Pasteurella multocida Toxin Activates the Inositol Triphosphate Signaling Pathway in Xenopus Oocytes via Gqalpha -coupled Phospholipase C-beta 1*

(Received for publication, October 17, 1996)

Brenda A. Wilson Dagger §, Xinjun Zhu Dagger , Mengfei Ho Dagger and Luo Lu

From the Dagger  Departments of Biochemistry and Molecular Biology and  Physiology and Biophysics, Wright State University School of Medicine, Dayton, Ohio 45435

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

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 PLCbeta 1, PLCbeta 2, PLCbeta 3, or PLCgamma 1 identified PLCbeta 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 alpha  subunits, or antibodies to different regions of G-protein beta  subunits established the involvement of a G-protein alpha  subunit in PMT-activation of PLCbeta 1. Injection with specific antibodies against the alpha -subunits of Gq/11, Gs/olf, Gi/o/t/z, or Gi-1/i-2/i-3 isoforms confirmed the involvement of Gq/11alpha . Preinjection of oocytes with pertussis toxin enhanced the PMT response. Overexpression of Gqalpha in oocytes could enhance the PMT response by 30-fold to more than 300-fold, whereas introduction of antisense Gqalpha cRNA reduced the response by 7-fold. The effects of various specific antibodies on the PMT response were reproduced in oocytes overexpressing Gqalpha .


INTRODUCTION

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 PLCgamma or G-protein activation of PLC beta -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 beta gamma subunits of PT-sensitive Go/i-proteins preferentially stimulate PLCbeta 3 > PLCbeta 2 > PLCbeta 1, whereas the alpha subunits of the PT-insensitive Gq family, including alpha q and alpha 11, stimulate PLCbeta >=  PLCbeta 3 >>  PLCbeta 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 GDPbeta S to permeabilized cells, but PMT does not increase tyrosine phosphorylation of PLCgamma (10). PMT action may thus involve a G-protein-dependent PLC activity, such as PLCbeta 1, PLCbeta 2, or PLCbeta 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 Gpanalpha , Gq/11alpha (C-terminal and N-terminal), Gi/o/t/zalpha , Gi-1/i-2/i-3alpha isoforms, Gs/olfalpha , Gpanbeta (C-terminal, internal, and N-terminal), PLCbeta 1, PLCbeta 2, PLCbeta 3, PLCgamma 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 Gqalpha protein in PMT activation of PLCbeta 1. The specific role of Gqalpha was further confirmed by over- and underexpression of mouse Gqalpha in Xenopus oocytes.


EXPERIMENTAL PROCEDURES

Materials

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 alpha  subunit of Gq/11 (anti-Gq/11alpha , QL) and against the common internal GTP-binding site of the alpha  subunit of G-proteins (anti-Gpanalpha , GA/1) were obtained from DuPont NEN. Affinity-purified rabbit polyclonal antibodies against the alpha  subunits of Gs/olf (anti-Gs/olfalpha , C-terminal, C-18, 377-394), Gq/11 (anti-Gq/11alpha , N-terminal, E-17, 13-29), and Gi/o/t/z (anti-Gi/o/t/zalpha , C-terminal, C-20, 325-344), the 1-4 Gbeta subunit isoforms (anti-Gpanbeta , C-terminal, T-20, 321-340), the 1-4 Gbeta subunit isoforms (anti-Gpanbeta , N-terminal, M-14, 1-14), PLCbeta 1 (anti-PLCbeta 1, C-terminal, G-12, 1204-1216), PLCbeta 2 (anti-PLCbeta 2, C-terminal, Q-15, 1170-1181), PLCbeta 3 (anti-PLCbeta 3, C-terminal, C-20, 1198-1217), and PLCgamma 1 (anti-PLCgamma 1, internal, 530-850) were obtained from Santa Cruz Biotechnology, Inc. Rabbit polyclonal antibodies against the alpha  subunits of Gi-1 (anti-Gi-1alpha , internal, 159-168), Gi-1/i-2 (anti-Gi-1/i-2alpha , C-terminal, 345-354), Gi-3 (anti-Gi-3alpha , C-terminal, 345-354), and the 1-4 Gbeta subunit isoforms (anti-Gpanbeta , 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 GDPbeta 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.

