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Differential signaling and regulation of apical vs. basolateral EGFR in polarized epithelial cells

Scott K. Kuwada1,2,3, Kirk A. Lund4, Xiu Fen Li1, Peter Cliften4, Kurt Amsler5, Lee K. Opresko6, and H. Steven Wiley3,6

Divisions of 1 Gastroenterology and 4 Hematology/Oncology, Departments of Medicine and of 6 Pathology, University of Utah, 2 Department of Veterans Affairs Medical Center, and 3 Huntsman Cancer Institute, Salt Lake City, Utah 84132; and 5 Department of Physiology and Biophysics, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Overexpression of the epidermal growth factor receptors (EGFR) in polarized kidney epithelial cells caused them to appear in high numbers at both the basolateral and apical cell surfaces. We utilized these cells to look for differences in the regulation and signaling of apical vs. basolateral EGFR. Apical and basolateral EGFR were biologically active and mediated EGF-induced cell proliferation to similar degrees. Receptor downregulation and endocytosis were less efficient at the apical surface, resulting in prolonged EGF-induced tyrosine kinase activity at the apical cell membrane. Tyrosine phosphorylation of EGFR substrates known to mediate cell proliferation, Src-homologous and collagen protein (SHC), extracellularly regulated kinase 1 (ERK1), and ERK2 could be induced similarly by activation of apical or basolateral EGFR. Focal adhesion kinase was tyrosine phosphorylated more by basolateral than by apical EGFR; however, beta -catenin was tyrosine phosphorylated to a much greater degree following the activation of mislocalized apical EGFR. Thus EGFR regulation and EGFR-mediated phosphorylation of certain substrates differ at the apical and basolateral cell membrane domains. This suggests that EGFR mislocalization could result in abnormal signal transduction and aberrant cell behavior.

tyrosine phosphorylation; beta -catenin; proliferation; epidermal growth factor receptor downregulation and endocytosis

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

POLARIZED EPITHELIAL CELLS display distinct sets of membrane proteins on their apical vs. basolateral surfaces (reviewed in Ref. 29). This asymmetric distribution is due to differential sorting of newly synthesized as well as recycling proteins. Signals for apical or basolateral targeting reside within the proteins themselves and are recognized by as yet poorly defined cellular mechanisms that operate at several different steps in the membrane-trafficking process. The correct localization of a number of membrane transporters is essential for the function of most polarized epithelial cells (29).

Receptors for growth factors and cytokines, such as epidermal growth factor (EGF), display a polarized distribution in epithelial cells, as do the autocrine ligands that activate these receptors (9). The EGF receptor (EGFR) is predominantly localized to the basolateral cell surface in various human epithelial tissues (5, 12, 19, 22, 25, 28, 33). Mislocalization of ion transporters and EGFR is observed in diseases such as polycystic kidney disease (PKD), and the abberant distribution of EGFR may be an etiological factor in the progression of PKD (1, 11, 36). An assumption underlying models relating abnormal receptor distribution with diseases is that there is a functional consequence of receptor mislocalization. In Caenorhabditis elegans, mislocalization of the EGFR homologue Let 23 in polarized PnP cells resulted in the lack of vulval development, suggesting that Let 23 must be colocalized with certain substrates for proper signal transduction to occur (30).

There is evidence that receptors that are normally expressed on both the apical and basolateral surfaces of polarized epithelial cells differ in their signaling and regulation. In polarized airway epithelial cells, stimulation of apical or basal purinoreceptors caused a membrane-specific generation and catabolism of inositol phosphates, which restricted calcium influxes ipsilateral to the stimulated receptors (24). In colonic epithelial cells, apical or basolateral adenosine receptor activation stimulated different levels of 3',5'-cyclic monophosphate accumulation and receptor downregulation (2). In addition, ANG II receptors expressed at the apical and basolateral cell surfaces of polarized renal epithelial cells exhibited different rates of receptor internalization and recycling (3). In polarized cultures of canine tracheal cells, submucosal (basal) bradykinin stimulated arachidonic acid release, whereas mucosal (apical) bradykinin did not (10).

Because several polarized human epithelial tissues express predominantly basolateral EGFR (5, 12, 19, 22, 25, 28, 33), we asked whether EGFR regulation and specific EGFR-mediated responses were compartmentalized as well. To explore this, we caused the mislocalization of EGFR to the apical cell membrane by overexpressing them in polarizing kidney epithelial cells that normally express predominantly basolateral EGFR. The formation of highly electrically resistant monolayers on permeable filters allowed us to independently stimulate apical or basolateral EGFR with EGF. We found significant differences in EGFR downregulation, endocytosis, tyrosine kinase activity, and signaling between basolateral and apical EGFR. We conclude that certain substrates involved in EGFR regulation and signaling are compartmentalized in polarized epithelial cells.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Overexpression of EGFR in LLC-PK1 cells. The full-length human EGFR gene was cloned into the vector RC/CMV (13), which was then used to transfect a clone (Cl4) of the LLC-PK1 cell line using the calcium phosphate method and 1.8 mg/ml G418 for selection. The stably transfected Cl4 (LLC-PK1) cells were called K2 cells. Transepithelial resistance was measured, and only wells measuring 350 Omega  · cm2 or higher were used for experiments.

