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Originally published In Press as doi:10.1074/jbc.M700424200 on March 5, 2007

J. Biol. Chem., Vol. 282, Issue 18, 13303-13315, May 4, 2007
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Consequences of Direct Versus Indirect Activation of Epidermal Growth Factor Receptor in Intestinal Epithelial Cells Are Dictated by Protein-tyrosine Phosphatase 1B*

Declan F. McCole1, Anh Truong, Michael Bunz, and Kim E. Barrett

From the Department of Medicine, University of California, San Diego, School of Medicine, La Jolla, California 92093

Received for publication, January 16, 2007 , and in revised form, February 28, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The epidermal growth factor receptor (EGFR) is an integral regulator of many cellular functions. EGFR also acts as a central conduit for extracellular signals involving direct activation of the receptor by EGFR ligands or indirect activation by G protein-coupled receptor (GPCR)-stimulated transactivation of the EGFR. We have previously shown that EGFR negatively regulates epithelial chloride secretion as a result of transforming growth factor-{alpha}-mediated EGFR transactivation in response to muscarinic GPCR activation. Here we show that direct activation of the EGFR by EGFR ligands produces a different pattern of EGFR tyrosine phosphorylation and downstream phosphatidylinositol 3-kinase recruitment than GPCR-stimulated transactivation of the EGFR occurring via paracrine EGFR ligand release. Moreover, we demonstrate that this differential signaling and its consequences depend on protein-tyrosine phosphatase 1B activity. Thus protein-tyrosine phosphatase 1B governs differential recruitment of signaling pathways involved in EGFR regulation of epithelial ion transport. Our findings furthermore establish how divergent signaling outcomes can arise from the activation of a single receptor.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
All cell types rely on a limited set of receptors and signaling intermediates to translate extracellular information into appropriate acute and chronic changes in cellular behavior. Furthermore, many receptors mediate their effects via the secondary recruitment of other receptor types, including by stimulating the release of ligands for the downstream receptor (1, 2). A well studied example of this paradigm relates to cross-talk between G-protein-coupled receptors and a variety of receptor tyrosine kinases. In some cases this receptor-tyrosine kinase "transactivation" results in different downstream consequences than does direct activation of the receptor with its endogenous ligand (3, 4). However, a fundamental question remains as to how a single receptor, after activation by binding of either exogenous or endogenous ligand, can recruit distinct sets of downstream effectors and associated physiological outcomes.

Epidermal growth factor receptor (EGFR)2 is one of four members of the ErbB receptor family, the other members being ErbB2, ErbB3, and ErbB4. Although ErbB2 has no known ligand of its own, it possesses the greatest enzymatic activity of the four isoforms and is the preferred dimerization partner for the other three receptors (5). The complexity of ErbB receptor activity is amplified by the large number of peptide ligands described for these receptors. All of these peptides originate as transmembrane precursors that are then enzymatically cleaved, thus releasing mature soluble ligands, including the EGFR ligands, EGF, and transforming growth factor {alpha} (TGF-{alpha}). The latter is the most abundant EGFR ligand found in the gastrointestinal tract (6, 7). After ligand binding, ErbB receptors undergo autophosphorylation and dimerization to form catalytically active homo- or heterodimers (8). The composition of these receptor dimers dictates the recruitment of signaling pathways, adding another level of complexity to transduction of extracellular signals by this receptor family (5, 9). Downstream interaction with SH2 domain-containing molecules depends on the type of dimer formed, reflected in the ultimate recruitment of different signaling effectors such as mitogen-activated protein kinases and phosphatidylinositol 3-kinase (PI3K). In addition, after binding to the EGFR, different EGFR ligands have variable effects on the ErbB receptor isoforms that are recruited and activated. This diversifies dimer formation and subsequent downstream signaling even though the ligands bind to the same receptor (6, 10).

Intestinal epithelial cells form a continuous monolayer that lines the intestinal tract. These cells are responsible for the uptake of nutrients, for forming a barrier against pathogens and environmental toxins, and for the tightly regulated absorption and secretion of ions and water. The active secretion of chloride ions provides the driving force for intestinal fluid secretion. The critical importance of regulatory mechanisms governing fluid secretory processes is highlighted by pathological conditions where regulation is impaired. The most dramatic example of excessive chloride and fluid secretion is cholera (11). Complex mechanisms regulate intestinal ion transport, and our laboratory has identified a critical role for the EGFR as a central regulator that limits ion transport stimulated by calcium-dependent agents such as the muscarinic agonist, carbachol (CCh).


Figure 1
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  		FIGURE 1.
TGF-{alpha} and EGF have different potencies in causing EGFR and ErbB2 phosphorylation. Polarized T84 cell monolayers were incubated basolaterally with EGF (0.167–33.4 nM; 5 min), CCh (100 µM; 2 min), or TGF-{alpha} (1.67–50.1 nM; 5 min), and cell lysate supernatants were immunoprecipitated (IP) with anti-EGFR or anti-ErbB2. A, representative Western blot showing phosphorylated (p-) EGFR tyrosine phosphorylation after treatment of cells with EGF (16.7 nM), CCh (100 µM), or TGF-{alpha} (1.67–50.7 nM). Con, control. B, the Western blot in A was stripped and reprobed to show comparable levels of total EGFR protein in each sample. C, densitometric analysis of EGFR phosphorylation (n = 4). D and E, representative Western blots for anti-phosphotyrosine and total EGFR in EGFR immunoprecipitates from T84 cell lysate supernatants after treatment of cells with TGF-{alpha} (33.4 nM; 5 min), carbachol (CCh, 100 µM; 2 min), or EGF (0.167–16.7 nM; 5 min). F, densitometric analysis of four similar blots to that in D showing EGFR tyrosine phosphorylation. G and H, representative Western blots of immunoprecipitated ErbB2 showing phosphorylated (G) and total (H) ErbB2 after treatment of cells with EGF(16.7 nM), CCh (100 µM), or TGF-{alpha} (8.35–167 nM). I, densitometric analysis of four similar blots to that in G showing ErbB2 tyrosine phosphorylation. J and K, representative Western blots for anti-phosphotyrosine and total ErbB2 in ErbB2 immunoprecipitates from T84 cell lysate supernatants after treatment of cells with TGF-{alpha} (33.4 nM), CCh (100 µM), or EGF (0.167–16.7 nM). L, densitometric analysis showing ErbB2 phosphorylation (n = 5). Results are presented as the means ± S.E. for levels of phosphorylation expressed as arbitrary units (a.u.). Asterisks represent significant differences from control/untreated cells (*, p < 0.05; **, p < 0.01; ***, p < 0.001), and # represent significant differences from cells treated with TGF-{alpha} (33.4 nM), (#, p < 0.05; ###, p < 0.001).