Synthesis of cRNA from Gqalpha cDNA

Complementary DNA coding for mouse Gq protein alpha  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.

Oocyte Preparation

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 Clamp

Two 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 Microinjections

Microinjections 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.

Gqalpha Protein Expression

In the experiments over- or underexpressing Gqalpha , 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 Gqalpha , 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.


Fig. 2. The effect of multiple injections of PMT on the Ca2+-dependent Cl- currents in Xenopus oocytes. A, dose-dependence of the PMT-induced response in normal oocytes, using PMT concentrations of 0.07 ng/oocyte (ntotal = 12), 0.14 ng/oocyte (ntotal = 6), 0.28 ng/oocyte (ntotal = 15), 0.42 ng/oocyte (ntotal = 9), and 0.56 ng/oocyte (ntotal = 9). The mean of the peak inward Cl- current (nA) induced by injection with PMT (from Sigma) is shown for each data point; bars, S.E. An EC50 value of 0.28 ng/oocyte was determined from the data using Sigma Plot. B, voltage-clamped oocytes were treated with multiple injections of 10 ng of IP3 (upper, representative of 10). Voltage-clamped oocytes were injected with 0.5 ng of PMT, followed by a second injection with 0.5 ng of PMT and then an injection with 10 ng of IP3 (middle, representative of 5); the gap between the two traces indicates a time lapse of ~10 min. In oocytes overexpressing Gqalpha , an additional injection of PMT (0.05 ng/injection) elicited a greatly diminished response after the initial injection of PMT (lower, representative of 16). This oocyte is representative of oocytes eliciting an overwhelming response to PMT (>3000 nA); the inset shows an amplification of the second response (~30 nA).
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Fig. 5. The effect of specific antibodies to key signal transduction proteins potentially involved in PMT action in Xenopus oocytes overexpressing mouse Gqalpha . Experiments were performed as described in Fig. 3, Bars, S. E. Anti-toxA28-42 (1:1 ratio, as in Fig. 1), N = 2, n = 5, 7 [12] (p < 0.06); anti-PLCgamma 1, N = 3, n = 3, 4, 5 [12] (p > 0.8); anti-PLCbeta 1, N = 2, n = 3, 5 [8] (p < 0.05); anti-PLCbeta 2, N = 2, n = 3, 4 [10] (p > 0.4); anti-PLCbeta 3, N = 2, n = 3, 4 [7] (p > 0.3); anti-Gpanalpha , N = 4, n = 3, 3, 3, 4 (p < 0.04); anti-Gs/olfalpha , N = 3, n = 3, 4, 4 [13] (p > 0.2); anti-Gi/o/t/zalpha , N = 3, n = 2, 3, 4 [13] (p > 0.1); anti-Gq/11alpha , N = 4, n = 4, 4, 4, 4 [16] (p < 0.09); anti-Gpanbeta (C-terminal), N = 2, n = 4, 4 [21] (p < 0.03).
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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.


Fig. 1.

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).


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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 Gqalpha , revealing a major band due to Gqalpha (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.


RESULTS

PMT Activation of the Ca2+-dependent Cl- Current in Xenopus Oocytes

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).


