Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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ABSTRACT |
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We previously reported that both carbachol and epidermal growth
factor (EGF) are potent inducers of the extracellular signal-regulated protein kinases (ERKs) in isolated gastric canine parietal cells and
that induction of these kinases leads to acute inhibitory and chronic
stimulatory effects on gastric acid secretion. In this study we
investigated the molecular mechanisms responsible for
these effects. Both carbachol (100 µM) and EGF (10 nM) induced Ras
activation. The role of Ras in ERK2 induction was examined by
transfecting parietal cells with a vector expressing hemoagglutinin (HA)-tagged ERK2 (HA-ERK2) together with a dominantly expressed mutant
(inactive) ras gene. HA-ERK2 activity was quantitated by in-gel
kinase assays. Dominant negative Ras reduced carbachol induction of
HA-ERK2 activity by 60% and completely inhibited the stimulatory
effect of EGF. Since Ras activation requires the assembly of a
multiprotein complex, we examined the effect of carbachol and EGF on
tyrosyl phosphorylation of Shc and its association with Grb2 and the
guanine nucleotide exchange factor Sos. Western blot analysis of
anti-Shc immunoprecipitates with an anti-phosphotyrosine antibody
demonstrated that both carbachol and EGF induced tyrosyl phosphorylation of a major 52-kDa shc isoform. Grb2 association with Shc was demonstrated by blotting Grb2 immunoprecipitates with an
anti-Shc antibody. Probing of anti-Sos immunoprecipitates with an
anti-Grb2 antibody revealed that Sos was constitutively bound to Grb2.
To examine the functional role of Sos in ERK2 activation, we
transfected parietal cells with the HA-ERK2 vector together with a
dominantly expressed mutant (inactive) sos gene. Dominant negative Sos did not affect carbachol stimulation of HA-ERK2 but inhibited the stimulatory effect of EGF by 60%. We then investigated the role of -subunits in carbachol induction of HA-ERK2. Parietal cells were transfected with the HA-ERK2 vector together with a vector
expressing the carboxy terminus of the
-adrenergic receptor kinase
1, known to block signaling mediated by
-subunits. In the
presence of this vector, carbachol induction of HA-ERK2 was inhibited
by 40%. Together these data suggest that, in the gastric parietal
cells, carbachol activates the ERKs through Ras- and
-dependent
mechanisms that require guanine nucleotide exchange factors other than Sos.
Ras; protein kinases; GTP-binding proteins; Grb2; Shc; mitogen-activated protein/extracellular signal-regulated protein kinases
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INTRODUCTION |
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RECENT STUDIES HAVE INDICATED that mammalian parietal cells contain multiple members of recently discovered families of protein kinases known as mitogen-activated protein kinases (MAPKs) or extracellular signal-regulated protein kinases (ERKs) (5, 27, 35).
The mechanisms responsible for the induction of the MAPK pathway appear to be complex and to require the assembly of multiple adapter proteins and the sequential activation of several protein kinases. Some of these protein-protein interactions occur through modular binding domains known as SH2 and SH3 domains (6, 7). These domains were initially described in the Rous sarcoma virus oncogene src. The SH2 domains have the ability to recognize and to stably associate with phosphorylated tyrosine residues, whereas the SH3 domains do not bind to phosphotyrosyl proteins but recognize proline-rich sequences, thus establishing a different and complementary system that allows protein-protein interactions within cells (7).
Recent studies have suggested that upon ligand stimulation the SH2
domain-containing adapter protein Shc gets phosphorylated on tyrosine
residues by Src-like cytoplasmic protein tyrosine kinases, allowing Shc
to interact with the adapter protein Grb2. This leads to
the recruitment of guanine nucleotide exchange factors with
proline-rich SH3 domain-binding sites to the membrane in proximity to
the isoprenylated small GTP-binding protein Ras (6, 7, 37, 38).
Exchange factors such as Sos promote the association of Ras with GTP,
and the GTP-bound, activated form of Ras binds members of the Raf
family of protein kinases, which are then recruited to the plasma
membrane and activated (6, 7, 13, 22, 38). MAPK kinases (MEKs) are
the downstream targets of Raf (6, 13, 19). These enzymes
phosphorylate both tyrosine and threonine residues on the MAP kinases,
leading to their phosphorylation and activation (6, 13,
19). In addition, -subunits of heterotrimeric
GTP-binding proteins have been recently shown to play an important role
in linking seven transmembrane receptors to the Ras/Raf/MAPK pathway
through only partially characterized mechanisms (10, 14, 21).
