Membrane receptor location defines receptor interaction with signaling proteins in a polarized epithelium

Kurt Amsler1 and Scott K. Kuwada2

1 Department of Physiology and Biophysics, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5635; 2 Division of Gastroenterology, Department of Medicine, University of Utah, Department of Veterans Affairs Medical Center, and Huntsman Cancer Institute, Salt Lake City, Utah 84132

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Signal transduction from receptors is mediated by the interaction of activated receptors with proximate downstream signaling proteins. In polarized epithelial cells, the membrane is divided into subdomains: the apical and basolateral membranes. Membrane receptors may be present in one or both subdomains. Using a combination of immunoprecipitation and Western blot analyses, we tested the hypothesis that a tyrosine kinase growth factor receptor, epidermal growth factor receptor (EGFR), interacts with distinct signaling proteins when present at the apical vs. basolateral membrane of a polarized renal epithelial cell. We report here that tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma ) was induced only when basolateral EGFR was activated. In contrast, tyrosine phosphorylation of several other signaling proteins was increased by activation of receptor at either surface. All signaling proteins were distributed diffusely throughout the cytoplasm; however, PLC-gamma protein also displayed a concentration at lateral cell borders. These results demonstrate that in polarized epithelial cells the array of signaling pathways initiated by activation of a membrane receptor is defined, at least in part, by the membrane location of the receptor.

epidermal growth factor receptor; src homology 2 domain-containing proteins; polycystic kidney disease; carcinoma

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

A DEFINING CHARACTERISTIC of polarized epithelial cells is the separation of the plasma membrane into subdomains containing different complements of proteins and lipids (27). For example, most membrane transporters, such as Na-K-ATPase, are expressed in a single membrane domain. The asymmetrical distribution of membrane transporters is essential for carrying out net transepithelial solute and water transport.

Many membrane receptors for growth factors and hormones also exhibit a polarized distribution in epithelial cells, being expressed at the basolateral membrane, where they are in contact with the serosal fluid compartment (27). However, several receptors have been detected at both the apical and basolateral membranes of epithelia. Renal inner medullary collecting duct cells express natriuretic peptide receptors at both the apical and basolateral membranes (26). Type 1 ANG II receptors are detected at both the apical and basolateral membranes of renal proximal tubule cells (30). Receptors for both insulin-like growth factor I and insulin-like growth factor II are also detected at both the apical and basolateral surfaces of proximal tubule cells (16). alpha 2A-Adrenergic receptors are detected only at the apical membrane of a renal epithelial cell line, Madin-Darby canine kidney cells (22). Nasal airway epithelia express purinoceptors at both cell surfaces (24). An intestinal epithelial cell line, T84, expresses adenosine receptors at both the apical and basolateral surfaces (3).

The location of some membrane receptors is altered during tissue development and in disease. The Notch membrane receptor shifts location during early sea urchin development (31). The epidermal growth factor (EGF) receptor (EGFR) was detected at the apical membrane of some renal epithelial cells during kidney development before assuming its typical basolateral location in the mature cells (35). In several forms of polycystic kidney disease, EGFRs are expressed at both the basolateral and apical membranes of epithelial cells lining the renal cysts (13, 23, 35, 36). Normal colonic epithelial cells express EGFR exclusively at the basolateral membrane. In contrast, the colon carcinoma cell line HT29-D4 expressed EGFR at both the apical and basolateral membranes, whereas insulin-like growth factor I receptor was expressed almost exclusively at the basolateral membrane (19). Apical EGFR was also detected in primary cultures of human colorectal carcinoma cells (33).

The implications of membrane location for receptor function in the polarized epithelial cell can be divided into two alternatives. In the first scenario, an asymmetrical receptor distribution would simply restrict access of receptor to its activating ligand. Colocalization of receptor and ligand to the same compartment would result in receptor activation, whereas separation of receptor and ligand would prevent receptor activation even though both components were expressed in the same tissue. The receptors at both surfaces would behave identically with respect to activation of signaling pathways and receptor regulation.