 
We have previously shown that the G protein-coupled receptor agonist, CCh, which stimulates chloride secretion across monolayers of T84 colonic epithelial cells, also transactivates the EGFR. This leads in turn to activation of extracellular signal-regulated kinases and a later reduction in the level of chloride secretion (4, 12). Exogenously added EGF can also inhibit CCh-stimulated chloride secretion (4, 13). The inhibitory effects of CCh-stimulated transactivation versus EGF-dependent activation of the EGFR are, however, attributable to different downstream mechanisms. Divergent signaling evoked by these two stimuli appears to occur at the level of the EGFR, since direct binding of EGF to EGFR causes recruitment of the ErbB2 receptor to form a heterodimer with the EGFR, whereas CCh-stimulated transactivation does not recruit ErbB2 (4). In addition, the inhibitory effect of EGF on CCh-stimulated Cl– secretion is mediated by activation of PI3K; however, CCh-induced activation of the EGFR does not cause an increase in the lipid kinase activity of PI3K (14). We have also shown that CCh transactivation of EGFR is mediated by the extracellular release of TGF-{alpha} (15). This led us to hypothesize that TGF-{alpha} and EGF may have different signaling properties in colonic epithelial cells despite both being ligands for EGFR. The aim of this study, therefore, was to investigate whether differences between EGF- and TGF-{alpha}-stimulated ErbB activation and downstream signal recruitment explain the previously observed signaling divergence between direct and indirect activation of the EGFR by EGF and CCh. respectively. In fact, we find that EGFR responses to TGF-{alpha} and their downstream signaling consequences are modified by the signaling milieu that exists in CCh-stimulated cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Carbachol (Sigma), LY294002, recombinant human transforming growth factor {alpha}, Tyrphostin AG825, wortmannin (Calbiochem), recombinant human epidermal growth factor (Genzyme, Cambridge, MA), rabbit anti-phospho-Akt1, rabbit anti-Akt1, mouse anti-human EGF receptor (clone LA1) and mouse anti-phosphotyrosine antibodies (Upstate%20Biotechnology">Upstate Biotechnology Inc., Lake Placid, NY), phosphorylation site-specific EGFR tyrosine antibodies (BioSource, Camarillo, CA), and Tris-glycine electrophoresis gels (Bio-Rad) were obtained from the sources noted. Rabbit polyclonal anti-Neu (ErbB2) and rabbit polyclonal anti-human EGFR (1005) antibody, used to measure total EGFR, were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All other reagents were of analytical grade and obtained commercially.

Cell Culture—All experiments were performed using monolayers of the T84 human colonic crypt carcinoma cell line. Methods for maintenance of T84 cells in culture were described previously (14). Briefly, T84 cells were grown in Dulbecco's modified Eagle's/F-12 medium (JRH, Lenexa, Kansas) supplemented with 5% newborn calf serum. For voltage clamp experiments, 5 x 105 cells were seeded onto 12-mm Millicell-HA filters. For Western blotting experiments, 106 cells were seeded onto 30-mm Millicell-HA filters. Cells were cultured for 10–15 days before use. When grown on filters, T84 cells are known to acquire the polarized phenotype of native colonic epithelia (16). In accordance with the known distribution of their receptors on intestinal epithelia, CCh, EGF, and TGF-{alpha} were added basolaterally in all experiments (17, 18).

Electrophysiological Studies—T84 cell monolayers were mounted in Ussing chambers (window area = 0.6 cm2) and bathed in oxygenated (95% O2, 5%CO2) Ringers' solution at 37 °C. The composition of the Ringer's solution was 140 mM Na+, 5.2 mM K+, 1.2 mM Ca2+, 0.8 mM Mg2+, 120 mM Cl–, 25 mM Formula, 2.4 mM Formula, 0.4 mM Formula, and 10 mM glucose. Monolayers were voltage-clamped to zero potential difference by the application of short-circuit current (Isc). Under these conditions, changes in Isc ({Delta}Isc) in response to agonists are wholly reflective of electrogenic chloride secretion (19).

Immunoprecipitations and Western Blotting—T84 cell monolayers were washed (x3) with Ringer's solution, equilibrated for 30 min at 37 °C, and then stimulated with agonists (with and without antagonists) as appropriate. The reaction was stopped by washing in ice-cold phosphate-buffered saline (PBS) and lysing in ice-cold lysis buffer (1% Triton X-100, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml antipain, 100 µg/ml phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, 1 mM sodium fluoride, and 1 mM EDTA in PBS) for 45 min. Cells were then scraped from the filters into microcentrifuge tubes and spun at 12,000 rpm for 10 min. Cell lysate supernatants were assayed for protein content (Bio-Rad protein assay kit) and adjusted so that each sample contained an equal amount of protein. For immunoprecipitation studies, cell lysate supernatants were incubated with immunoprecipitating antibody for 1 h at 4 °C followed by 1 h with protein A-Sepharose beads. The beads were washed in ice-cold phosphate-buffered saline (x3) and resuspended in 2x gel loading buffer (50 mM Tris, pH 6.8, 2% SDS, 200 mM dithiothreitol, 40% glycerol, 0.2 bromphenol blue). Samples were separated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene membranes (PerkinElmer Life Sciences), and immunoreactive proteins were detected by chemiluminescence (Roche Applied Science) of horseradish peroxidase-conjugated secondary antibodies (anti-mouse or anti-rabbit IgG; Transduction Laboratories, Lexington, KY). Densitometric analysis of Western blots was carried out using NIH Image software.