To measure receptor number, cells were plated at 50,000 cells/well onto polycarbonate filters (0.45-µm Transwell filters; Costar) in alpha -MEM (ICN) with 10% fetal calf serum (Hyclone), penicillin, streptomycin, and glutamine. At 14 days in culture, the medium was changed to cold alpha -MEM-HB (1 g/l BSA and 20 mM HEPES buffer) for 30 min. EGF (Preprotech) was labeled with 125I to ~150,000 counts · min-1(cpm) · ng-1 as described in Ref. 34. K2 cells (408,000/Transwell filter) were incubated to equilibrium at 0°C with serial half-dilutions of 125I-labeled EGF (12 nM) in DMEM-HB that were applied to either the apical or basolateral sides of the monolayers. Nonspecific binding was measured in the presence of a 100-fold excess of unlabeled EGF. The cells were incubated at 4°C for 24 h. The cells were washed five times with ice-cold WHIPS-saline [20 mM HEPES (pH 7.4), 130 mM NaCl, 5 mM KCl, 0.5 mM MgCl2, 1 mM CaCl2, and 1 mg/ml polyvinyl propylene (PVP)] and stripped in 1 ml of stripping buffer [50 mM glycine-HCl, 2 M urea (pH 3), 100 mM NaCl, and 2 mg/ml PVP] for 1 min. The stripping buffer was collected and counted in a gamma counter. EGFR affinity and number per cell were determined by Scatchard analysis. Parameters were estimated by nonlinear regression using the Levenberg-Marquardt algorithm and were fit using the Profit program (Quantum Soft).

Mitogenic response of polarized K2 cells. Polarized and confluent (14 days in culture) monolayers of K2 cells in culture on Transwell filters (24 mm) were treated with 20 ng/ml EGF in complete media (alpha -MEM + 2 mM L-glutamine, 10% fetal calf serum, and 1.8 mg/ml G418) that were added to the apical or basolateral compartments. Control cells received no EGF. After 18 h, the media in the apical and basolateral compartments were replaced with Hank's balanced salt solution containing 2 µCi/ml [3H]thymidine. After 2 h at 37°C, the solution was replaced with 10% TCA, and the acid-insoluble radioactivity was measured. In some cases, 10 µg/ml of anti-EGFR antagonistic monoclonal antibody (MAb 225) was added ipsilateral or contralateral to the compartment containing EGF to demonstrate the specificity of EGFR activation and the integrity of the cell monolayers. In addition, cell counts were used to corroborate the changes in thymidine incorporation: K2 cells were plated at 20,000 cells/24-mm Transwell filter and cultured for 14 days, at which time EGF (20 ng/ml) was added to either the apical or basolateral compartments. Control cells received no EGF. The cells were cultured for another 5 days and then dispersed with trypsin and counted using a hemocytometer.

Downregulation of EGFR from apical and basolateral surfaces. Polarized monolayers of K2 cells on 12-mm Transwell filters (~500,000 cells/Transwell filter) were incubated with 100 ng/ml unlabeled EGF added to either the apical or basolateral chamber at 37°C for the indicated periods of time. Cells were rinsed twice with PBS and then incubated for 2 h on ice with 100 ng/ml of 125I-EGF added to the same chamber as the unlabeled EGF. Membranes were then rinsed six times with ice-cold PBS, removed with a no. 4 cork borer, and solubilized in 2% SDS before counting. The results were calculated as the amount of radiolabeled EGF bound as a percentage of that bound to untreated cells.

Internalization of EGFR from apical and basolateral surfaces. Polarized monolayers of K2 cells on 12-mm Transwell inserts (~500,000 cells/Transwell filter) were incubated with 10 ng/ml of 125I-EGF (150,000 cpm/ng) added to either the apical or basolateral chamber for up to 5 min at 37°C. At 1-min intervals, the inserts were rinsed in ice-cold PBS six times and the membranes were removed using a no. 4 cork borer. Surface radioactivity was removed by placing the membranes in 1.5 ml of acid-strip solution [50 mM glycine-HCl (pH 2.5), 150 mM NaCl, and 2 M urea] for 8 min. Internalized radioactivity was solubilized using 2.5 ml of 1 N NaOH. The relative amount of internalized and surface-associated EGF was converted to internalization plots as previously described (35). Nonspecific EGF binding was determined in parallel using a 1,000-fold excess of unlabeled EGF. LLC-PK1 cells were transfected with the pRC-CMV vector containing the c'973 EGFR and a G418 resistance gene (7) by the calcium phosphate method. Polarized monolayers were evaluated for internalization from the apical vs. basolateral surfaces as described above.