In the second scenario, the same receptor at the two membrane surfaces would behave differently. Apical and basolateral receptors may be coupled to distinct signaling pathways and/or may be differently regulated by the cell. In support of this latter alternative, ANG II receptors expressed at the basolateral and apical membranes of proximal tubule cells inhibited adenylate cyclase and activated phospholipase A2, respectively (28). Activation of apical and basolateral ANG II receptors in proximal tubule cells induced opposite effects on transepithelial sodium transport (17, 28). Also, transforming growth factor-alpha , a ligand for EGFR, stimulated mitogenesis when added to either surface of polarized populations of a rat mammary epithelial tumor cell line, Con8, but disrupted monolayer integrity and localization of the tight junction-associated protein ZO-1 only when added to the basolateral compartment (8). Apical and basolateral ANG II receptors also displayed different rates of ligand-induced endocytosis (4).

The EGFR is postulated to play an important role in regulating the growth and differentiation of a variety of epithelial cell types (5, 11, 12). EGFR is located at the basolateral membrane of most mature, polarized epithelial cells (5, 13, 18, 21), but this localization is altered during renal development (35), in human and mouse forms of polycystic kidney disease (13, 23, 35, 36), in colorectal carcinoma cells (33), and in a mammary tumor epithelial cell line (8). This raises the question of whether EGFR function is different when the receptor is expressed at the apical and basolateral surfaces of a polarized epithelial cell.

We have recently described a polarized renal epithelial cell model system in which substantial EGFR is expressed at both the apical and basolateral membranes (Ref. 18a and see Table 2 and Fig. 1). We found that specific aspects of receptor behavior, e.g., receptor internalization and downregulation, were different for EGFR expressed at the apical vs. basolateral membrane. Activation of EGFR at either surface, however, induced cell proliferation and tyrosine phosphorylation of the src homology 2 (SH2) domain-containing signaling protein SHC. In this article, we pursue the question of whether equivalent access to apical and basolateral EGFR is a general feature for SH2 domain-containing signaling proteins or whether one or more signaling proteins exhibit differential access to EGFR expressed at the two surfaces of the polarized epithelial cells. Our results indicate that a limited subset of SH2 domain-containing signaling proteins exhibit differential access to apical vs. basolateral EGFR.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cells, constructs, and materials. The Clone 4 (Cl4) cell line is a random clonal line derived from the wild-type LLC-PK1 cell line (1). The wild-type human EGFR cDNA construct was a gift from Dr. G. Gill (University of California, San Diego, CA). A monoclonal anti-phospholipase C-gamma (PLC-gamma ) antibody, both monoclonal and polyclonal anti-SHC antibodies, a monoclonal anti-phosphatidylinositol 3-kinase (PI3K) regulatory subunit antibody, a monoclonal anti-EGFR antibody, and a peroxidase-conjugated recombinant anti-phosphotyrosine (RC20H) antibody were obtained from Transduction Laboratories (Lexington, KY). A polyclonal anti-PI3K regulatory subunit antibody was obtained from Upstate Biotechnology (Lake Placid, NY). A monoclonal anti-pp60src antibody was obtained from Calbiochem (Cambridge, MA). A function-blocking, human-specific, monoclonal anti-EGFR antibody (no. 225) was a gift from Dr. S. Wiley (University of Utah Medical School, Salt Lake City, UT). Peroxidase-conjugated anti-mouse or anti-rabbit IgG was obtained from Sigma (St. Louis, MO). EGF was obtained from Becton Dickinson (Franklin Lakes, NJ). Fetal bovine serum was obtained from HyClone Laboratories (Ogden, UT) and Gemini Bio-Products (Calabasas, CA). G418 was obtained from Life Technologies (Gaithersburg, MD).

The monoclonal anti-EGFR antibody from Transduction Laboratories recognized a protein of ~180 kDa, the expected molecular mass of EGFR, in Western blots of lysates of transfected cells expressing wild-type human EGFR but not in lysates of untransfected cells. This antibody did not, however, efficiently immunoprecipitate a protein of similar size from cell lysates. The human-specific, monoclonal anti-EGFR (no. 225) efficiently immunoprecipitated a protein of similar molecular mass from whole cell lysates of cells transfected with wild-type human EGFR but not from untransfected cells. This antibody was relatively insensitive for detection of the protein by Western blotting.