In a recent study we reported that carbachol is the most potent inducer of ERK2 activity in isolated gastric canine parietal cells, whereas gastrin and EGF were found to have weaker stimulatory effects (35).
Our understanding of the function and physiological role of the ERK pathway in the parietal cells has been improved significantly by the use of PD-98059, a selective inhibitor of the upstream ERK activator MEK (1). We recently tested the effect of this compound on carbachol-stimulated uptake of [14C]aminopyrine in the gastric parietal cells and noted that acute inhibition of the ERKs by PD-98059 led to a small increase in [14C]aminopyrine uptake and to a complete reversal of the acute inhibitory effect of EGF on [14C]aminopyrine uptake induced by either carbachol or histamine (35). In contrast, exposure of the cells to PD-98059 for 16 h reversed the chronic stimulatory effect of EGF on [14C]aminopyrine uptake induced by carbachol, suggesting that, although the acute effect of the ERKs on gastric acid secretion appears to be inhibitory, the activation of transcription factors and of early gene expression could be responsible for the ERK's chronic stimulatory effects (35).
In this study we sought to investigate the molecular mechanisms
responsible for induction of the ERKs in the gastric parietal cells. In
particular, we examined if carbachol induction of the ERKs would
require activation of Ras through a signaling pathway involving Shc,
Grb2, and Sos and whether this effect would involve -subunits of
heterotrimeric GTP-binding proteins.
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MATERIALS AND METHODS |
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Plasmids.
pLNCML-ET, expressing HA-ERK2 (19), was a gift of M. J. Weber
(Charlottesville, VA). RasAsn-17 expressing a dominantly
mutant (inactive) ras gene was obtained from Kunliang Guan (Ann
Arbor, MI). pcDNA-CD8-ARK-c, expressing a peptide derived from the
carboxy terminus of the
-adrenergic receptor kinase 1 (8), was a
gift of S. Gutkind (Bethesda, MD). SRa
SOS, expressing a dominantly
mutant (inactive) mSOS1 gene (32), was a gift of David Bowtell
(Melbourne, Australia). G
transducin, expressing the
-subunit of
bovine transducin gene, was obtained from American Type Culture
Collection (Manassas, VA). Plasmids were purified using Qiagen
(Chatsworth, CA) plasmid purification kits according to the
manufacturer's instructions.
Primary parietal cell preparation and culture.
For preparation of primary parietal cells, we used a modification of a
previously described method (3, 34). The mucosal layer of freshly
obtained canine gastric fundus was bluntly separated from the submucosa
and rinsed in Hank's balanced salt solution containing 0.1% BSA. The
cells were then dispersed by sequential exposure to collagenase (0.35 mg/ml) and 1 mM EDTA, and parietal cells were enriched by centrifugal
elutriation using a Beckman JE-6B elutriation rotor. Our best
preparations contained 70% parietal cells as determined by hematoxylin
and eosin and periodic acid-Schiff reagent staining. The parietal cells
were further purified by centrifugation through density gradients
generated by 50% Percoll (Pharmacia Biotech, Piscataway, NJ) at 30,000 g for 20 min. The cell fraction at = 1.05 consisted of
virtually 100% parietal cells as determined by staining with a
specific mouse monoclonal antibody against the hog
H+-K+-ATPase
-subunit (a gift of A. Smolka,
Charleston, SC) (33). The isolated parietal cells
(2 × 106 cells/well) were cultured for 16 h according
to the method of Chew et al. with some modifications (4, 26). Briefly,
the cells were cultured in Ham's F-12-DMEM (1:1) containing 50 mg/ml gentamycin, 50 U/ml penicillin G, and 2% DMSO (Sigma, St. Louis, MO)
on 12-well culture dishes (Coster, MA) coated with 150 ml of H2O-diluted (1:5) growth factor-reduced Matrigel (Becton
Dickinson, Bedford, MA). For our studies, the parietal cells were
incubated with either carbachol (Sigma) (100 µM) or EGF (Becton
Dickinson) (10 nM) for various time periods.