Phosphotyrosine immunoblotting. K2 cells were seeded at 50,000 cells/ml onto 24-mm Transwell filters and cultured for 14 days, at which time they were serum starved for 18 h. EGF (50 µg/ml) in serum-free medium was added to either the apical or the basolateral chambers of three Transwell filters (24 mm) containing confluent K2 cell monolayers for 15 min. At various time points over a 20-h period, the cells were lysed in ice-cold lysis buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM Na2PO4, 1 mM Na3VO4, 10% glycerol, 1% Triton X-100, aprotinin, leupeptin, chymostatin, and pepstatin), sonicated for 10 s, then clarified at 10,000 rpm at 4°C for 15 min. Sixty micrograms (total protein) of each lysate were resolved by SDS-PAGE (7.5% polyacrylamide). After the blot was transferred to nitrocellulose, it was blocked in 1% TTBS (100 mM Tris · Cl, 0.9% NaCl, 0.1% Tween, and 1% BSA) overnight at 4°C. The blot was then incubated with a 1:2,500 dilution of MAb PY20 (Transduction Laboratories) in 1% TTBS for 2 h on a rocker and washed twice with 1% TTBS for 20 min. The blot was incubated with a 1:5,000 dilution of rabbit anti-mouse MAb (Zymed), washed twice for 20 min each wash, and incubated with 50 ng/ml of 125I-labeled protein A in 1% TTBS. The blot was washed twice and scanned with a Molecular Imager storage phosphor device (Bio-Rad Laboratories).

Differential phosphorylation of EGFR substrates. Polarized monolayers of K2 cells on Transwell filters (24 mm) were treated with or without 100 ng/ml of EGF added to either the apical or basolateral chambers for 15 min. Triplicate groups of cells were lysed in ice-cold lysis buffer, sonicated for 10 s, then clarified at 14,000 rpm at 4°C for 15 min. The pellets were stored at -70°C until further use. Src-homologous and collagen protein (SHC) MAb (2 µg; Transduction Laboratories) or 4 µg of focal adhesion kinase (FAK) MAb (Transduction Laboratories) were added to the supernatants and incubated on a rocker at 4°C overnight. Fifty microliters of a 50% slurry of protein A/G- Sepharose immunopure beads (Pierce) were then added to each lysate and incubated on a rocker at 4°C for 2 h. The beads were washed three times in 1 ml of lysis buffer then boiled in SDS-reducing sample buffer. The samples were divided in half, resolved on SDS-7% acrylamide gels, and transferred to nitrocellulose. The blots were blocked in 1% TTBS (100 mM Tris · Cl, 0.9% NaCl, 0.1% Tween, 1% BSA) overnight and then probed with either a 1:2,500 dilution of anti-phosphotyrosine MAb PY20 (Transduction Laboratories), a 1:5,000 dilution of MAbs to SHC (Transduction Laboratories), or a 1:2,000 dilution of FAK (Transduction Laboratories) in 1% TTBS for 2 h. The blots were washed twice for 20 min each wash and then incubated with a 1:5,000 dilution of rabbit anti-mouse MAb (Zymed); the blots were washed twice and then detected with 50 ng/ml 125I-protein A; and the blots were washed twice and then scanned and quantified using a Molecular Imager storage phosphor device (Bio-Rad Laboratories).

For beta -catenin, 2 µg of E-cadherin MAb (Transduction Laboratories) were used to coprecipitate beta -catenin from the Triton X-soluble fraction using the same immunoprecipitation method as that described above. beta -Catenin MAb (2 µg; Transduction Laboratories) was then used to immunoprecipitate beta -catenin from the Triton X-soluble fractions after immunodepletion of E-cadherin. beta -Catenin MAb (2 µg; Transduction Laboratories) was used to immunoprecipitate beta -catenin from the Triton X-insoluble pellet fractions after solubilization in 0.1% SDS in lysis buffer (pellets were boiled for 3 min and sonicated before immunoprecipitation). Phosphotyrosine and beta -catenin were detected by the Western blot protocol described in Phosphotyrosine immunoblotting.