Cell culture and isolation of transfected cells. The original renal epithelial cell line, LLC-PK1, was obtained from Dr. R. N. Hull (Eli Lilly Laboratories, Indianapolis, IN). Stock cultures of a random clone of the LLC-PK1 cells, Clone 4 (Cl4) (1), transfected with pRC vector, which contains the neomycin resistance gene, with or without cDNA inserts were maintained at a subconfluent density in complete medium (alpha -modification of MEM supplemented with 2 mM L-glutamine and 10% fetal bovine serum) containing 1.5 mg/ml G418 (selection medium). Cells were passaged by detachment with trypsin-EDTA solution and reseeding in the same medium. For experiments, cells were seeded at a density of ~3 × 104 cells/cm2 onto Falcon cell culture inserts (1-µm pore diameter; Becton Dickinson) in complete medium. Populations were refed every 2-3 days for 14 days, at which time they were ~10 days postconfluence.

The wild-type human EGFR cDNA was incorporated into the multiple cloning site of the pRC mammalian expression vector, and proper orientation was confirmed by sequencing (15). Transcription of the cDNA construct was driven by the cytomegalovirus promoter, which is highly active in these renal epithelial cells. Subconfluent Cl4 cell populations were transfected with ~10 µg of vector containing the wild-type human EGFR cDNA using the modified calcium phosphate precipitation procedure (9). Transfected cells were selected by incubation in selection medium for at least 2 wk. Cells surviving selection were pooled and grown for a further 7 days in selection medium. Surviving cells were detached, labeled with rhodamine-conjugated human-specific anti-EGFR antibody (no. 225), which recognizes an extracellular epitope of the receptor, sorted by fluorescence-activated cell sorting, and cloned.

Western blot analysis. Cell populations, grown as described above, were refed with HEPES-buffered complete medium (complete medium containing 0.2% BSA and 20 mM HEPES-Tris, pH 7.40). After an overnight incubation, EGF (final concentration 100 ng/ml) was added to either the apical or basolateral compartment and populations were incubated at 37°C for 15 min. In some experiments, EGFR blocking antibody (no. 225) was added 30 min before EGF addition. After a rinsing, cell proteins were solubilized in immunoprecipitation solution (150 mM NaCl, 10 mM Tris · HCl, pH 7.40, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 5 µg/ml pepstatin A, 2 mM NaF, 2 mM Na3VO4, and 2 mM tetramisole) for 30 min at room temperature with rocking. In some experiments, cells were first solubilized in Triton X-100 solution (immunoprecipitation solution without sodium deoxycholate and SDS) for 30 min at room temperature with rocking. Insoluble proteins were then solubilized in immunoprecipitation solution for 30 min at room temperature with rocking. Insoluble material from each fraction was removed by centrifugation, and solubilized proteins were incubated overnight at 4°C with primary antibodies [anti-PLC-gamma antibody (1:125 dilution), anti-SHC antibody (1:125 dilution), anti-pp60src antibody (1:50 dilution), or anti-PI3K antibody (1:200 dilution)] plus 50 µl of a 50% suspension of protein A-Sepharose beads in Hanks' balanced salt solution (HBSS). Each immunoprecipitation was performed using the cells derived from a single 25-mm filter. In experiments involving cell extraction, equal cell equivalents were immunoprecipitated and separated in each lane. After extensive rinsing, beads were collected by centrifugation and immunoprecipitated proteins were detached by boiling for 5 min in Laemmli sample buffer. Released proteins from a single filter were divided into two equal aliquots, separated by polyacrylamide gel electrophoresis, and transferred to nitrocellulose. Protein content or phosphotyrosine content was measured on these parallel gels by Western blotting as described previously (2). Immunoreactive proteins were detected by enhanced chemiluminescence (Amersham, Arlington Heights, IL).