Figure 2
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  		FIGURE 2.
The inhibitory effect of TGF-{alpha} on CCh-stimulated Cl– secretion does not require ErbB2 activation but is dependent in part on PI3K activation. A, T84 cell monolayers, mounted in Ussing chambers, were pretreated basolaterally for 20 min with a range of concentrations of TGF-{alpha} below those capable of activating ErbB2 (0.0167–16.7 nM). Cells were then stimulated basolaterally with CCh (100 µM), and changes in short-circuit current ({Delta}Isc) were measured (n = 4). B, T84 cells were pretreated with the ErbB2 kinase inhibitor, AG825 (1 or 10 µM), for 30 min before basolateral treatment with TGF-{alpha} (33.4 nM; 5 min). ErbB2 immunoprecipitates (IP) were probed for phosphotyrosine levels then stripped and reprobed for total ErbB2 levels to show comparable ErbB2 levels in each sample. C, representative blot of total ErbB2. Con, control. D, representative blot of EGFR phosphorylation (p) after treatment with TGF-{alpha} in the presence or absence of AG825. E, densitometric analysis of four similar experiments to that in B. F, T84 cells were pretreated with the ErbB2 kinase inhibitor, AG825 (1 or 10µM), for 30 min before basolateral treatment with EGF (16.7 nM; 5 min). EGFR immunoprecipitates were probed for phosphotyrosine (p-Tyr) levels then stripped and reprobed for total EGFR to show comparable EGFR levels in each sample. Blots are representative of four separate experiments. G, monolayers of T84 cells were mounted in Ussing chambers and incubated for 30 min with AG825 (0, 1, or 10 µM). Monolayers were then incubated basolaterally with TGF-{alpha} (33.4 nM; 20 min) and stimulated basolaterally with CCh (100 µM), and changes in {Delta}Isc were measured (n = 4). Results are presented as the means ± S.E. Asterisks represent significant differences from control/unstimulated cells (**, p < 0.01; ***, p < 0.001), x represents differences from CCh alone (x, p < 0.05; xx, p < 0.01; xxx, p < 0.001), # signifies differences from TGF-{alpha} treatment (##, p < 0.01; ###, p < 0.001).

 


Figure 3
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  		FIGURE 3.
The inhibitory effect of TGF-{alpha} on CCh-stimulated Cl– secretion is dependent in part on PI3K activation. A, after treatment of cells with TGF-{alpha} (1.67–33.4 nM; 5 min), CCh (100 µM; 2 min), or EGF (16.7 nM; 5 min), cell lysate supernatants were probed for phosphorylated and total Akt1, as shown in representative blots. Con, control. B, densitometric analysis of blots from four similar experiments to A. C and D, T84 monolayers were preincubated with the PI3K inhibitors, wortmannin (Wort; 50 nM; n = 3), or LY294002 (20 µM; n = 4) for 30 min before administration of TGF-{alpha} (1.67 nM; 20 min) and subsequent measurement of CCh (100 µM)-stimulated Cl– secretion. Results are presented as the means ± S.E. Asterisks represent significant differences from control/unstimulated cells (*, p < 0.05; **, p < 0.01; ***, p < 0.001), and @ represents differences from TGF-{alpha} plus CCh treatment (@, p < 0.05; @@, p < 0.01).

 
Small Interfering RNA (siRNA) Transfection—T84 cells were plated at a density of 2 x 106 cells in T75 flasks 72 h before the first transfection and allowed to reach 50–70% confluency. Protein-tyrosine phosphatase (PTP) 1B SMARTpool siRNA reagent containing 4 pooled siRNA duplexes was purchased from Upstate%20Biotechnology">Upstate Biotechnology/Dharmacon (Chicago, IL), and 100 pmol of siRNA was transfected into T84 cell suspension using the Amaxa nucleofector system (Amaxa Inc., Gaithersburg, MD) as per the manufacturer's instructions. Cells were seeded onto permeable supports and incubated for 66 h. After culture, cells were stimulated with CCh, EGF, or Ringer's solution for the indicated periods of time. A nonspecific control siRNA SMARTpool (100 pmol) was used as negative control (Upstate).

Statistical Analysis—All data are expressed as the means ± S.E. for a series of n experiments. Student's t tests or analysis of variance with the Student-Newman-Keul's post hoc test were used to compare mean values as appropriate. p values <0.05 were considered to represent significant differences.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
EGF and TGF-{alpha} Have Different Potencies in Stimulating Phosphorylation of the EGFR and ErbB2 in the Colonic Epithelial Cell Line, T84—We first set out to investigate whether the EGFR agonists EGF and TGF-{alpha} were equipotent in inducing EGFR phosphorylation in T84 human colonic epithelial cells. Polarized T84 cells, grown on permeable supports, were treated basolaterally with CCh (100 µM; 2 min), EGF (16.7 nM; 5 min), or a range of concentrations of TGF-{alpha} (1.67–50.1 nM; 5 min). The duration of CCh or growth factor treatment has previously been shown by our group to be optimal for stimulation of EGFR phosphorylation in this system (12). In addition, the concentration of CCh used is physiologically relevant and is well established as the prototypic stimulus of GPCR-induced calcium-dependent chloride secretion across intestinal epithelial cells by our group and others (13, 20, 21). Western blot analysis of cell lysate supernatants immunoprecipitated with an anti-EGFR antibody and probed for phosphotyrosine showed that CCh and EGF induced EGFR phosphorylation as expected (Fig. 1A). Total EGFR levels are shown in Fig. 1B. TGF-{alpha} also induced EGFR phosphorylation in a concentration-dependent manner, with 8.35 nM being the lowest effective dose. However, densitometric analysis revealed that a 3-fold higher dose of TGF-{alpha} (50.1 nM) was required to induce EGFR phosphorylation at levels equivalent to that induced by EGF (16.7 nM)(p < 0.05; n = 4; Fig. 1C). This observation was further explored in a separate set of experiments where T84 cells were treated basolaterally with CCh (100 µM; 2 min), TGF-{alpha} (33.4 nM; 5 min), or a range of EGF concentrations (0.167–16.7 nM; 5 min) (Fig. 1D). EGF was not tested at concentrations higher than 16.7 nM due to the marked potency of this concentration compared with higher concentrations of TGF-{alpha}, as shown in Fig. 1C. EGF induced EGFR phosphorylation in a concentration-dependent manner (Fig. 1D). Total EGFR levels are shown in Fig. 1E. Densitometric analysis showed that EGFR tyrosine phosphorylation stimulated by EGF at 8.35 nM was comparable with that induced by a 4-fold higher concentration of TGF-{alpha} (33.4 nM) (Fig. 1F). Furthermore, EGF induced significant EGFR phosphorylation at 1.67 nM (p < 0.05; n = 4; Fig. 1F), whereas the lowest concentration of TGF-{alpha} that stimulated EGFR phosphorylation was 8.35 nM. These data indicate that EGF is significantly more potent than TGF-{alpha} at inducing EGFR phosphorylation in colonic epithelial cells.