Confocal immunofluorescence imaging. K2 cells were grown on 12-mm Transwell filters until a transepithelial resistance reading of >350 Omega  · cm2 (~8-9 days) was obtained. The media were aspirated, the monolayers were rinsed twice with PBS, and the cell monolayers were fixed in 4% paraformaldehyde-PBS for 20 min at room temperature. The monolayers were washed twice with PBS and then incubated in a blocking buffer [1% BSA (Bio-Rad) in PBS, pH 7.4] for 30 min. The monolayers were then incubated with a 1:500 dilution of beta -catenin MAb (Transduction) in blocking buffer for 30 min. The cells were washed twice with blocking buffer and then incubated with a 1:1,000 dilution of FITC-rabbit anti-mouse (Jackson ImmunoResearch) in blocking buffer for 30 min. The monolayers were washed twice with blocking buffer, and then the filters were cut out of their plastic inserts, mounted on glass slides in 4 µl of anti-fade solution (Vector Laboratories), and covered with a square coverslip that was sealed using clear nail polish. X-Y plane images were collected using a ×60 objective microscope (Zeiss) fitted with an MRC-1024ES confocal laser scanning system (Bio-Rad). Serial images were obtained at 0.5-µm spacings, which were then reconstructed with the LaserSharp program (Bio-Rad) into X-Z plane interpolated images.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Overexpression of EGFR causes missorting of EGFR to the apical cell surface. EGFR were overexpressed in a clone (Cl4) of the LLC-PK1 polarized porcine kidney epithelial cell line. The parental Cl4 cells expressed ~30,000 EGFR/cell (~22,000 basolateral and ~5,000 apical EGFR/cell) as determined by 125I-EGF binding studies at 0°C (Fig. 1). Transfection of Cl4 cells with a vector containing the full-length human EGFR cDNA resulted in stable transfectants (K2 cells) expressing ~1.4 × 106 EGFR per cell basolaterally and 5 × 105 apically, as determined by ligand-binding analysis at 0°C (Fig. 1). Thus K2 cells expressed many more EGFR apically than the total receptor complement of the parental cell line. EGFR overexpression did not diminish the formation of highly electrically resistant monolayers. The measured transepithelial resistance of confluent K2 monolayers was typically >600 Omega  · cm2. Because the ratio of apical-to-basolateral EGFR for K2 cells (1:3) was greater than that for the parental cell line (1:4.4), the overexpression of EGFR in K2 cells resulted in mislocalization of receptors to the apical cell surface. The apical and basolateral EGFR expressed by the parental Cl4 and transfected K2 cells comprised both low [dissociation constant (Kd) = 10-7 M] and high (Kd = 10-9 M) affinity classes of receptors.


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Fig. 1.   125I-labeled epidermal growth factor (EGF) Scatchard binding analysis of confluent 14-day-old parental wild-type LLC-PK1 clone Cl4 (A) and of Cl4 cells transfected with a full-length human EGF receptor (EGFR) construct (K2; B). Wild-type cells (160,000 cells/filter) expressed ~22,000 basolateral and 5,000 apical EGFR/cell. K2 cells (408,000 cells/filter) expressed ~1,444,000 basolateral and 474,000 apical EGFR/cell. The apical EGFR expressed by K2 cells were of high (Kd = 1.5 × 10-9 M) and low affinity (2.4 × 10-7 M). Basolateral EGFR of K2 cells were of high (Kd = 3.6 × 10-8 M) and low affinity (1.1 × 10-7 M) as well.

Activation of apical or basolateral EGFR causes cell proliferation. We wished to determine whether the EGFR expressed on the apical or basolateral cell surfaces possessed biological activity. To do this, we examined cell proliferation, perhaps the best-known response of EGFR activation, in confluent cell monolayers following the addition of EGF to the apical or basolateral compartment. Similar levels of [3H]thymidine incorporation were observed in response to either apical or basolateral EGFR activation over an 18-h period (Fig. 2A). The simultaneous addition of EGF and an antagonistic anti-EGFR antibody to the ipsilateral side of the monolayer resulted in a significantly diminished mitogenic response. Addition of anti-EGFR antibody contralaterally to EGF had no effect. This demonstrated the specificity of EGFR-mediated proliferation and that there was no significant leakage of EGF through the polarized epithelial monolayer.


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Fig. 2.   A: [3H]thymidine incorporation, expressed as a percentage over control (incorporation in absence of EGF) of K2 cell monolayers after addition of apical or basolateral EGF for 24 h. As a control, monoclonal antibody 225 (MAb 225), an EGFR-blocking antibody, was added to the ipsilateral side or contralateral to EGF to demonstrate the specificity of apical or basolateral EGFR stimulation. This also demonstrated the lack of significant leakage or translocation of EGF from one side of the monolayer to the other. Results are expressed as averages of triplicate experiments ± SE. B: cell densities of K2 cell monolayers 5 days after addition of apical or basolateral EGF. Cells were plated at equal cell density at time zero and cultured on permeable filters for 14 days, at which time EGF (20 ng/ml) was added to the monolayers. Cells were counted 5 days after addition of EGF. Control cells received no EGF. Results expressed are averages of triplicate experiments ± SE.

The addition of EGF to the apical or basolateral compartments of K2 cell monolayers increased cell numbers as well (Fig. 2B). There was a 1.6-fold increase in cell number above control following apical EGF and a 1.4-fold increase following basolateral EGF. This corroborated the increased thymidine incorporation by K2 cells following either apical or basolateral EGF addition, with an actual increase in cell numbers.

EGFR downregulation is more efficient from the basolateral cell surface. Previous studies have shown that a major mechanism for attenuating EGFR-mediated cell proliferative signaling is through internalization of EGFR (7). In fact, kinase-active EGFR mutants incapable of internalization caused unregulated growth, which ultimately resulted in cell transformation (32). Furthermore, ANG II receptors at the apical and basolateral cell surfaces of polarized renal epithelial cells demonstrated different rates of downregulation and endocytosis (3), suggesting that the machinery for receptor downregulation may differ between the apical and basolateral membrane surfaces. This led us to examine the efficiency of EGF-induced downregulation of apical and basolateral EGFR.