Immunofluorescence microscopy. Cells, grown as described above, were rinsed and then fixed with 4% paraformaldehyde in PBS for 30 min. Some samples were extracted with Triton X-100 solution before fixation. After extensive rinsing, all samples were permeabilized with 0.5% Triton X-100 in PBS. Reactive groups were blocked by sequential incubation with sodium borohydride and NH4Cl solutions. Protein binding sites were blocked by incubation for 60 min with PBS containing 0.2% BSA plus 0.2% gelatin (PBS-BG). Samples were incubated with primary antibodies (1:50-1:200 dilution) for 30 min in PBS-BG. After being rinsed, samples were incubated with fluorescein-conjugated secondary antibody (1:250; Vector Laboratories) for 30 min in PBS-BG. Samples were mounted in anti-quench solution (10% 0.1 M Tris, pH 8.6, 90% glycerol, and 2% triethylenediamine) and viewed using a Nikon Diaphot microscope equipped for epifluorescence. Photographs were taken using a ×100 objective under oil immersion.

Measurement of Na-hexose symport activity. Na-hexose symport activity was quantitated by measuring the uptake of the specific symporter substrate alpha -methyl-D-glucose (AMG), as described previously (1). Briefly, populations of Cl4 cells transfected with wild-type human EGFR were grown on permeable membrane filters for 14 days. Medium was replenished every 2-3 days. For uptake measurements, medium was aspirated. Populations were rinsed twice with warm (37°C) HBSS, and HBSS was added to the apical (0.9 ml) and basolateral (1.8 ml) compartments. Populations were incubated for 2 min at 37°C. [14C]AMG in HBSS was added to either the apical (0.1 ml) or the basolateral (0.2 ml) compartment to a final concentration of 100 µM (0.2 µCi/ml), and populations were incubated for 60 min at 37°C. To stop uptake, solution was aspirated from both compartments, and populations were rinsed rapidly six times with ice-cold balanced salt solution (150 mM NaCl and 2 mM Tris · HCl, pH 7.4). Cells were solubilized with 0.2% SDS. Radioactivity was measured by liquid scintillation counting, and protein content was measured by fluorometry. Previous work demonstrated that uptake of AMG by cells not expressing Na-hexose symport activity was ~0.4 nmol AMG · mg cell protein-1 · 60 min-1 using 100 µM AMG. Data are expressed as means ± SD of triplicate, independent samples. All experiments were performed at least three times with identical results.

Measurement of ouabain binding. To measure [3H]ouabain binding, cell populations grown on permeable membrane filters for 14 days were rinsed twice with warm (37°C) K-free HBSS, and K-free HBSS was added to the apical (0.9 ml) and basolateral (1.8 ml) compartments. Populations were incubated at 37°C for 2 min. Various concentrations of [3H]ouabain (final concentrations 0.1-20 µM) were added to the apical (0.1 ml) or basolateral (0.2 ml) compartment in K-free HBSS, and populations were incubated at 37°C for 15 min. To stop binding, solution was aspirated, and populations were rinsed rapidly six times with ice-cold balanced salt solution. Cells were solubilized with 0.2% SDS. Radioactivity was quantitated by liquid scintillation counting, and protein was quantitated by fluorometry. Nonspecific binding was determined by measuring binding in the presence of 100 µM nonradioactive ouabain. Nonspecific binding was subtracted from total binding for each sample to calculate "specific" (ouabain-displaceable) binding. Specific ouabain binding, but not nonspecific binding, was displaceable by K. All data are expressed as means ± SE of triplicate samples. Data are representative of at least three separate experiments performed using different cell preparations.