Ras activation assay. Analysis of Ras activation was performed by an immunoprecipitation assay as previously described (30). The parietal cells were labeled with [32P]orthophosphate at 0.1 mCi/ml in phosphate-free DMEM for 8 h and incubated with either carbachol or EGF for 5 min. Cells were lysed in 500 µl of lysis buffer [50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 1.5 mM MgCl2, 1 mM Na3VO4, 10 mM NaF, 10 mM Na4P2O7 · 10 H2O, 1 mM 4-(2-aminoethyl)-benzenesulfonylfluoridehydrochlorine (ICN-Biomedicals, Aurora, OH), 1 µg/ml leupeptin, and 1 µg/ml aprotinin], transferred into Microfuge tubes, and spun at 16,000 g for 20 min at 4°C. Ras was immunoprecipitated from the parietal cell lysates with a specific anti-Ras antibody precoupled to protein A-agarose (Oncogene Science, Cambridge, MA). Nucleotide was eluted with 2 mM dithiothreitol, 0.2% SDS, 0.5 mM GTP, and 0.5 mM GDP at 68°C for 20 min. Separation of the eluted nucleotide was performed on PEI-cellulose plates (Sigma) run in 1.2 M ammonium formate and 0.8 M HCl. The position of the GTP and GDP standards was determined by migration of [3H]GDP and [32P]GTP, respectively.
Transfection of primary cultured parietal cells. Before transfection the cells were washed once with 1 ml of Opti-MEM I serum-reduced media (GIBCO) and fed with 400 µl of Opti-MEM I media supplemented with 2% DMSO. The cells were transfected with 2-4 µg of the expression vectors. Transfections were carried out using Lipofectin (GIBCO) as previously described (26, 34). The day after transfection the media were removed, and the cells were fed with serum-free media for 24 h and then incubated with the test substances.
Immunoprecipitations and in-gel ERK2 assay.
Immunoprecipitations and in-gel ERK2 assays were performed according to
previously described techniques (31, 35) with minor modifications. The
parietal cells were lysed as previously described in Ras activation
assay. Equal amounts of proteins from each treatment group (1,000 µg) were incubated with 1 µl of an anti-HA specific mouse
monoclonal antibody (Babco) and mixed on a rotating
platform for 3 h at 4°C. Equal aliquots of protein G-agarose (50 µl) (Santa Cruz Biotechnology, Santa Cruz, CA) were then added to the
tubes, and the solutions were mixed for an additional 1 h. Protein
concentrations were measured by the Bradford method (2). After
centrifugation the pellets were washed twice with lysis buffer. The
samples were resuspended in 20 µl of electrophoresis buffer [for 10 ml:1 ml glycerol, 0.5 ml 2-mercaptoethanol, 3 ml 10% SDS, 1.25 ml of 1 M Tris buffer, 2 ml of 0.1% bromphenol blue, and 0.6 g urea], boiled
for 5 min, and applied to a 10% SDS-polyacrylamide gel containing 0.5 mg/ml myelin basic protein (Sigma). After electrophoresis the gel was
washed with two changes of 20% 2-propanol in 50 mM Tris (pH 8.0) for 1 h and then with two changes of 50 mM Tris (pH 8.0) containing 5 mM
2-mercaptoethanol for 1 h. The enzyme was denatured by incubating the
gel with two changes of 6 M guanidine-HCl for 1 h and then renatured
with five changes of 50 mM Tris (pH 8.0) containing 0.04% Tween 40 and
5 mM 2-mercaptoethanol for 1 h. The kinase reaction was performed in
conditions inhibitory to cyclic nucleotide-dependent protein kinase and
Ca2+-dependent protein kinases by incubating the gel at
25°C for 1 h with 40 mM HEPES (pH 8.0) containing 0.5 mM EGTA, 10 mM
MgCl2, 2 µM cAMP-dependent protein kinase inhibitor
peptide (Sigma), 40 µM ATP, and 2.5 µCi/ml of
[32P]ATP (6,000 Ci/mmol) (Amersham Life
Science, Arlington Heights, IL). After incubation, the gel was washed
with a 5% (wt/vol) TCA solution containing 1% (wt/vol) sodium
pyrophosphate, dried, and subjected to autoradiography.
Western blots.