Fig. 3. The effect of specific antibodies to key signal transduction proteins potentially involved in PMT action in normal Xenopus oocytes. The peak inward Cl- current was used to measure the effect of specific antibodies on the PMT-induced response in oocytes, as described under "Experimental Procedures." The data are shown as the mean for each set of experiments (N groups), using untreated oocytes as reference; bars, S.E. For those experiments in which N = 1, the S.E. shown is for the data within that group. p (t test) values were determined for each of the N groups. The range of the p values for each experiment is given in parentheses. The total number of oocytes tested, including those giving overwhelming (but not those giving weak) responses, is indicated in brackets. A, the effect of specific antibodies to PLCbeta 1, PLCbeta 2, PLCbeta 3, PLCgamma 1, the N-terminal peptide28-42 of PMT, and the C-terminal peptide1239-1253 of PMT on the PMT-induced Cl- currents in normal Xenopus oocytes. Anti-toxA28-42 (1:1 ratio, as in Fig. 1), N = 2, n = 5, 5 [10] (p < 0.0006); anti-toxA1239-1253 (1:1 ratio, as in Fig. 1), N = 2, n = 7, 8 [15] (p > 0.5); anti-PLCgamma 1, N = 4, n = 2, 3, 3, 4 [12] (p > 0.6); anti-PLCbeta 1, N = 3, n = 6, 6, 7 [19] (p < 0.004); anti-PLCbeta 2, N = 3, n = 4, 5, 5 [14] (p > 0.4); anti-PLCbeta 3, N = 3, n = 4, 5, 5 [16] (p > 0.1). B, the effect of specific antibodies to Gpanalpha , Gq/11alpha (N-terminal, C-terminal), Gs/olfalpha , Gi-1alpha , Gi-1alpha , Gi-1/i-2alpha , Gi-3alpha , Gi/o/t/zalpha , and Gpanbeta (N-terminal, internal, C-terminal) on the PMT-induced Cl- currents in normal Xenopus oocytes. a, anti-Gpanalpha , N = 2, n = 4, 4 [8] (p < 0.002); anti-Gs/olfalpha , N = 2, n = 3, 3 [10] (p > 0.6); anti-Gi/o/t/zalpha , N = 4, n = 2, 3, 3, 4 [12] (p > 0.4); anti-Gi-1alpha , N = 1, n = 8 [8] (p < 0.2); anti-Gi-1/i-2alpha , N = 1, n = 10 [10] (p < 0.3); anti-Gi-3alpha , N = 1, n = 12 [12] (p < 0.004); anti-Gq/11alpha (C-terminal), N = 4, n = 4, 4, 4, 5 [17] (p < 0.006); antiGq/11alpha (N-terminal), N = 2, n = 5, 6 [11] (p > 0.1). b, anti-Gpanbeta (C-terminal), N = 3, n = 8, 9, 10 [33] (p < 0.0003); anti-Gpanbeta (internal), N = 1, n = 9 [9] (p < 0.0001); anti-Gpanbeta (N-terminal), N = 2, n = 6, 7 [13] (p > 0.1).
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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.

Dose-Response and Effect of Multiple Doses of PMT on the Ca2+-dependent Cl- Currents in Xenopus Oocytes

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 Gqalpha -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.

Effects of Specific Antibodies to PLC Isoforms on the PMT-induced Response in Normal Oocytes

Specific antibodies against each of the PLC isoforms, beta 1, beta 2, beta 3, or gamma 1, were microinjected into normal oocytes 3 h prior to microinjection with PMT. Antibodies against PLCgamma 1 and PLCbeta 2 did not block (p > 0.4 for both) the Ca2+-dependent Cl- current induced by PMT; in fact, anti-PLCbeta 2 appeared to cause a 25% enhancement of the response. On the other hand, antibodies directed against PLCbeta 1 greatly diminished the PMT-mediated response (p < 0.01), similar to the effect of anti-toxA28-42, strongly supporting a direct role for PLCbeta 1 in PMT action. Antibodies against PLCbeta 3 showed a 25% reduction (p >=  0.3) in the PMT-induced response, suggesting the possibility that PLCbeta 3 may play a minor role in the PMT response. The results are summarized in Fig. 3A.