Measurement of transepithelial [14C]polyethylene glycol transport. The transepithelial movement of [14C]polyethylene glycol (PEG) was measured using a modification of the procedure described above for measuring cellular uptake of AMG. Cells were grown and prepared for measurement of PEG transport as described above, except that the HBSS contained 5 mM D-glucose and 2 mM L-glutamine and the basolateral compartment received 2 ml HBSS. [14C]PEG was added to the apical compartment (0.1 ml; final concentration 100 µM; 0.2 µCi/ml). Samples of the basolateral fluid (0.02 ml) were withdrawn periodically over a 4-h incubation period. Radioactive content of the solution was measured by liquid scintillation counting, and total transported PEG was calculated, taking into account the progressive decrease in basolateral fluid volume. The same procedure was performed using filters without cells. Data are expressed as means ± SD of triplicate samples, and the results presented are representative of at least three independent experiments.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cl4 cells expressing wild-type EGFR form a permeability barrier. Cells transfected with wild-type EGFR form typical epithelial monolayers composed of densely packed cells with a cobblestone appearance. Several pieces of evidence indicated that these cells formed typical junctional complexes that impeded paracellular solute movement. First, populations of transfected cells grown on plastic surfaces formed "domes" (data not shown), which are raised areas of the cell monolayer under which is trapped fluid and solutes. The fluid and solutes were transported from the medium to underneath the cell monolayers. The ability to trap fluid underneath the monolayer requires formation of circumferential tight junctions and adherens junctions that impede paracellular fluid and solute movement. Consistent with formation of typical adherens junctions, localization of the junction-associated protein beta -catenin was normal in Cl4 cells expressing wild-type human EGFR, and this localization was not disrupted dramatically by EGF treatment (18a).

Second, confluent monolayers of cells transfected with wild-type human EGFR restricted the transepithelial movement of ions when grown on permeable membrane filters. The transepithelial resistance of populations of these cells was 460 ± 130 Omega  · cm2. This was similar to the transepithelial resistance of untransfected cell populations and was substantially greater than the resistance of filters without cells (<50 Omega  · cm2).

Third, monolayers of cells transfected with wild-type human EGFR impeded the movement of uncharged solutes between the fluid compartments separated by the cell layer. The rate of apical-to-basolateral movement of [14C]PEG across confluent monolayers of cells (~0.30 nmol PEG · cm-2 · h-1) was substantially slower than the rate of movement of this compound across filters without cells (Table 1). The rate of PEG movement in the basolateral-to-apical direction was similar (data not shown). The cell monolayers also dramatically slowed [14C]mannitol movement between the fluid compartments (data not shown).

                              
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Table 1.   Transepithelial movement of [14C]PEG

These results indicate that the transfected cells form epithelial monolayers capable of separating fluid compartments and impeding the movement of ionic and nonionic solutes between the fluid compartments. The properties of these monolayers are not different from those of monolayers of untransfected cells.

Cl4 cells expressing wild-type human EGFR exhibit a polarized distribution of membrane proteins. The ability of confluent populations of transfected cells to form domes suggested that these cells expressed membrane proteins in a polarized manner, another characteristic of normal polarized epithelia. This point is particularly relevant because the transfected cells exhibit a relatively nonpolarized distribution of EGFR, i.e., EGFR is present at substantial levels at both membrane surfaces (Table 2; also see Fig. 1 and Ref. 18a). The nonpolarized distribution of EGFR could reflect either a specific lack of polarized expression of EGFR or a generalized loss of membrane protein polarization in the transfected cells. To examine this question directly, we determined the membrane localization of a marker basolateral membrane protein, Na-K-ATPase, and a marker apical membrane protein, Na-hexose symporter.

                              
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Table 2.   Membrane localization of a basolateral membrane marker (Na-K-ATPase), an apical membrane marker (Na-hexose symporter), and EGFR

Na-K-ATPase was localized by measuring specific [3H]ouabain binding (ouabain-displaceable, K-inhibitable binding) to each surface of confluent monolayers of transfected cells. Specific ouabain binding to the basolateral membrane was ~20-fold greater than binding to the apical membrane (Table 2).

Na-hexose symporter localization was determined by measuring the Na-dependent uptake of the specific Na-hexose symporter substrate AMG from the two surfaces of polarized cell monolayers. Specific AMG uptake was ~20-fold greater from the apical compartment than from the basolateral compartment (Table 2).

These results indicate that postconfluent populations of Cl4 cells expressing high levels of wild-type human EGFR were able to localize a basolateral membrane protein, Na-K-ATPase, and an apical membrane protein, Na-hexose symporter, to the appropriate membrane surfaces in a highly polarized manner. Thus the polarized expression of membrane proteins per se is not disrupted by expression of high levels of wild-type EGFR. Activation of EGFR at either surface did not alter the polarized distribution of membrane proteins or maintenance of the permeability barrier over the time course of these experiments (data not shown).