K2 cells were incubated with high concentrations of EGF, and the number of receptors remaining on the cell surface as a function of time was determined by 125I-EGF binding at 0°C. Basolateral EGFR showed a continuous decrease in numbers for the entire incubation period, reaching ~20% of initial numbers (Fig. 3). In contrast, the apical EGFR displayed an initial drop but then remained constant at 60% of the initial receptor levels. These data suggest that EGFR downregulation is less efficient from the apical than from the basolateral cell surface. This inefficiency of downregulation of apical EGFR might be due to a decreased rate of ligand-induced disappearance of EGFR from the apical cell surface. We tested this by examining the rates of ligand-induced endocytosis of apical and basolateral EGFR. Endocytosis of EGFR was significantly faster at the basolateral than at the apical cell surface (Fig. 4A). Previous studies have demonstrated that ligand-induced endocytosis of EGFR is dependent on a specific cytoplasmic domain of EGFR (7). To ensure that the faster rate at the basolateral surface did not simply reflect a different net rate of endocytosis from the two surfaces, a mutated EGFR, c'973, was expressed in Cl4 cells. This receptor mutant possesses intrinsic tyrosine kinase activity but lacks the domains necessary for ligand-induced endocytosis (24, 25). There were no differences in apical vs. basolateral endocytosis of c'973 receptors (Fig. 4B).


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Fig. 3.   EGFR downregulation shown as a percentage of initial 125I-EGF binding to K2 cells. open circle , Cells pretreated with apical EGF; bullet , cells pretreated with basolateral EGF. 125I-EGF binding was observed for 60 min.


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Fig. 4.   EGFR endocytosis plotted as internalized EGF vs. integral surface EGF. open circle , Cells pretreated with apical EGF; bullet , cells pretreated with basolateral EGF. Slopes of lines represent endocytic rates. A: typical plot showing endocytosis of full-length EGFR by K2 cells after apical or basolateral EGF addition. This experiment was performed 3 times, yielding an average endocytic rate constant of 0.15 ± 0.022 (SE) for cells pretreated with apical EGF and 0.03 ± 0.014 (SE) for cells pretreated with basolateral EGF. B: typical plot showing EGF-induced endocytosis of apical or basolateral c'973 EGFR mutant constructs transfected into parental cell line (Cl4). This experiment was performed 3 times and yielded an average endocytic rate constant (Ke) of 0.04 ± 0.007 (SE) for cells pretreated with apical EGF and a Ke of 0.04 ± 0.006 (SE) for cells pretreated with basolateral EGF.

EGFR tyrosine kinase activity differs between the apical and basolateral surfaces. Because there were spatial and temporal differences between apical and basolateral EGFR regulation, we wondered whether apical EGF would cause greater EGFR tyrosine kinase activity than basolateral EGF. Confluent monolayers of K2 cells grown on permeable filter inserts were serum starved for 18 h, after which EGF (50 ng/ml) was added at time zero to either the apical or basolateral compartment. At various time points during a 20-h duration following EGF addition, EGFR phosphotyrosine levels were determined by Western blot analysis (Fig. 5). EGFR tyrosine phosphorylation was greater in response to basolateral than to apical EGF through 60 min, which paralleled the greater numbers of basolateral than apical EGFR per cell. However, the attenuation of EGFR tyrosine kinase activity differed between the apical and basolateral cell membrane surfaces, starting at 4 h. There was a significant decline in detectable tyrosine-phosphorylated basolateral EGFR by 4 h and none were detected by 20 h, whereas tyrosine-phosphorylated apical EGFR were still present 20 h after EGF stimulation. The delayed attenuation of tyrosine-phosphorylated apical relative to basolateral EGFR could explain why EGFR-mediated proliferation was stimulated to similar levels 18 h following apical or basolateral EGF, even though there was a nearly 3:1 ratio of basolateral-to-apical EGFR.


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Fig. 5.   Anti-phosphotyrosine blot of EGFR at various time points following addition of apical (Ap) or basolateral (BL) EGF (50 ng/ml) to confluent K2 cell monolayers. Band representing EGFR was determined in a parallel Western blot (not shown) and is the most prominent tyrosine-phosphorylated band seen following EGF stimulation.

EGF-induced tyrosine phosphorylation of substrates mediating cell proliferation. Because cell proliferation was similarly induced by apical and basolateral EGF, we asked whether levels of activation of EGFR substrates involved in cell proliferation were also similar.

The signal transduction cascade mediating EGFR-induced mitogenesis has been characterized (6, 8, 17, 23). The protein SHC is a known substrate of EGFR and is important in EGFR-mediated mitogenesis via activation of the Ras-mitogen-activated protein (Ras-MAP) kinase pathway (16). The phosphotyrosine content of SHC was determined by Western blotting of SHC immunoprecipitates from K2 monolayers on filters, following the addition of EGF to the apical or basolateral compartment. SHC tyrosine phosphorylation increased following either apical or basolateral EGFR activation (Fig. 6). In addition, we examined the phosphotyrosine content of extracellularly regulated kinase 1 (ERK1) and ERK2, which are substrates at the downstream end of the EGFR-Ras-MAP kinase signal transduction pathway (6, 8, 17, 23). Both ERK1 and ERK2 immunoprecipitates were tyrosine phosphorylated to a similar extent in response to apical or basolateral EGFR activation by Western blot analysis (data not shown). These results were consistent with the similar levels of cell proliferation following either apical or basolateral EGFR stimulation and demonstrated a lack of compartmentation of EGFR substrates involved in EGFR-mediated cell proliferation.