Figure 4
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  		FIGURE 4.
Indirect activation of EGFR by carbachol produces a different pattern of receptor tyrosine phosphorylation than direct activation by EGF or TGF-{alpha}. T84 cells were incubated basolaterally with EGF (16.7 nM; 5 min), CCh (100 µM; 2 min), or TGF-{alpha} (1.67–33.4 nM; 5 min). Cell lysate supernatants were analyzed by Western blotting using phospho-specific antibodies against individual tyrosine residues of the EGFR. A, representative blots of phosphorylated (p-) EGFR tyrosine residues Tyr-1173, Tyr-1086, and Tyr-1068 and a stripped blot probed for total EGFR to demonstrate equal loading of protein. B–D, densitometric analysis of Western blots detecting phosphorylation of EGFR tyrosines Tyr-1173 (B), Tyr-1086 (C), and Tyr-1068 (D). E, representative blots of phosphorylated EGFR tyrosine residues Tyr-1148, Tyr-992, and Tyr-845 and a stripped blot probed for total EGFR to demonstrate equal loading of protein. F–H, densitometric analysis of Western blots detecting phosphorylation levels of EGFR tyrosines Tyr-1148 (F), Tyr-992 (G), and Tyr-845 (H). Results are presented as the means ± S.E. Asterisks represent significant differences from control/unstimulated cells (*, p < 0.05; **, p < 0.01; ***, p < 0.001; n = 3–7).

 
Having shown that EGF is a more potent inducer of EGFR activation than TGF-{alpha}, we next investigated whether this difference in potency also applied to ErbB2 receptor phosphorylation. Confluent monolayers of polarized T84 cells were treated basolaterally with CCh (100 µM; 2 min), EGF (16.7 nM; 5 min), or a range of concentrations of TGF-{alpha} (8.35–167 nM). As reported previously, EGF, but not CCh, induced ErbB2 phosphorylation (Fig. 1G). Furthermore, TGF-{alpha} was unable to cause ErbB2 phosphorylation at concentrations below 33.4 nM (Figs. 1, G and I). Thus, concentrations of TGF-{alpha} that were capable of inducing EGFR phosphorylation (cf. Fig. 1C) did not phosphorylate ErbB2 (Fig. 2I). This apparent difference in potency was more fully investigated by testing the ability of a range of EGF concentrations to induce ErbB2 phosphorylation in T84 cells (representative blots for phosphorylated and total ErbB2 shown in Figs. 1, J and K). EGF induced significant ErbB2 phosphorylation at a concentration of 8.35 nM (p < 0.05 versus control; n = 4; Fig. 1L). The level of phosphorylation induced by EGF at this concentration was significantly greater than that induced by 33.4 nM TGF-{alpha} (p < 0.05; n = 4). These data suggest that EGF is also markedly more potent than TGF-{alpha} at inducing ErbB2 phosphorylation.

TGF-{alpha} Inhibition of Ca2+-dependent Cl– Secretion Does Not Require ErbB2 Activation—We next explored the significance of our data for regulation of Ca2+-dependent transepithelial chloride secretion. We investigated whether EGFR-mediated inhibition of Ca2+-dependent chloride secretion and, specifically, inhibition produced by TGF-{alpha} required activation of the ErbB2 receptor. Confluent monolayers of T84 cells were mounted in Ussing chambers. Basolateral application of CCh (100 µM) stimulated a rapid transepithelial Cl– transport response (Fig. 2A). Maximal inhibition of CCh-stimulated Isc was achieved after pretreatment with TGF-{alpha} at a concentration of 1.67 nM (p < 0.001; n = 4). This concentration of TGF-{alpha} is well below the concentration required to stimulate ErbB2 receptor phosphorylation (cf. Fig. 1I), suggesting that ErbB2 is unlikely to be involved in TGF-{alpha}-stimulated inhibition of Cl– secretion.

Having shown that TGF-{alpha} could maximally inhibit CCh-stimulated Isc at concentrations below those required to activate the ErbB2 receptor, we set out to confirm these findings by blocking ErbB2 activation and examining any effect on the ability of TGF-{alpha} to inhibit secretory responses to CCh in T84 cells. Our approach was to use a pharmacologic inhibitor of ErbB2 activation, tyrphostin AG825 (22). First, we confirmed that AG825 could block TGF-{alpha} stimulation of ErbB2 phosphorylation. Fig. 2B shows a representative Western blot of immunoprecipitated ErbB2 prepared from cell lysate supernatants of cells stimulated with TGF-{alpha} (33.4 nM) with or without pretreatment with AG825 (1 or 10 µM; 30 min) and probed for phosphotyrosine. AG825 inhibited TGF-{alpha}-induced ErbB2 phosphorylation at both 1 µM (42 ± 12% inhibition; p < 0.01; n = 4) and 10 µM (65 ± 13% inhibition; p < 0.001; n = 4; Fig. 2E). AG825 had no effect on the ability of TGF-{alpha} to induce EGFR phosphorylation (Fig. 2D). Furthermore, AG825 did not affect the ability of EGF to induce phosphorylation of the EGFR itself (Fig. 2F).

Because AG825 can effectively block TGF-{alpha} stimulation of ErbB2 activation, we next investigated whether ErbB2 activation is required for the inhibitory effect of a high concentration of TGF-{alpha} on CCh-stimulated Isc.T84 cell monolayers were mounted in Ussing chambers and preincubated bilaterally for 30 min with AG825 at 1 or 10 µM or with vehicle control. TGF-{alpha} significantly inhibited the Isc response to CCh whether or not AG825 was present (Fig. 2G). These data combined with the data in Fig. 2A suggest that ErbB2 activation is not required for TGF-{alpha}-mediated inhibition of the chloride secretory response to CCh.