EGFRs at both surfaces are functional. We have developed a polarized renal epithelial cell system that expresses EGFR at both the apical and basolateral membranes (see above and Ref. 18a). Addition of EGF to the apical and basolateral compartments induced receptor tyrosine phosphorylation (Fig. 1). To confirm that we can selectively activate apical or basolateral EGFR, we determined the ability of an EGFR antibody that blocks EGF activation of the receptor (no. 225) to inhibit EGF-induced protein tyrosine phosphorylation when added to the compartment ipsilateral or contralateral to EGF. Postconfluent populations of transfected cells were exposed to EGF (100 ng/ml) in either the apical or basolateral compartment in the presence of blocking EGFR antibody in either the apical or basolateral compartment. Tyrosine phosphorylation of cell proteins was assessed by Western blot analysis.


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Fig. 1.   Effect of epidermal growth factor (EGF) addition (100 ng/ml) to either apical (AP) or basolateral (BL) compartment on tyrosine phosphorylation of cellular proteins (A) or EGF receptor (EGFR; B) derived from postconfluent populations of Cl4 cells transfected with wild-type human EGFR. A: to determine sidedness of receptor activation, a receptor-blocking antibody (Ab; no. 225; 10 µg/ml) was added to either apical or basolateral compartment 30 min before EGF addition. Control populations received no Ab (Ab NO) and/or no EGF (EGF NO). Equivalent numbers of cells were added to each well. B: EGFR was immunoprecipitated with Ab no. 225 after treatment of cell populations with EGF added to either apical or basolateral compartment. Immunoprecipitated proteins were then blotted for phosphotyrosine content. Control populations received no EGF before EGFR immunoprecipitation. Each lane represents receptor protein immunoprecipitated from a single membrane filter, and similar amounts were present in each well (data not shown). Molecular masses are shown at left. Blots are representative of 4 (A) and 6 (B) independent experiments.

In the absence of EGF, there was a weak reactivity of several cell proteins with the phosphotyrosine antibody (Fig. 1A, lane 1). Addition of EGF to either the apical (Fig. 1A, lane 4) or the basolateral (lane 7) compartment markedly increased the phosphotyrosine content of a protein of ~180 kDa, the expected molecular mass of the wild-type human EGFR. The phosphotyrosine content of multiple other cell proteins, with molecular masses ranging from ~40 to ~150 kDa, was also increased by addition of EGF to either compartment. Immunoprecipitation of EGFR and immunodetection of receptor phosphotyrosine content confirmed that EGFR phosphotyrosine content increased substantially upon addition of EGF to either the apical (Fig. 1B, lane 2) or basolateral (lane 3) compartment compared with the receptor phosphotyrosine content of untreated cell populations (lane 1). Addition of EGF to the basolateral compartment induced a greater increase in EGFR phosphotyrosine content than did addition to the apical compartment, consistent with the difference in 125I-labeled EGF binding capacity at each surface (see Table 2 and Ref. 18a). Addition of EGF to either the apical or basolateral compartment of postconfluent populations of cells transfected with vector alone or cells transfected with kinase-inactive human EGFR (Lys721-to-Met substitution) did not markedly increase the phosphotyrosine content of the ~180-kDa protein or any other cell protein detected in whole cell lysates (data not shown).

To confirm that EGF stimulated EGFR at the expected cell surface, cells were preincubated with EGFR function-blocking antibody (no. 225) in the apical or basolateral compartment for 30 min before addition of EGF to one or the other compartment. Addition of antibody to the basolateral compartment markedly inhibited the ability of basolateral EGF to increase tyrosine phosphorylation of the ~180-kDa protein, whereas addition of antibody to the apical compartment did not inhibit this effect (Fig. 1A, compare lanes 7-9). Likewise, apical but not basolateral antibody inhibited the ability of apical EGF to increase tyrosine phosphorylation of the ~180-kDa protein (compare Fig. 1A, lanes 4-6). These results indicate that addition of EGF to one fluid compartment stimulates tyrosine kinase activity almost exclusively of receptor located at the same surface.