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Fig. 6.   Anti-phosphotyrosine (PY) Western (W) blot of Src-homologous and collagen protein (SHC) immunoprecipitated following addition of apical or basolateral EGF to K2 cell monolayers. Each immunoprecipitation (IP) was performed on an equal number of K2 cells. Prominent tyrosine-phosphorylated band at top of the blot is EGFR, which coprecipitated with SHC protein. Numbers at bottom represent densitometry results, normalized to control (no EGF), of bands representing tyrosine-phosphorylated SHC.

EGF-induced tyrosine phosphorylation of substrates in cell adhesions. Because of previous studies demonstrating differences in apical and basolateral receptor signaling in polarized epithelial cells, we furthered our search for differences in apical and basolateral EGFR signaling. We chose to study EGF-induced tyrosine phosphorylation of substrates in cell adhesion structures, since their localization in polarized epithelial cells is well defined. Focal adhesions are localized to the basal cell surfaces of cells, where they provide connections between the extracellular matrix and cytoskeleton. Focal adhesions contain several cytoplasmic proteins, including FAK, which can be tyrosine phosphorylated in response to various growth factors (14, 18, 26). Adherens junctions are formed along the lateral cell surfaces of polarized epithelial cells at cell-cell junctions, where they mediate cell-cell adhesion. beta -Catenin, a known EGFR substrate (15), is an important component of adherens junctions. The highly localized distributions of FAK and beta -catenin in polarized epithelial cells made them likely candidates for differential phosphorylation by apical and basolateral EGFR. To test this prediction, we examined EGF-induced tyrosine phosphorylation of FAK and beta -catenin.

EGF was added for 15 min to either the apical or basolateral compartments of K2 monolayers, after which phosphotyrosine levels of immunoprecipitated FAK and beta -catenin were determined by Western blotting. An increase in FAK tyrosine phosphorylation above the control (no EGF) was stimulated by basolateral EGFR activation (Fig. 7). Constitutive FAK tyrosine phosphorylation was seen in the absence of EGF, probably reflecting autocrine growth factor production. There was no increase in FAK tyrosine phosphorylation above constitutive levels following apical EGF. Paxillin, another focal adhesion protein, showed only low constitutive levels of tyrosine phosphorylation, which failed to increase significantly following either apical or basolateral EGFR stimulation (data not shown).


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Fig. 7.   Anti-phosphotyrosine blot of focal adhesion kinase (FAK) immunoprecipitated from K2 cell monolayers treated with apical or basolateral EGF. Each immunoprecipitation was performed on an equal number of cells.

In striking contrast, beta -catenin immunoprecipitated from total cell lysates was tyrosine phosphorylated to a much greater degree in response to apical than to basolateral EGFR activation (Fig. 8A, left). The loading controls (Fig. 8A, right) demonstrated two bands immunodetected as beta -catenin. The lower band corresponded exactly to tyrosine-phosphorylated beta -catenin on the anti-phosphotyrosine blot and the upper band corresponded to non-tyrosine-phosphorylated beta -catenin. Only the lower (tyrosine-phosphorylated) band was seen in any of the lanes on the anti-phosphotyrosine Western blot (Fig. 8A, left). What was apparent from the two Western blots was that much more tyrosine-phosphorylated beta -catenin was immunoprecipitated after apical than after basolateral EGFR stimulation from whole cell lysates. This led us to examine the various cell fractions for the presence of tyrosine-phosphorylated beta -catenin.


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Fig. 8.   Anti-phosphotyrosine (left) and beta -catenin immunoblots (right) of beta -catenin immunoprecipitated after addition of apical or basolateral EGF to K2 cell monolayers. All immunoprecipitations were performed on an equal number of cells. A: beta -catenin was immunoprecipitated from whole cell lysates and detected with either anti-phosphotyrosine (left) or beta -catenin antibodies (right). Two protein bands were detected on the beta -catenin immunoblots. Top band, unphosphorylated form; bottom band, tyrosine-phosphorylated beta -catenin. Numbers appearing under tyrosine-phosphorylated bands are densitometry results normalized to control (no EGF). B: beta -catenin was coimmunoprecipitated with an E-cadherin-specific monoclonal antibody from the Triton-soluble cell fraction and probed with anti-phosphotyrosine (left) or beta -catenin (right) monoclonal antibodies. C: beta -catenin was immunoprecipitated from Triton-soluble cell fractions that were immunodepleted of E-cadherin and probed with antiphosphotyrosine (left) or beta -catenin (right) monoclonal antibodies. D: beta -catenin was immunoprecipitated from Triton-insoluble cell fraction and probed with anti-phosphotyrosine (left) or beta -catenin (right) monoclonal antibodies.