Expression of HA-ERK2 and SOS in parietal cells transfected with the
pLNCML-ET and SRa
SOS vectors were measured by Western blots using
anti-HA and anti-Sos1 (Santa Cruz Biotechnology) antibodies. In some
experiments, Grb2 and Shc were immunoprecipitated from each treatment
group using specific anti-Grb2 (Santa Cruz Biotechnology) and anti-Shc
(Transduction Laboratories, Lexington, KY) antibodies. Equal amounts of
proteins were loaded on 10% SDS-polyacrylamide minigels and run at 200 V for 1 h. The gels were transferred on a Immobilon-P transfer membrane
(Millipore) in 25 mM Tris, 150 mM glycine, and 20% methanol. After
transfer the membranes were blocked in 10 ml of TBST (20 mM Tris, 0.15M
NaCl, 0.3% Tween), 5% dry milk for 2 h and then incubated for 1 h at
37°C in TBST, 5% dry milk, containing a specific antibody against
Shc, HA, and Sos. At the end of the incubation period the membranes
were washed in TBST for 30 min at room temperature and then incubated
for 1 h in TBST, 5% dry milk, containing either protein A directly conjugated to horseradish peroxidase (HRP) (Amersham Life Science) (1:2,500) or HRP-conjugated anti-mouse IgG (Transduction Laboratories) (1:2,000) for the anti-HA Western blots. Tyrosine phosphorylation of
Shc or the association of Grb2 with tyrosine-phosphorylated Shc were
examined using anti-phosphotyrosine Western blots. In these studies the
membranes were blocked in 10 ml of TBST, 5% BSA for 2 h and incubated
for 20 min at 37°C in TBST, 1% BSA, containing a specific
anti-phosphotyrosine antibody directly conjugated to HRP (RC20,
Transduction Laboratories) (1:2,500). At the end of the incubation
period the membranes were washed in TBST for 15 min at room temperature
and then exposed to the Amersham ECL detection system according to the
manufacturer's instructions.
Immunohistochemistry.
The parietal cells were cultured on slides and fixed in 4%
Formalin-PBS. The slides were blocked for 30 min with 20% donkey serum
and incubated for 2 h with either the mouse monoclonal anti-HA antibody
(1:500) or with the mouse monoclonal
anti-H+-K+-ATPase -subunit antibody (1:500)
(33). The cells were rinsed with PBS and a 1:150 dilution of the donkey
anti-mouse IgG secondary antibody [either FITC- or Cy-5-conjugated
(Jackson ImmunoResearch Laboratories, West Grove, PA)] was added for 1 h. After a wash with PBS, the cells were mounted in Vectashield (Vector
Laboratories, Burlingame, CA) and visualized by fluorescence
microscopy. In control experiments the parietal cells were "mock"
transfected (subjected to the transfection procedure with a
transfection solution that did not contain any HA-ERK2 DNA) and
incubated with both the primary (anti-HA antibody) and with either the
FITC- or the Cy-5-conjugated secondary antibodies.
Data analysis. Data are expressed as means ± SE, wherein n is equal to the number of separate dog preparations from which the parietal cells were obtained. Statistical analysis was performed using Student's t-test. P < 0.05 was considered to be significant.
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RESULTS |
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We have analyzed the purity of our parietal cell preparation by
staining the cultured cells with a well-characterized and specific
mouse monoclonal antibody raised against the hog
H+-K+-ATPase -subunit (33). As shown in Fig.
1A (cells incubated with the
H+-K+-ATPase antibody and with a donkey
anti-mouse FITC-conjugated secondary antibody) and Fig. 1B
(cells incubated with the H+-K+-ATPase antibody
and with a donkey anti-mouse Cy-5-conjugated secondary antibody),
virtually all cells stained positive with the
anti-H+-K+-ATPase
-subunit antibody,
confirming that our preparation contains almost exclusively parietal
cells. Transfection with Lipofectin led to a change in the morphology
of the parietal cells that became clustered as a consequence of the
transfection procedure (Fig. 1, C and D). However, in spite
of these morphological changes, both the transfected and the
nontransfected cells exhibited identical responses to carbachol
stimulation as indicated by their ability to uptake the weak base
aminopyrine (data not shown). We then demonstrated that the parietal
cells could be successfully transfected with the HA-ERK2 expression
vector. Figure 1F depicts parietal cells that stained positive
after incubation with a specific mouse monoclonal anti-HA antibody and
with a donkey anti-mouse FITC-conjugated secondary antibody. The
specificity of the immunofluorescence was confirmed by the observation
that no staining was detected in mock-transfected cells (Fig.
1E). Identical results were observed in the presence of a
donkey anti-mouse Cy-5-conjugated secondary antibody (data not shown).
Examination of 100 cells in three separate slides revealed that 11 ± 1% (n = 3) of the cells were successfully transfected with
the HA expression vector.