Interestingly, in the absence of EGF, addition of antibody to either the apical or basolateral compartment increased slightly the tyrosine phosphorylation of a few cell proteins, including a protein of ~180 kDa, the expected molecular mass of EGFR, and a protein of ~100 kDa (Fig. 1A, compare lanes 1-3). This effect was, however, much weaker than that produced by EGF. This suggests that the blocking antibody may itself activate EGFR to a small extent. We cannot, however, rule out the possibility that the blocking antibody is weakly activating another tyrosine kinase, although we have no independent evidence for this.

Basolateral but not apical EGFR interacts with PLC-gamma . To assess the accessibility of signaling proteins to apical vs. basolateral EGFR, we investigated the tyrosine phosphorylation of several SH2 domain-containing signaling proteins, PLC-gamma , SHC, PI3K, and pp60src, upon addition of EGF to the apical or basolateral compartment. As found previously (18a), addition of EGF to either the apical or basolateral compartment increased dramatically the tyrosine phosphorylation of two SHC antibody-reactive proteins of molecular masses ~46 and ~52 kDa (Fig. 2B, left), the expected molecular masses of the two smaller SHC isoforms (24). Basolateral EGF addition produced a greater increase in SHC tyrosine phosphorylation than did apical EGF, consistent with the greater functional receptor content of the basolateral membrane (see above and Ref. 18a). Two larger tyrosine-phosphorylated proteins, ~120 and ~180 kDa, were coprecipitated with SHC, although the relative levels varied among experiments (data not shown). Tyrosine-phosphorylated proteins of similar molecular masses were coimmunoprecipitated with PI3K regulatory subunit (see Fig. 2C, left). A 180-kDa protein was also coprecipitated with PLC-gamma , but no 120-kDa phosphoprotein was detected (see Fig. 2A, left). The 180-kDa protein was identified as EGFR by comigration with authentic EGFR and immunoreactivity with anti-EGFR antibody (data not shown). c-Cbl (~120 kDa) was reported to become tyrosine phosphorylated and to associate with EGFR upon receptor activation (32). However, the 120-kDa protein coprecipitated with PI3K regulatory subunit from lysates of the Cl4 cells was not immunoreactive with an anti-c-Cbl antibody, which detected a protein of ~120 kDa in the Cl4 cells, nor was EGFR or PI3K coprecipitated with the anti-c-Cbl antibody under these conditions (data not shown). Interestingly, neither phosphoprotein was coprecipitated with pp60src, possibly because tyrosine phosphorylation of pp60src was not increased significantly by EGF treatment (see Fig. 2D, left).


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Fig. 2.   Effect of EGF addition (100 ng/ml) to apical or basolateral compartment of postconfluent cell populations on tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma ; A), SHC (B), phosphatidylinositol 3-kinase (PI3K; C), and pp60src (D). Control populations did not receive EGF. Left: detection of tyrosine-phosphorylated proteins. Right: detection of specific protein content in each lane is presented to demonstrate equivalent protein loading. Arrows, where present, indicate positions of immunoprecipitated proteins. Arrowheads indicate positions of coimmunoprecipitated proteins (see text). Lower bands detected in B and D, right, are immunoglobulin. Solubilized, immunoprecipitated proteins from a single filter were split into 2 equal aliquots and electrophoresed on parallel polyacrylamide gels. Protein content and phosphotyrosine content were detected on these parallel gels. Molecular masses are shown for C, left. Molecular masses for other detected proteins are described in text (see MATERIALS AND METHODS). Same Ab was used for immunoprecipitation and immunodetection of PLC-gamma and pp60src. For immunodetection of SHC, a monoclonal anti-SHC Ab (1:200 dilution; Transduction Laboratories) was used. For detection of PI3K, a monoclonal anti-PI3K regulatory subunit Ab (1:2,000 dilution; Transduction Laboratories) was used. Results are representative of at least 7 independent experiments for each protein examined.