ErbB2 Activation Is Not Required for Activation of PI3K Signaling—Although CCh transactivates the EGFR via the release of TGF-{alpha} (15), we hypothesized that the differential potency of EGF and TGF-{alpha} could account for the ability of EGF and CCh to recruit different signals downstream of EGFR. We have previously observed that inhibition of calcium-dependent Cl– secretion by exogenous EGF involves PI3K activation, whereas inhibition of Cl– secretion mediated by CCh-stimulated transactivation of the EGFR likely does not (14). Therefore, to determine whether ErbB2 activation was required for PI3K recruitment, we next investigated whether inhibition of Cl– secretion by exogenous TGF-{alpha}, at concentrations that do not activate ErbB2, involved PI3K activation. Western blot analysis showed that TGF-{alpha} could activate PI3K signaling, as measured by phosphorylation of the PI3K downstream target, Akt1, on threonine 308. This effect occurred at TGF-{alpha} concentrations below those required to phosphorylate ErbB2 (Fig. 3, A and B). These data indicate that TGF-{alpha} can likely activate PI3K independent of ErbB2. The involvement of PI3K in TGF-{alpha} induced inhibition of Cl– secretion was confirmed by studies demonstrating that the PI3K inhibitors wortmannin (50 nM) and LY294002 (20 µM) partially reversed the inhibitory effect of a low concentration of TGF-{alpha}, 1.67 nM, on chloride secretion (Fig. 3, C and D). Therefore, because TGF-{alpha} inhibits Cl– secretion via PI3K activation, albeit in an ErbB2-independent fashion, differences in ErbB2 activation likely do not account for differential recruitment of PI3K in the negative regulation of Cl– secretion by direct versus indirect activation of EGFR.

Indirect Activation of EGFR Induces a Different Pattern of Tyrosine Phosphorylation than Direct Activation—Because the signaling divergence between direct versus indirect EGFR activation was apparently not mediated at the level of ErbB2 kinase recruitment, we investigated whether the basis for differential signal recruitment by direct versus indirect EGFR activation occurred at the level of EGFR itself. EGFR activation and recruitment of downstream signaling pathways is dependent on phosphorylation of key EGFR tyrosine residues (10). EGFR contains four major autophosphorylation sites, Tyr-1173, Tyr-1148, Tyr-1086, and Tyr-1068. EGFR Tyr-1086 and Tyr-1068 are also binding sites for the adaptor protein, Grb2, which is involved in recruitment of Ras-mitogen-activated protein kinase pathways (23, 24). In addition, we have previously shown that Grb2 associates with the EGFR during CCh-stimulated transactivation (12). Tyr-992 is a minor autophosphorylation site and also serves as a binding site for phospholipase-{gamma} (25, 26). Tyr-845 is directly phosphorylated by Src kinase and is located in the kinase domain of EGFR (27).

We characterized EGFR tyrosine phosphorylation patterns after stimulation with EGF, CCh, or TGF-{alpha} to determine whether differences in EGFR tyrosine phosphorylation could account for differential recruitment of ion transport regulatory pathways by EGF versus CCh. T84 monolayers were treated with CCh, EGF, or TGF-{alpha}, and lysate supernatants were run on gels and transferred to polyvinylidene difluoride membranes. Blots were probed with antibodies directed against the phosphorylated forms of EGFR tyrosine residues. EGF, CCh, and TGF-{alpha} all increased phosphorylation of the major autophosphorylation sites, Tyr-1173, Tyr-1086, and Tyr-1148, although EGF was a more potent stimulus than TGF-{alpha} (Fig. 4, A, B, C, E, and F). However, although both EGF and TGF-{alpha} increased phosphorylation of the other major autophosphorylation residue, Tyr-1068, CCh failed to significantly increase phosphorylation of this residue (Fig. 4D). Similarly, treatment of cells with EGF and TGF-{alpha}, but not CCh, increased phosphorylation of Tyr-992 (Fig. 4G). On the other hand, all three stimuli increased phosphorylation of Tyr-845, indicating Src recruitment (Fig. 4H). Table 1 summarizes these findings. They indicate that both EGF and TGF-{alpha}, albeit with different potencies, are capable of phosphorylating all six of the EGFR tyrosines probed, whereas CCh increases phosphorylation of only four of these residues with no significant increase in the phosphorylation of EGFR residues Tyr-1068 or Tyr-992. We, therefore, investigated whether CCh activates some other regulatory component that modifies EGFR tyrosine phosphorylation produced by autocrine TGF-{alpha}. This could account for differential activation of PI3K, since both Tyr-992 and Tyr-1068 of EGFR have been implicated in activation of this enzyme (28).


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  		TABLE 1
Summary of EGFR tyrosine phosphorylation data

 
Tyrosine Phosphatase Activity Accounts for Divergent Recruitment of PI3K by Direct Versus Indirect Activation of the EGFR—Regulation of EGFR activation involves not only increasing EGFR tyrosine phosphorylation but also dephosphorylation of tyrosine residues by protein-tyrosine phosphatases to switch off/redirect activation. Consequently, we investigated whether PTP activity could be involved in the differential phosphorylation of EGFR tyrosines and subsequent activation of PI3K by direct (EGF) or indirect (CCh) activation of EGFR. In the presence of the PTP inhibitor, Na3VO4 (500 µM), phosphorylation of Tyr-992 and Tyr-1068 in response to CCh was uncovered compared with CCh or Na3VO4 alone (Fig. 5, A–C). Interestingly, Na3VO4 pretreatment did not augment CCh-induced phosphorylation of Tyr-1148 above the significant level of phosphorylation induced by CCh alone (p < 0.05, n = 4, Fig. 5, A and D). However, Na3VO4 pretreatment did significantly increase total EGFR tyrosine phosphorylation induced by CCh (p < 0.05, n = 8, Fig. 5E). Our findings with Na3VO4 were confirmed with a structurally distinct PTP inhibitor, phenylarsine oxide (PAO). PAO (100 µM) also uncovered the ability of CCh to induce phosphorylation of EGFR Tyr-992 (Fig. 6, A and B) and EGFR Tyr-1068 (Fig. 6, A and C) compared with CCh alone, whereas PAO pretreatment had no effect on CCh-stimulated phosphorylation of Tyr-1148 (p < 0.05 versus control, n = 4, Fig. 6, A and D). Cells were also treated with EGF as a positive control, and interestingly, PAO pretreatment caused a significant reduction in EGF-stimulated phosphorylation of Tyr-1068 (Fig. 6C) and Tyr-1148 (Fig. 6D) compared with EGF alone. PAO pretreatment also uncovered a significant level of Akt1 phosphorylation in response to CCh (Fig. 6, E and F), indicating that CCh is capable of recruiting PI3K signaling if protein-tyrosine phosphatase activity is blocked. Moreover, because CCh transactivates EGFR by the release of the EGFR ligand, TGF-{alpha}, but does not phosphorylate all of the same EGFR tyrosines or recruit the same signaling molecules that respond to exogenous TGF-{alpha}, these data suggest that direct versus indirect activation of the EGFR may be associated with altered activity of phosphatases that dephosphorylate certain key EGFR residues.