In a separate experiment, EGF was added apically or basolaterally to K2 monolayers for 15 min, after which they were lysed in a 1% Triton X-100-containing buffer. The pellet fraction was isolated from the supernatant (Triton-soluble) fraction by centrifugation and resolubilized in an SDS-based buffer. beta -Catenin forms complexes with E-cadherin at the adherens junction and in the cytoplasm (21). Therefore, beta -catenin was coprecipitated with E-cadherin from the Triton X-soluble fraction (Fig. 8B). Equal amounts of E-cadherin were immunoprecipitated from the Triton-soluble fraction (data not shown). Similar levels of beta -catenin coprecipitated with E-cadherin, regardless of the degree of beta -catenin tyrosine phosphorylation (compare Fig. 8B, left and right). However, tyrosine phosphorylation of the coprecipitated beta -catenin was much greater after apical than after basolateral EGF (Fig. 8B, left). No tyrosine phosphorylation of E-cadherin was seen in response to EGF stimulation (data not shown). The same Triton X-soluble fractions that had been immunodepleted of E-cadherin (data not shown) were then used to immunoprecipitate beta -catenin. Again, only apical EGFR stimulation led to tyrosine phosphorylation of beta -catenin (Fig. 8C). This further suggests that not all beta -catenin was complexed with E-cadherin in the Triton-soluble fraction.

beta -Catenin was immunoprecipitated from the Triton-insoluble fraction under denaturing conditions. Again, tyrosine phosphorylation of beta -catenin was greater after apical than after basolateral EGFR stimulation (Fig. 8D, left). Furthermore, more beta -catenin protein was found in the Triton-insoluble fraction after stimulation of apical than after basolateral EGFR (Fig. 8D, right).

In K2 cells, beta -catenin was found to be localized to the lateral cell membrane by X-Z-plane confocal microscopy (Fig. 9A), but specific cytoplasmic staining was seen as well on X-Y plane scans (Fig. 9B). beta -Catenin tyrosine phosphorylation did not change its lateral membrane localization, as determined by confocal microscopy, in response to apical or basolateral EGFR activation compared with control cells (no EGF; data not shown).


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Fig. 9.   Confocal laser microscopy of beta -catenin in confluent K2 monolayers in the absence of exogenous EGF. A: X-Z plane reconstructions of scans of K2 monolayers stained for beta -catenin and detected with an FITC-labeled secondary antibody. beta -Catenin localized to the lateral cell membranes, and no apical or basal staining were seen. B: X-Y plane scans of same cells showing phosphotyrosine staining (left) as a reference and beta -catenin staining (right). Note the presence of specific cytoplasmic staining for beta -catenin.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

EGFR is predominantly localized to the basolateral cell surface in various human epithelial tissues (5, 12, 19, 22, 25, 28, 33), and it is the basolateral rather than apical EGFR that mediate growth factor-induced cell proliferation in polarized epithelial cells (5, 33). It is generally felt that growth factors found in luminal fluids bathing the apical surfaces of polarized epithelium exert their effects following translocation to the basolateral cell surface (28). In certain disease states, such as PKD and cancer, the basolateral localization of EGFR is lost (1, 11, 31, 36). Mislocalized apical EGFR in the cysts of polycystic kidneys are biologically active and mediate cell proliferation (11). This implies that the substrates involved in EGFR-mediated cell proliferation are not compartmentalized with respect to apical and basolateral EGFR. However, there is a generalized disruption of cell polarity in the abnormal epithelial cells lining the cysts in PKD. Thus we wondered whether EGFR substrates are spatially organized in normal polarized epithelial cells. The mislocalization of apical EGFR in the polarizing kidney epithelial cell line LLC-PK1, which normally expresses predominantly basolateral EGFR, allowed us to study the spatial organization of EGFR substrates. Our approach was to study EGF-induced regulation and signaling by independently stimulating apical or basolateral EGFR.

Both the apical and basolateral EGFR were biologically active in the K2 cells, and they both stimulated cell proliferation to a similar degree. We were interested in EGFR regulation, since it is a principal mechanism of attenuating activated EGFR (7). EGF-induced EGFR downregulation was very inefficient at the apical-vs.-basolateral cell membrane. We then looked specifically at the process of endocytosis, since it has been previously demonstrated that inhibition of this process in the presence of EGF caused constitutive tyrosine kinase activity, leading to unregulated cell proliferation and, ultimately, cell transformation (32). The c'973 EGFR mutant, which lacks the domain necessary for internalization, failed to demonstrate ligand-induced endocytosis at either the apical or basolateral cell surface, demonstrating that there was no difference in net endocytosis at the apical and basolateral cell membranes. However, the full-length EGFR construct underwent EGF-induced endocytosis to a much greater extent from the basolateral cell membrane. These results suggest that the differences in the endocytosis rates, and hence downregulation, of EGFR at the apical and basolateral membranes might be due to a differential distribution of the substrates(s) necessary for EGFR internalization. The inefficient downregulation and endocytosis of apical EGFR resulted in a more prolonged level of EGF-induced apical than basolateral receptor tyrosine kinase activity at steady state. This probably accounted for the similar degrees of EGFR-mediated mitogenesis 18 h after apical or basolateral EGF addition, despite a 3:1 ratio of basolateral-to-apical EGFR per cell. Therefore, overexpression or mislocalization of EGFR in polarized epithelium leads to inefficient and delayed attenuation of EGFR signaling.

