 24, 25, 26, 27, 30, 42). On the other hand, the enhancement of the PMT
response due to anti-Gpan
(C-terminal) could
be accounted for by antibody sequestration of the G
subunits,
causing dissociation of G
from the G
heterotrimer. This is
consistent with the finding in normal oocytes that antibodies to an
internal region of Gpan
also enhanced the PMT response,
whereas antibodies to the N terminus of Gpan
had little
effect on the response (Fig. 3B). The preferred substrate
for PT is the heterotrimeric Gi/o/t
complex (63), and ADP-ribosylation of the GDP-bound
subunit locks the complex in
its inactive heterotrimeric form (52, 64). To test if sequestration of
the G
subunits to release more Gq
might enhance
the PMT response, we examined the time-dependent effect of
PT on the PMT response. Preinjection of PT enhanced the subsequent PMT
response in a time-dependent manner, with a more than
20-fold increased PMT response at 3.5 h after PT injection (Fig.
6). These combined results suggest that the direct target of PMT action
is the free, monomeric form of Gq
, which agrees with the
enhancement of the response observed in oocytes overexpressing
Gq
(Fig. 4, A and B).
Although there was a greater than 300-fold enhancement in the response
to a 20-fold lower dose of PMT due to overexpression of
Gq (Fig. 2B, lower trace), the Western blot
of such oocyte lysates showed at most a 2-3-fold increase in total
Gq
protein expression (Fig. 4C). For those
oocytes showing only moderate increase in response (3-fold enhancement
with 10-fold lower PMT dose), little difference in total
Gq
expression was observed (data not shown). Likewise,
the antisense Gq
cRNA-treated oocytes showed a 7-fold
decrease in response (Fig. 4A, middle trace), whereas the
Western blot indicated only a slight reduction in total
Gq
protein levels (Fig. 4C). These findings
are consistent with the hypothesis that the magnitude of the
PMT-induced response is dependent on the level of free, monomeric
Gq
protein, instead of the total amount of
Gq
protein present in the oocytes.
In light of our findings and the above discussion, we propose a
possible mechanism for PMT action in Xenopus oocytes, in
which PMT acts on free Gq, possibly the GDP-bound form,
and converts Gq
into an active form, which stimulates
PLC
1. The activated PLC
1 causes PIP2 hydrolysis,
leading to IP3 release and eventual Ca2+
mobilization that results in the Ca2+-dependent
Cl
current. The PMT-induced, Gq
-mediated
PLC
1 activation appears to be transient, and the presumably modified
Gq
involved in this activation is not readily available
for further PMT action.
We are grateful to Dr. Clarence Chrisp for
generously providing us with highly purified samples of native PMT and
to Dr. Petra Schnabel for the generous gift of mouse Gq
cRNA. We thank Yunfei Huang for technical assistance in oocyte and cRNA
preparation and John Peterson in performing Western blot analysis.