Effects of Specific Antibodies to Different G-protein alpha  and beta  Subunits on the PMT-induced Response in Normal Oocytes

We investigated the ability of specific antibodies against different G-protein subunits, Gpanalpha , Gs/olfalpha , Gi/o/t/zalpha , Gi-1alpha , Gi-1/i-2alpha , Gi-3alpha , Gq/11alpha , or Gpanbeta to block the PMT-induced currents. Specific antibodies against the common GTP-binding region of most G-protein alpha  subunits (Gpanalpha ) greatly diminished the PMT-mediated response (p < 0.07). Specific antibodies to the C-terminal regions of Gs/olfalpha , Gi/o/t/zalpha , or to the N-terminal region of Gqalpha had no significant effect (p > 0.4) on the PMT-induced Cl- currents. Antibodies to the C-terminal regions of Gi-1alpha , Gi-1/i-2alpha , or Gi-3alpha only slightly increased the PMT response (p < 0.3). On the other hand, antibodies directed against the unique C-terminal region of Gq/11alpha caused a pronounced reduction (p < 0.006) in the PMT response, strongly supporting a direct role for Gqalpha -coupled PLCbeta 1 in PMT action. To determine whether release of the beta gamma subunits from the alpha  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 Gbeta (anti-Gpanbeta ) 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 Gpanbeta had little effect on the PMT response (p > 0.1). The results are summarized in Fig. 3B.

Effects of Over- and Underexpression of Gqalpha on the PMT-induced Response

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 Gqalpha 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 Gqalpha 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, GDPbeta S (final intracellular concentration was estimated to be 500 µM), even at a dose of 1.0 ng/oocyte. Preinjection with antisense Gqalpha 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 Gqalpha 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 Gqalpha -underexpressed oocytes, only a slight reduction in Gqalpha protein level was noticed. The effects of various specific antibodies on the PMT-induced response were also investigated in sense Gqalpha cRNA-treated oocytes. The results, summarized in Fig. 5, were consistent with those found for normal oocytes (Fig. 3).


Fig. 4.

The effect of injection of sense and antisense mouse Gqalpha 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 Gqalpha 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 Gqalpha cRNA (middle), and oocytes injected with sense Gqalpha 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 Gqalpha protein and injected with 0.1 ng and 1.0 ng of PMT, respectively. Also shown are results from oocytes overexpressing Gqalpha but first injected with GDPbeta 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 Gqalpha cRNA, N = 4, n = 3-4 (ntotal = 14 [14]) (p < 0.01); oocytes injected with sense Gqalpha cRNA, N = 11, n = 2-6 (ntotal = 37 [69]) (p < 0.001); oocytes injected with sense Gqalpha cRNA and GDPbeta S, N = 3, n = 3 (ntotal = 9 [9]) (p < 0.01). C, Western analysis of over- and underexpression of mouse Gqalpha protein in Xenopus oocytes. The entire content of each oocyte lysate was analyzed by 10% SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-Gqalpha 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 Gqalpha 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 Gqalpha 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.


[View Larger Version of this Image (12K GIF file)]


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).


Fig. 6. The effect of PT on the PMT-induced Cl- currents in normal Xenopus oocytes. A, normal oocytes were first injected with PT (1 ng) at -80 mV to give a PT-induced conductance, which upon recovery of a stable membrane potential after ~2 h, was followed by injection of PMT (0.5 ng) at 3.5 h to evoke an enhanced PMT response (representative of 4); the time shown indicates the time lapsed between the end of the first recording and the beginning of the second. B, time course of the effect of PT on the PMT response. PMT was injected into normal oocytes at various time intervals after PT injection, as in A. The data are shown as the mean for each experiment using control oocytes not preinjected with PT as reference (N = 1); bars, S.E. For control oocytes, ntotal = 6; 2 h, ntotal = 11 (p > 0.6); 2.5 h, ntotal = 8 (p < 0.01); 3 h, ntotal = 14 (p < 0.0001); 3.5 h, ntotal = 10 (p < 0.0001).
[View Larger Version of this Image (13K GIF file)]