Inhibition of PTP1B Expression Permits CCh-induced EGFR Tyrosine 992 and 1068 Phosphorylation and PI3K Signaling— The experiments discussed above implicated tyrosine phosphatase activity in differential EGFR signaling but did not identify a specific molecular target. PTP1B is a well recognized modulator of EGFR tyrosine phosphorylation and EGFR activity (29, 30). In addition, PTP1B activity can be inhibited by Na3VO4 and PAO (31, 32). Therefore, we next investigated whether inhibiting PTP1B expression could alter the EGFR phosphorylation profile and downstream signal recruitment in response to CCh treatment. Small interfering RNA technology was used to knock down PTP1B expression in T84 cells. A PTP1B siRNA SMARTpool was transfected into T84 cells, whereas a control pool of scrambled RNA sequences was used to control for nonspecific effects of transfection. PTP1B expression was dramatically reduced in PTP1B siRNA-transfected T84 monolayers with a maximal reduction of 86 ± 6% (Fig. 7A; n = 4). PTP1B siRNA had no nonspecific effect on cellular protein expression as demonstrated by equivalent levels of the nuclear envelope protein, lamin A/C, in both control and PTP1B siRNA-transfected cells as well as total EGFR protein levels, as shown in Fig. 7A. T84 knockdown of PTP1B facilitated CCh-induced phosphorylation of Tyr-1068 on the EGFR (p < 0.01; n = 4), whereas T84 cells transfected with control siRNA sequences showed no change in Tyr-1068 phosphorylation after CCh treatment (Fig. 7B). Furthermore, PTP1B knockdown significantly enhanced the ability of CCh to induce phosphorylation of Tyr-992 on the EGFR (Fig. 8A; p < 0.05; n = 4).


Figure 5
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  		FIGURE 5.
Inhibition of protein-tyrosine phosphatase activity facilitates phosphorylation of a full complement of EGFR tyrosine residues by carbachol. T84 cells were preincubated with Na3VO4 (Van; 500 µM) for 30 min before treatment with CCh (100 µM; 2 min) or EGF (16.7 nM; 5 min). A, Western blots prepared from cell lysate supernatants were probed with phospho (p-)-specific antibodies against EGFR tyrosines Tyr-992, Tyr-1068, and Tyr-1148, whereas EGFR immunoprecipitates (IP) from the same supernatants were probed for total EGFR phosphorylation. Blots were then stripped and probed for total EGFR. Con, control. B–D, densitometric analysis of Western blots detecting phosphorylation levels of EGFR tyrosines (B) Tyr-992 (n = 3), (C) Tyr-1068 (n = 5), and (D) Tyr-1148 (n = 4). E, densitometric analysis of total EGFR phosphorylation (n = 8). All data are expressed as means ± S.E. for a series of n experiments. Asterisks represent significant differences from control (*, p < 0.05), whereas # represents differences from treatment with CCh alone (#, p < 0.05).

 
Lysate supernatants from the same samples were also analyzed for PI3K signaling, as measured by activation of the downstream target, Akt1. CCh treatment caused an increase in Akt1 phosphorylation in cells subjected to PTP1B siRNA transfection, whereas cells transfected with control siRNA showed no response to CCh (Fig. 8B; p < 0.05; n = 3). These data, combined with those in Figs. 5 and 6, indicate that protein-tyrosine phosphatase activity, accounted for wholly or partially by PTP1B, prevents CCh-stimulated EGFR transactivation from phosphorylating the full complement of EGFR tyrosine residues that are phosphorylated by exogenous EGFR ligand.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
GPCR-induced transactivation of the EGFR and direct activation of EGFR with its natural ligand, EGF, both result in inhibition of calcium-dependent chloride secretion across intestinal epithelial cells. However, different signaling pathways downstream of the EGFR are recruited by these two routes of EGFR activation. The present study expands upon these previous findings to demonstrate that the signaling differences between direct and indirect activation of EGFR are likely accounted for by differential effects on protein-tyrosine phosphatase activity.


Figure 6
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  		FIGURE 6.
Protein-tyrosine phosphatase inhibition by phenylarsine oxide uncovers carbachol-stimulated phosphorylation of EGFR tyrosine residues 992 and 1068 and activation of PI3K signaling. T84 cells were preincubated with either Ringer's physiological solution or PAO (100 µM) for 30 min before treatment with CCh (100 µM; 2 min) or EGF (16. 7 nM; 5 min). A, representative Western blots prepared from cell lysate supernatants were probed with phospho (p-)-specific antibodies against EGFR tyrosines Tyr-992, Tyr-1068, and Tyr-1148. Blots were then stripped and probed for total EGFR. Con, control. B–D, densitometric analysis of Western blots detecting phosphorylation levels of EGFR tyrosines Tyr-992 (n = 4) (B), Tyr-1068 (n = 9) (C), and Tyr-1148 (n = 4) (D). E, representative Western blots were probed for phosphorylation of the PI3K downstream molecule, Akt1 (Thr 308), and total Akt1. F, densitometric analysis of phosphorylated Akt1 blots (n = 3). All quantitative data are expressed as means ± S.E. for a series of n experiments. Asterisks represent significant differences from control (*, p < 0.05; **, p < 0.01; ***, p < 0.001), # represents differences from treatment with CCh alone (#, p < 0.05; ##, p < 0.01), and x signifies differences from EGF plus PAO treatment (xxx, p < 0.001).