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Induction of the ERKs is known to involve the activation of the small
GTP-binding protein Ras (6, 13, 38). Thus we examined whether carbachol
and EGF would induce Ras activation. Ras activation was determined by
measuring GTP loading in immunoprecipitated Ras from parietal cells
metabolically labeled with 32P. 32P-labeled GTP
and GDP were separated by thin-layer chromatography. As depicted in
Fig. 2, both carbachol and EGF induced Ras
activation.
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To confirm that activation of Ras leads to induction of ERK2 in the
gastric parietal cells, we performed experiments in which we
transfected the parietal cells with the HA-ERK2 expression vector
together with a dominantly expressed mutant (inactive) ras
gene. HA-ERK2 was immunoprecipitated with a specific anti-HA antibody,
and its activity was quantitated by in-gel kinase assays. As shown in
Fig. 3A, dominant negative Ras
reduced carbachol induction of HA-ERK2 activity by 60%, whereas it
almost completely inhibited the stimulatory effect observed in the
presence of EGF. Expression of HA-ERK2 was monitored by Western blots
with an anti-HA antibody, as depicted in Fig. 3B. A linear
transformation of the densitometric analysis of the autoradiograms is
depicted in Fig. 3C.
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We then examined whether carbachol induction of Ras activation would
require the formation of Shc-Grb2 complexes through interaction with
phosphotyrosine binding domains. First, we examined the pattern of
expression of Shc proteins in the parietal cells. As shown in Fig.
4A, Western blot analysis of
anti-Shc immunoprecipitates with an anti-Shc antibody demonstrated the
presence of two major bands of 52 and 46 kDa, respectively. In contrast
to what has been reported in other cell types, no expression of the
66-kDa Shc isoform was detected in the canine parietal cells. We then sought to investigate the effect of carbachol and EGF on Shc tyrosine phosphorylation and association with Grb2. Probing of anti-Shc immunoprecipitates with an anti-phosphotyrosine antibody demonstrated that both carbachol and EGF time dependently induced tyrosyl
phosphorylation of a major 52-kDa Shc isoform and, to a lesser extent,
of the 46-kDa Shc isoform, with EGF being more potent then carbachol (Fig. 4C). Probing of anti-Grb2 immunoprecipitates with an
anti-phosphotyrosine antibody confirmed that both carbachol and EGF
induced the association of Grb2 with tyrosyl-phosphorylated Shc (Fig.
4B). Association of Grb2 with tyrosyl-phosphorylated Shc was
demonstrated by blotting Grb2 immunoprecipitates with an anti-Shc
antibody as shown in Fig. 4D. Probing of immunoprecipitated Shc
with the anti-Grb2 antibody further confirmed the association of Shc
with Grb2 in response to parietal cell stimulation (data not shown).
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Since Grb2 is known to associate with the guanine nucleotide exchange
factor Sos, we examined if carbachol and EGF would induce interaction
of Grb2 with Sos. As shown in Fig. 5,
probing of anti-Sos immunoprecipitates with an anti-Grb2 antibody
revealed that Sos was constitutively bound to Grb2 in response to both
carbachol and EGF. Similar results were obtained by probing Grb2
immunoprecipitates with the anti-Sos antibody (data not shown).
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To examine the functional role of Sos in carbachol induction of ERK2
activity, we performed experiments in which we transfected the parietal
cells with the HA-ERK2 vector together with a dominantly expressed
mutant (inactive) sos gene. As shown in Fig.
6A, dominant negative Sos did not
affect carbachol-stimulated HA-ERK2 activity, but it inhibited the
stimulatory effect of EGF by 60%. Expression of the HA-ERK2 protein
was documented by Western blots using an anti-HA antibody as indicated
in Fig. 6B. Western blot analysis of lysates from parietal
cells transfected with the dominant negative Sos gene using an
anti-Sos antibody documented the expression of both endogenous Sos and
of dominant negative Sos, which appeared as a smaller protein of ~110
kDa (data not shown). A linear transformation of the densitometric
analysis of the autoradiograms is depicted in Fig. 6C.
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Since -subunits of heterotrimeric GTP-binding proteins are known
to induce ERK activation in response to ligand stimulation (10, 14,
21), we sought to investigate whether carbachol induction of the ERKs
would be mediated, at least in part, by
-subunits. For these
experiments we transfected the parietal cells with the HA-ERK2 vector
together with a vector expressing a peptide derived from the carboxy
terminus of the
-adrenergic receptor kinase 1 (
ARKct
peptide), which specifically blocks signaling mediated by
-subunits (8). As shown in Fig.