DISCUSSION

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 PLCbeta 1, PLCbeta 2, PLCbeta 3, or PLCgamma 1 on the PMT-induced response. Each of the anti-PLCbeta 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-PLCgamma 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 PLCgamma 1 activation via phosphorylation (20). As summarized in Fig. 3A, only antibodies to PLCbeta 1 were able to block the PMT-induced response in a manner similar to anti-toxA28-42. Anti-PLCgamma 1 had no effect on the PMT-induced response, consistent with reported results showing no tyrosine phosphorylation-dependent activation of PLCgamma in PMT-treated cultured fibroblasts (10). Anti-PLCbeta 2 actually showed a 25% increased response, whereas anti-PLCbeta 3 showed a 25% decrease. From these results, it is clear that PLCbeta 1 is the primary mediator of the PMT-induced IP3 release; however, PLCbeta 3 might also have a minor role (see discussion below for overexpressed Gqalpha experiments).

Activation of PLCbeta 1 has been shown to be primarily mediated through alpha  subunits of the Gq family (16, 19, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41), whereas co-transfection of Goaalpha , Gobalpha , Gtalpha , or Gzalpha with PLCbeta 1 in COS-7 cells failed to increase IP3 formation (39). PLCbeta 3 can also be activated by alpha  subunits of the Gq family to an equal or lesser extent than PLCbeta 1 (40). Overexpression of Gqalpha , G11alpha , Goaalpha , or Gobalpha in Xenopus oocytes has been shown to enhance the receptor-initiated Ca2+-dependent Cl- currents, but overexpression of Gsalpha , Golfalpha , or Gtalpha did not (19). Another mechanism for activation of PLCbeta 2 and PLCbeta 3 isoforms is through the beta gamma subunits of the PT-sensitive Gi- or Go-proteins (23, 24, 25, 26, 27, 30, 42). Specific antibodies against Gpanalpha or Gpanbeta were used to distinguish between the two possible mechanisms of G-protein activation of PLCbeta isoforms. Anti-Gpanalpha antibodies were directed against the common GTP-binding region of Galpha proteins. Anti-Gpanbeta antibodies were against the common C-terminal, internal, or N-terminal regions of the Gbeta 1-4 isoforms. As summarized in Fig. 4, anti-Gpanalpha nearly abolished the PMT-induced response in a manner similar to anti-toxA28-42, implicating a Galpha -dependent pathway. Anti-Gpanbeta did not block, but instead enhanced, the response by as much as 4-fold (see discussion below).

To determine which of the Galpha families is involved in activating the PLCbeta isoforms leading to IP3 release, the effects of specific antibodies against Gs/olfalpha , Gi/o/t/zalpha , Gi-1alpha , Gi-1/i-2alpha , Gi-3alpha , or Gq/11alpha on PMT action were examined. Anti-Gq/11alpha completely blocked the PMT-induced response, similar to that observed for anti-Gpanalpha , anti-PLCbeta 1, or anti-toxA28-42. AntiGs/olfalpha , anti-Gi-1alpha , anti-Gi-1/i-2alpha , anti-Gi-3alpha , and anti-Gi/o/t/zalpha did not block the PMT-induced response, consistent with the known non-PLC effector targets of the respective Galpha 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/11alpha -dependent activation of PLCbeta 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 Gqalpha shares 96% identity to that from mouse (43, 57) and retains all of the characteristics that distinguish Gqalpha from other G-protein alpha  subunits (43, 57). To confirm that Gqalpha is involved in PMT action, we overexpressed the mouse Gqalpha subunit in Xenopus oocytes. Overexpressing Gqalpha in oocytes could increase the PMT-dependent Cl- current 30- to more than 300-fold (Fig. 2B), whereas oocytes treated with antisense Gqalpha cRNA showed a 7-fold decreased response (Fig. 4, A and B). Western blot analysis confirmed an enhancement of Gqalpha production in the Gqalpha cRNA-treated oocytes (Fig. 4C). GDPbeta S, a known inhibitor of Gqalpha - and other Galpha -mediated signaling pathways (10, 17, 34, 58), blocked the PMT-induced response in Gqalpha -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 Gqalpha (Fig. 2B, lower trace). These findings further support the direct involvement of Gqalpha in PMT-mediated signaling pathways.