 
Although EGF and TGF-{alpha} bind to the same receptor, there is some evidence to suggest that these ligands bind with different affinities depending upon the system studied. Chicken EGFR binds human TGF-{alpha} with 100-fold greater affinity than human EGF (33). Differences in EGF versus TGF-{alpha} potency have also been described in a number of other cellular systems (34, 35). Differences in affinity may partly be explained by evidence indicating that EGF and TGF-{alpha} bind to different epitopes on the extracellular ligand binding domain of the EGFR. Studies by Lenferink et al. (35) suggest that this is governed by the flexible C-terminal linear domains of EGF and TGF-{alpha}, which bind to separate amino acid sequences on EGFR. In addition, Winkler et al. (36) characterized an antibody against human EGFR, designated 13A9, that could inhibit TGF-{alpha} but not EGF binding, suggesting that binding of these related ligands was not identical. In our studies EGF and TGF-{alpha} had different potencies for inducing both EGFR and ErbB2 phosphorylation (Fig. 1L). In the present study, electrophysiological data suggest that ErbB2 activation is not required for TGF-{alpha} inhibition of Ca2+-dependent chloride secretion across colonic epithelial cells. These results are also in keeping with our previous findings showing that, (i) CCh-induced transactivation of the EGFR can inhibit subsequent Ca2+-dependent chloride secretion, (ii) CCh does not cause ErbB2 recruitment to the EGFR, and (iii) CCh stimulates the release of TGF-{alpha} at concentrations that are likely much lower than those required to cause ErbB2 phosphorylation (12, 13, 15).

The most striking area of signaling divergence between direct versus indirect EGFR activation and subsequent regulation of epithelial Cl– secretion, however, is the differential activation of PI3K. We initially thought that the levels of TGF-{alpha} released during CCh-stimulated transactivation of the EGFR might be insufficient to activate ErbB2 and thereby recruit PI3K. Our data showed, however, that exogenous TGF-{alpha} concentrations below those capable of activating ErbB2 were not only capable of inhibiting CCh-stimulated Cl– secretion but could also activate PI3K signaling as measured by activation of the PI3K downstream target, Akt1. In addition, the inhibitory effect of low doses of TGF-{alpha} on CCh-stimulated Cl– secretion was, at least in part, PI3K-dependent, implying again that low doses of exogenous TGF-{alpha} could activate PI3K signaling. Because CCh transactivation of the EGFR is TGF-{alpha}-dependent, this begged the question as to why direct application of exogenous EGF or TGF-{alpha} produced a different signaling phenotype than indirect activation of the EGFR mediated by autocrine TGF-{alpha}.


Figure 7
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  		FIGURE 7.
PTP1B siRNA increases CCh-stimulated EGFR Tyr-1068 phosphorylation. Transfection of T84 cells with PTP1B siRNA was used to reduce expression of PTP1B in cultured T84 monolayers. A, Western blot of whole cell lysate supernatants from T84 monolayers transfected with either control or PTP1B siRNA. The transfected monolayers were treated with Ringer's, CCh, or EGF, and then supernatants were probed for PTP1B (A and B) or phosphorylation of EGFR Tyr-1068 (C and D). The nuclear envelope protein, lamin A/C, was probed as a loading control. B, densitometric analysis of Western blots detecting PTP1B levels (n = 4). C, representative Western blot prepared from cell lysate supernatants was probed with a phospho (P-)-specific antibody against EGFR tyrosine Tyr-1068. D, densitometric analysis of Western blots detecting phosphorylation levels of EGFR Tyr-1068 (n = 4). Data in B and D are expressed as the means ± S.E. for a series of n experiments. Asterisks represent significant differences from control siRNA (**, p < 0.01; ***, p < 0.001), and # represents differences from CCh treatment of control siRNA-transfected cells (#, p < 0.05; ##, p < 0.01; ###, p < 0.001).

 
We suspected that the answer lay in the pattern of phosphorylation of key tyrosine residues on the EGFR involved in receptor activation and signal recruitment. Indeed, we demonstrated that, whereas EGF and TGF-{alpha} phosphorylated all six of the tyrosine residues probed, CCh did not phosphorylate Tyr-992 or Tyr-1068, which are binding sites for phospholipase C{gamma} and Grb-2, respectively. Protein-tyrosine phosphorylation is regulated by the competing actions of protein-tyrosine kinases and phosphatases. Disturbances in the normal balance between the functions of tyrosine phosphatases and kinases alters the tyrosine phosphorylation pattern of proteins and have been implicated in the onset of cancer, inflammation, and diabetes (37). We, therefore, turned our attention to the possible involvement of protein-tyrosine phosphatases, which are capable of dephosphorylating tyrosines, in the regulation of EGFR phosphorylation status after direct versus indirect EGFR activation. Protein-tyrosine phosphatase inhibition uncovered the ability of CCh to phosphorylate Tyr-992 and Tyr-1068 and, importantly, to activate PI3K signaling. Intriguingly, protein-tyrosine phosphatase inhibition also reduced the ability of EGF to phosphorylate Tyr-1068 and Tyr-1148, indicating that EGF may require activation of protein-tyrosine phosphatases such as SHP-2 (38, 39) to fully phosphorylate certain EGFR tyrosine residues. These data suggested that CCh may either enhance or fail to inhibit the activity of phosphatases involved in dephosphorylating EGFR tyrosine residues. We focused on PTP1B (29) as a likely candidate phosphatase because it is the prototypic member of the PTP family and a ubiquitously expressed non-transmembrane tyrosine phosphatase that can regulate signaling induced by a number of growth factor receptors (40–42). Moreover, PTP1B has been shown to have a high specificity for Tyr-992 on the EGFR (43, 44).


Figure 8
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  		FIGURE 8.
Decreased PTP1B expression permits CCh stimulation of EGFR Tyr-992 phosphorylation (P-) and PI3K signaling. T84 cell monolayers transfected with PTP1B siRNA or control siRNA sequences were treated with Ringer's, CCh (100 µM), or EGF (16. 7 nM) for 5 min. Whole cell lysate supernatants were probed by Western blot for phosphorylation of EGFR Tyr-992 (representative blot, A) and phospho-Akt1 (representative blot, C). B, densitometric analysis of Western blots detecting phosphorylation levels of EGFR Tyr-992 (n = 4). D, densitometric analysis of Western blots detecting phospho-Akt1 levels (n = 3). Total Akt1 was probed as a loading control (C). Data in B and D are expressed as the means ± S.E. for a series of n experiments. Asterisks represent significant differences from control siRNA-transfected cells treated with Ringer's solution (*, p < 0.05; **, p < 0.01), and # represents differences from CCh treatment of control siRNA transfected cells (#, p < 0.05; ##, p < 0.01). a.u., arbitrary units.