We then looked for differences in apical vs. basolateral EGFR-mediated tyrosine phophorylation of known EGFR signaling substrates. The tyrosine phosphorylation of EGFR substrates known to mediate EGF-induced proliferation (SHC, ERK1, and ERK2) (6, 8, 17, 23) occurred in response to both apical and basolateral EGFR activation. This was expected, since apical or basolateral EGFR stimulation caused cell proliferation. These results demonstrate that EGFR substrates known to mediate EGF-induced cell proliferation are not compartmentalized in polarized epithelial cells.

EGFR substrates in cell adhesion complexes seemed to be likely targets for differential tyrosine phosphorylation by apical and basolateral EGFR, since they are well localized in polarized epithelial cells with respect to the apical and basolateral cell membrane. The focal adhesion protein FAK demonstrated increased tyrosine phosphorylation in response to basolateral EGF but not following apical EGF. These results were expected, since FAK is localized to focal adhesion plaques in the basal cell membrane in polarized epithelial cells. We did not find differences in EGF-induced tyrosine phosphorylation of the focal adhesion protein paxillin, but this may have been due to the high levels of constitutive tyrosine phosphorylation of these proteins in K2 cells. Tyrosine phosphorylation of beta -catenin, which was predominantly localized to the lateral cell membrane in K2 cells, was much greater after apical than after basolateral EGFR stimulation in whole cell lysates. Because EGFR are normally basolaterally localized in polarized epithelial cells, the greater level of tyrosine phosphorylation of beta -catenin after apical than after basolateral EGF stimulation in K2 cells demonstrated compartmentalization of beta -catenin.

Analysis of EGF-induced tyrosine phosphorylation of beta -catenin in various cell fractions revealed that the missorted apical EGFR had much greater access to a Triton-soluble pool of beta -catenin than basolateral EGFR. Specifically, apical EGFR had access to beta -catenin either associated or not associated with E-cadherin in the Triton-soluble fraction. Our confocal microscopy results demonstrated the presence of specific but relatively lower levels of cytoplasmic than lateral cell membrane beta -catenin staining. It has been previously reported that increased tyrosine phosphorylation of beta -catenin led to dissociation with E-cadherin at adherens junctions (4). However, we did not see any decrease in beta -catenin at the lateral cell membrane in response to either apical or basolateral EGFR activation by X-Z plane confocal microscopy, and coprecipitation of beta -catenin with E-cadherin occurred independently of the level of tyrosine phosphorylation of beta -catenin. The higher degree of tyrosine phosphorylation of beta -catenin by apical EGFR did produce a greater appearance of beta -catenin protein in the Triton-insoluble cell fraction than that produced by basolateral EGFR stimulation. beta -Catenin translocates to the nucleus where it can form a transcription complex (16, 20, 27). Although it is tempting to say that the increased appearance of tyrosine-phosphorylated beta -catenin in the Triton-insoluble pellet may represent nuclear translocation, we do not have any evidence to support this.

The differential tyrosine phosphorylation of beta -catenin in K2 cells is likely due to the overexpression of EGFR per se rather than to perturbation of beta -catenin localization as a result of EGFR overexpression. This was evident by the fact that beta -catenin was well localized to the lateral cell membrane in K2 cells (see Fig. 9), where it is also localized in the parental cell line (data not shown). Furthermore, overexpression of EGFR did not disrupt the formation of highly electrically resistant monolayers, implying the presence of well-formed intercellular junctions.

Our studies revealed that EGFR regulation and EGFR-mediated tyrosine phosphorylation of beta -catenin are compartmentalized in polarized epithelial cells. This implies a higher-order level of regulation of EGFR signaling in polarized epithelial cells based on the spatial compartmentalization of specific substrates. Thus mislocalization of EGFR in polarized epithelium, through EGFR overexpression or the loss of cell polarity, as occurs in certain diseases, may contribute to the pathogenesis of those disorders by resulting in aberrant EGFR signaling.

    ACKNOWLEDGEMENTS

We thank Gordon Gill for the EGFR constructs and Virginia Hill for her technical assistance.

    FOOTNOTES

This work was supported in part by grants from the Glaxo Insitute of Digestive Health, the American Cancer Society, the Huntsman Cancer Institute, and National Institute of Diabetes and Digestive and Kidney Diseases Grant KO8-DK-02531 (to S. K. Kuwada); by grants from the American Heart Association, National Affiliate, and the American Heart Association, New Jersey Affiliate (to K. Amsler); and by Grant DAMD17-94-J-444 from the Department of Army Medical Research Acquisition Activity (to H. S. Wiley).

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: S. K. Kuwada, Univ. of Utah, Division of Gastroenterology, 50 N. Medical Dr., Salt Lake City, Utah 84132.

Received 5 May 1998; accepted in final form 17 September 1998.

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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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