Furthermore, the effects of specific antibodies, anti-PLCbeta 1, anti-Gpanalpha , anti-Gq/11alpha , or anti-toxA28-42 on the PMT-induced Cl- currents in normal oocytes were reproduced in Gqalpha -overexpressing oocytes (Fig. 5). In the Gqalpha -overexpressing oocytes, anti-PLCbeta 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-PLCbeta 3 was reproduced in the Gqalpha -overexpressing oocytes (Figs. 3A and 5). This effect of anti-PLCbeta 3 suggested the possibility of a minor role for PLCbeta 3 in a partial Gqalpha - or Gbeta gamma -mediated pathway (40, 42). However, anti-Gpanbeta (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-Gpanbeta may not be effective in blocking the activation of PLCbeta 1 or PLCbeta 3 mediated by the Gbeta gamma subunits, the anti-Gpanbeta used in this study was directed against the C-terminal 20 amino acids of Gbeta 1-4. This region has been shown to be important for Galpha association with Gbeta gamma (52, 59, 60, 61, 62), as well as dimerization of Gbeta gamma , which is important for Gbeta gamma -dependent activation of PLCbeta 2 and PLCbeta 3 (23, 24, 25, 26, 27, 30, 42). On the other hand, the enhancement of the PMT response due to anti-Gpanbeta (C-terminal) could be accounted for by antibody sequestration of the Gbeta gamma subunits, causing dissociation of Galpha from the Galpha beta gamma heterotrimer. This is consistent with the finding in normal oocytes that antibodies to an internal region of Gpanbeta also enhanced the PMT response, whereas antibodies to the N terminus of Gpanbeta had little effect on the response (Fig. 3B). The preferred substrate for PT is the heterotrimeric Gi/o/talpha beta gamma complex (63), and ADP-ribosylation of the GDP-bound alpha  subunit locks the complex in its inactive heterotrimeric form (52, 64). To test if sequestration of the Gbeta gamma subunits to release more Gqalpha 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 Gqalpha , which agrees with the enhancement of the response observed in oocytes overexpressing Gqalpha (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 Gqalpha (Fig. 2B, lower trace), the Western blot of such oocyte lysates showed at most a 2-3-fold increase in total Gqalpha 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 Gqalpha expression was observed (data not shown). Likewise, the antisense Gqalpha 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 Gqalpha 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 Gqalpha protein, instead of the total amount of Gqalpha 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 Gqalpha , possibly the GDP-bound form, and converts Gqalpha into an active form, which stimulates PLCbeta 1. The activated PLCbeta 1 causes PIP2 hydrolysis, leading to IP3 release and eventual Ca2+ mobilization that results in the Ca2+-dependent Cl- current. The PMT-induced, Gqalpha -mediated PLCbeta 1 activation appears to be transient, and the presumably modified Gqalpha involved in this activation is not readily available for further PMT action.


FOOTNOTES

*   This work was supported in part by an Ohio Research Challenge Grant and Grant AI38396 from the National Institutes of Health/NIAID (to B. A. W.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed. Tel.: 937-775-4803; Fax: 937-775-3730; E-mail: bwilson{at}desire.wright.edu.
1    The abbreviations used are: PMT, Pasteurella multocida toxin; PLC, phospholipase C; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5-triphosphate; PT, pertussis toxin; G-protein, GTP-binding protein; GDPbeta S, guanosine 5'-O-(2-(thio)diphosphate).
2    B. A. Wilson and X. Zhu, unpublished observations.

Acknowledgments

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 Gqalpha cRNA. We thank Yunfei Huang for technical assistance in oocyte and cRNA preparation and John Peterson in performing Western blot analysis.


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