 


Figure 9
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  		FIGURE 9.
Model of PTP1B-mediated effects on differential signal-dependent EGFR activation and recruitment of signaling pathways involved in regulation of epithelial chloride secretion. The muscarinic agonist, CCh, stimulates epithelial chloride secretion via a mechanism requiring elevations in intracellular calcium (1). However, the magnitude and duration of secretory responses to CCh are intrinsically limited by a signaling pathway incorporating CCh-stimulated transactivation of the EGFR and activation of the extracellular signal-regulated kinase mitogen-activated protein kinase isoforms. Preincubation of T84 cells with EGF or TGF-{alpha} can also inhibit CCh-stimulated Cl– secretion via extracellular signal-regulated kinase activation. CCh-stimulated transactivation of the EGFR requires the matrix metalloproteinase (MMP) dependent proteolytic release of membrane-bound TGF-{alpha} (2). The binding of TGF-{alpha} to the EGFR appears to be the major mechanism by which the EGFR becomes activated after CCh stimulation. Activation of the EGFR leads to downstream extracellular signal-regulated kinase (Erk) activation (3), which results in an overall decrease in epithelial chloride secretion by as yet undetermined mechanisms (4). Please note that the signaling pathway downstream of extracellular signal-regulated kinase leading to an apical chloride channel is not necessarily intended to imply a direct effect of these mechanisms on a chloride channel protein but, rather (for simplicity), an overall negative effect on chloride secretion. This could also involve effects on basolateral membrane transport pathways, such as potassium channels. CCh transactivation of the EGFR leads to a different pattern of EGFR tyrosine phosphorylation and fails to recruit PI3K signaling compared with exogenous TGF-{alpha} or EGF (5). Furthermore, EGF recruits activated ErbB2 whereas CCh does not (6). The differential effects of indirect EGFR activation by CCh and direct activation by exogenous EGF or TGF-{alpha} on EGFR tyrosine phosphorylation (P-) and PI3K activation appear to be mediated by protein-tyrosine phosphatases, including PTP1B, which dephosphorylates tyrosine residues on the EGFR linked to PI3K signaling (7). CCh-stimulated signals are represented by solid lines, whereas signaling pathways activated by exogenous EGF or TGF-{alpha} are represented by broken lines.

 
We demonstrated that when expression of PTP1B was decreased, CCh treatment significantly increased phosphorylation of Tyr-992 and Tyr-1068 on the EGFR. Moreover, CCh was also able to activate PI3K signaling in PTP1B-deficient cells. These data support our findings generated with pharmacological inhibitors of PTPs and indicate a key role for these enzymes, including PTP1B, in mediating differential outcomes of EGFR signaling in response to different modes of receptor activation. Future studies will attempt to elaborate on the mechanisms involved in PTP1B regulation and determine the effects of EGF, TGF-{alpha}, and CCh, possibly acting through muscarinic GPCR beta{gamma} subunits, on PTP1B activity. Studies in other systems have demonstrated that the binding of EGF to the EGFR can induce mechanisms to suppress phosphatase activity, possibly through generation of reactive oxygen species (42, 45). In turn, this would cause enhanced EGFR tyrosine phosphorylation, resulting in recruitment of signaling pathways that cannot be recruited in the absence of phosphatase inhibition. This likely has implications for inflammatory conditions where suppressors of phosphatase activity, such as reactive oxygen species, are generated in excess. Dysregulated ErbB signaling and expression have also been implicated in a number of epithelial cancers arising both from chronic inflammatory conditions such as ulcerative colitis (46, 47) and after activation by GPCRs such as proteinase-activated receptors and insulin receptors (48, 49). Of direct relevance to our studies, EGFR regulation of ion transport and recruitment of ion transport regulatory signaling pathways is altered compared with normal intestine in the setting of intestinal inflammation (50). The involvement of other protein-tyrosine phosphatases in our system cannot, however, be excluded as several protein-tyrosine phosphatases interact with the EGFR (51, 52). A schematic model of this differential signaling mechanism and its role in the negative regulation of epithelial chloride secretion is shown in Fig. 9.

In conclusion, the central role of EGFR in the regulation of numerous cell functions and its involvement in a number of pathologies such as epithelial cancers dictates that EGFR signaling must be tightly regulated in response to different stimuli. EGFR has been shown to be activated by GPCRs, UV light, stress, inflammatory mediators, and a variety of peptide ligands. Because EGFR acts as a central hub for diverse incoming and outgoing signals, the molecules involved in both appropriate propagation and termination of the flow of information from the extracellular environment are critical to efficient cell functioning. We have uncovered a heretofore unknown role for one such molecule, the key EGFR phosphatase, PTP1B, in the regulation of epithelial chloride secretion. Furthermore, we have demonstrated that phosphatase activity governs the differential recruitment of signaling pathways by direct versus indirect activation of the EGFR. Therefore, in addition to increasing our understanding of signal diversification via the EGFR, these findings may also suggest a therapeutic target in pathophysiological conditions characterized by dysregulated growth factor signaling.


   FOOTNOTES
 
* This research was funded by a Crohn and Colitis Research Fellowship Award (to D. F. M.) and National Institutes of Health Grant DK28305 (to K. E. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Division of Gastroenterology, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0063. Tel.: 858-534-2794; Fax: 858-534-3338; E-mail: dmccole{at}ucsd.edu.

2 The abbreviations used are: EGFR, epidermal growth factor (EGF) receptor; CCh, carbachol; GPCR, G protein-coupled receptor; PAO, phenylarsine oxide; PTP1B, protein-tyrosine phosphatase 1B; siRNA, small interfering RNA; TGF-{alpha}, transforming growth factor {alpha}; PI3K, phosphatidylinositol 3-kinase. Back



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 DISCUSSION
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