7A, expression of the
ARKct
peptide inhibited carbachol-stimulated HA-ERK2 activity by 40%,
suggesting that carbachol targets the ERKs via signaling pathways that
involve, at least in part,
-subunits. Similar results were
observed when the cells were transfected with the HA-ERK2 vector
together with a vector expressing the
-subunit of transducin,
another inhibitor of
-subunit-activated signal transduction
pathways (10) (data not shown). Expression of the HA-ERK2 protein was
documented by Western blots using an anti-HA antibody as indicated in
Fig. 7B. A linear transformation of the densitometric analysis
of the autoradiograms is depicted in Fig. 7C.
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DISCUSSION |
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The ERKs or MAPKs are key elements in a cascade of phosphorylation reactions that is triggered by the interaction of hormones, growth factors, and neurotransmitters with their specific cellular receptors (6, 8, 9, 12, 13, 29, 35). Numerous studies have shown that these enzymes are responsible for the regulation of a wide variety of physiological functions. Activation of the ERKs is in fact necessary for both the induction of cellular proliferation and for the expression of highly differentiated cellular phenotypes (23, 35, 36). Recent studies have shown that seven transmembrane receptors, known to interact with heterotrimeric GTP-binding proteins to transduce extracellular signals to downstream effector molecules (28), are able to activate Ras, Raf, MEK, the ERKs, and early response genes in a fashion similar to that employed by receptor tyrosine kinases (8-12, 14, 24, 25, 29-31, 35, 36).
The gastric parietal cell is a highly differentiated cell that secretes gastric acid in response to a broad range of physiological stimuli. Carbachol, gastrin, and histamine are among the major gastric acid secretagogues (15). Carbachol is known to interact with specific m3-muscarinic receptors present on the surface of the parietal cells, leading to the activation of multiple signaling pathways (15, 20, 35).
In a previous report we examined the physiological regulation of the ERKs in response to carbachol, gastrin, histamine, and EGF and found that, of these agents, carbachol was the most potent inducer of ERK2 (35). Thus, in this study, we dissected some of the signaling pathways that target ERK2 in response to carbachol stimulation.
Muscarinic receptors are known to activate Ras in multiple cell types (10, 24, 25). In our study we observed that, in the parietal cells, carbachol was as potent as EGF in inducing Ras activation. The functional significance of this observation was examined by transfecting the parietal cells with a dominant negative Ras gene together with a vector expressing an epitope-tagged ERK2. Interestingly, in these experiments, we noted that dominant negative Ras inhibited only partially the stimulatory action of carbachol, whereas it completely blocked the effect of EGF. A possible explanation for this phenomenon could be that carbachol induces multiple signaling pathways in order to activate Ras. A recent study has reported that, in some instances, Ras is the target of both tyrosine kinase-dependent and tyrosine kinase-independent protein kinase C (PKC)-dependent pathways and that PKC activation of Ras and of its downstream target Raf-1 is insensitive to inhibition by dominant negative Ras (22). Although in this study we did not analyze the role of PKC in Ras activation, it is intriguing to speculate that, in the parietal cells, carbachol could activate Ras via both tyrosine kinase-dependent and tyrosine kinase-independent PKC-dependent pathways. Further studies are needed to elucidate in more detail the molecular mechanisms responsible for Ras activation in the gastric parietal cells.
One of the first steps in the process of Ras activation involves the phosphorylation of Shc on tyrosine residues by Src-like cytoplasmic protein tyrosine kinases, allowing its interaction with the adapter protein Grb2 (6, 7, 11, 12, 30). Three major Shc isoforms of 66, 52, and 46 kDa, respectively, are known to be expressed in mammalian cells. In the canine parietal cells we were able to detect only the 52- and 46-kDa isoforms. Interestingly, comparable results were observed in the rat pancreatic acini where the 66-kDa isoform was only barely detectable (11). These data suggest that cell type-specific differences regulate the pattern of expression of Shc in mammalian tissues. In agreement with these observations, we noted that both carbachol and EGF stimulated the tyrosyl phosphorylation of the 52- and, to a lesser extent, of the 46-kDa Shc isoforms, leading to their association with the adapter protein Grb2. Similarly, numerous other reports have demonstrated identical patterns of Shc phosphorylation in response to gastrin (12), cholecystokinin (11), and thyrotropin-releasing hormone (29) in different tissues and tumor cell lines. In our study, we also observed that EGF was more potent then carbachol in inducing the phosphorylation of Shc and its association with Grb2, further supporting the notion that carbachol, but not EGF, might activate tyrosine kinase-independent pathways to activate Ras.
Another important component of the multiprotein complex involved in the activation of Ras is Sos, a guanine nucleotide exchange factor that is recruited to the plasma membrane in proximity to Ras through its ability to interact with Grb2 (6, 7, 17, 38). Sos leads to the activation of Ras by promoting the association of Ras with GTP (6, 7, 17, 38). In some cell types Sos appears to be constitutively bound to Grb2 (17). Indeed, in the parietal cells we noted that Sos was constitutively bound to Grb2, because the level of association of this adapter molecule with Sos did not change upon stimulation of the cells with either carbachol or EGF. However, when we examined the effect of a dominant negative sos gene on ERK2 induction, we observed that, although this construct potently inhibited the stimulatory action of EGF, it was unable to inhibit the effect of carbachol. Interestingly, recent reports have demonstrated that in addition to Sos, other exchangers can lead to Ras activation in several cell types (16, 18, 24). M1- and M2-muscarinic receptors transfected in NIH/3T3 and COS-7 cells have been shown to recruit a novel Ras exchange factor, Ras-GRF/CD25Mm, to activate MAPK (24). Although Ras-GRF is predominantly expressed in the brain, it can also be detected in other tissues and nonneuronal cell lines (24). In our study we performed Western blots with a specific anti-Ras-GRF antibody, but we were unable to detect any Ras-GRF in the parietal cells (data not shown). Although this negative finding could simply reflect the inability of our antibody to detect canine Ras-GRF, it is possible that this factor is not expressed in the stomach. Accordingly, we postulate that, in the gastric parietal cells, carbachol, but not EGF, induces the recruitment of a novel Ras exchanger possibly bound to Grb2, which stimulates Ras activation, leading in the end to ERK2 induction. Additional studies will be needed to characterize in more detail the exchange factor that leads to Ras activation in response to carbachol.
Another signaling pathway employed by seven transmembrane receptors to
signal to the ERKs is known to involve -subunits of GTP-binding
proteins (10, 14, 21). Although this pathway has been only partially
characterized, recent reports have suggested that it requires
activation of phosphoinositide 3-kinase (PI3K), leading to induction of
the ERKs through the recruitment of Shc, Grb2, Sos, and Ras.
Accordingly, we examined whether in the parietal cells carbachol
induction of ERK2 would be mediated, at least in part, by
-subunits. We demonstrated that transfection of the parietal
cells with vectors expressing proteins known to bind and inactivate
-subunits leads to inhibition of ERK2 induction in response to
carbachol. These observations are in agreement with studies conducted
in COS-7 cells transfected with both M1- and
M2-muscarinic receptors (10, 21). In this study we did not
examine the role of PI3K in the process of Ras activation, and
experiments will be needed to study the role of this kinase in
carbachol signaling.
In conclusion, in the parietal cells, carbachol targets the ERKs via
multiple signaling pathways, which appear to involve Ras, tyrosine
kinases, -subunits, and a Ras exchanger other than Sos.
Additional studies will be necessary to dissect in more detail the
complex interaction existing among these different signaling molecules
and to identify the specific Ras exchanger involved in the process of
Ras activation in response to muscarinic receptor activation.
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ACKNOWLEDGEMENTS |
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We thank Lara Post for assistance with parietal cell preparation, Judy Poore for the immunohistochemical studies, and Dr. Rebecca Van Dyke and Dr. John Williams for helpful advice.
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FOOTNOTES |
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This work was supported by National Institutes of Health (NIH) Grant RO1-DK-33500 and funds from the University of Michigan Gastrointestinal Peptide Research Center (NIH Grant P30-DK-34933). A. Todisco is a recipient of an American Gastroenterological Association Industry Research Scholar Award, a Clinical Investigator Award from the NIH (grant K08-DK-02336), and a grant from the Charles E. Culpeper Foundation Health Program.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. Todisco, 6520 MSRBI, Ann Arbor, MI 48109-0682 (E-mail: atodisco{at}umich.edu).
Received 13 October 1998; accepted in final form 17 March 1999.
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