Because SHC protein had equivalent access to apical and basolateral EGFR in these cells, we asked whether other SH2 domain-containing signaling proteins also exhibited an equivalent access to receptor located at each surface. Anti-PLC-gamma antibody immunoprecipitated a protein of ~165 kDa (Fig. 2A, right), similar to the expected molecular mass for PLC-gamma protein. In the absence of EGF, PLC-gamma protein exhibited minimal tyrosine phosphorylation (Fig. 2A, left). Addition of EGF to the apical compartment had little effect on PLC-gamma tyrosine phosphorylation, whereas basolateral EGF addition dramatically increased PLC-gamma tyrosine phosphorylation. This was paralleled by an increased coprecipitation of a tyrosine-phosphorylated protein of ~180 kDa, which was identified as EGFR by comigration with authentic EGFR and by reactivity with anti-EGFR antibody (data not shown). Similar amounts of PLC-gamma protein were immunoprecipitated from each sample (Fig. 2A, right).

The anti-PI3K regulatory subunit antibody immunoprecipitated a protein of ~83 kDa, the expected molecular mass for PI3K regulatory subunit, from lysates of the pig kidney cells (Fig. 2C, right). In the absence of EGF, minimal tyrosine-phosphorylated protein could be detected (Fig. 2C, left). Addition of EGF to either the apical or the basolateral compartment induced tyrosine phosphorylation of a protein of the same molecular mass (Fig. 2C, left). Basolateral EGF addition produced a greater increase in the phosphotyrosine content of this protein than did addition of EGF to the apical compartment. Two tyrosine-phosphorylated proteins (~180 and ~120 kDa) were coprecipitated with PI3K regulatory subunit. Significant amounts of tyrosine-phosphorylated ~120- and ~180-kDa proteins were coprecipitated with the PI3K regulatory subunit even in the absence of EGF. EGF addition to either compartment increased the phosphotyrosine signal of the ~120-kDa protein coprecipitated with regulatory subunit. Basolateral EGF produced a modestly greater increase. In some experiments, basolateral EGF addition increased the phosphotyrosine signal of the ~180-kDa protein coprecipitated with PI3K regulatory subunit, but this was not reproducibly observed (see Fig. 2B, left).

In the absence of EGF, pp60src exhibited a high level of tyrosine phosphorylation that was not reproducibly increased by addition of EGF to either compartment (Fig. 2D, left). We did not observe coprecipitation of either tyrosine-phosphorylated EGFR or the 120-kDa protein with pp60src (data not shown). Approximately equal amounts of each immunoprecipitated protein were present in each lane (Fig. 2, right).

Triton X-100 solubility of total and tyrosine-phosphorylated protein. Many proteins are present in both the detergent-soluble fraction (soluble proteins, membrane proteins) and detergent-insoluble fraction (cytoskeleton-associated proteins, nuclear matrix-associated proteins) of a cell. Accessibility of signaling proteins to apical vs. basolateral EGFR could be influenced by signaling protein partitioning between these two compartments. To investigate this possibility, the ability of apical vs. basolateral EGF addition to induce tyrosine phosphorylation of signaling proteins was examined in the fraction of cell proteins solubilized by 1% Triton X-100 and the fraction of proteins not solubilized by Triton X-100.

The vast majority of all four signaling proteins was recovered in the Triton X-100-soluble fraction (Fig. 3, right). Prolonged exposure of the blots revealed small amounts of pp60src and PLC-gamma in the Triton X-100-insoluble fraction (Figs. 4 and 5). The presence of large amounts of immunoglobulin heavy chain interfered with detection of SHC protein, but the phosphotyrosine blots support the presence of SHC protein in the Triton X-100-insoluble fraction (see Figs. 4 and 5). Although PI3K regulatory subunit protein was not detected in the Triton X-100-insoluble fraction, even after very long exposure of the Western blots (Fig. 4C, right), the phosphotyrosine blot (Fig. 4C, left) and the immunofluorescence micrograph (Fig. 5g) suggest that a small amount of regulatory subunit was present in this fraction.


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Fig. 3.   Effect of apical vs. basolateral EGF (100 ng/ml) addition to postconfluent cell populations on tyrosine phosphorylation of PLC-gamma (A), SHC (B), PI3K (C), and pp60src (D) contained in Triton X-100-soluble cell fraction. Left: detection of phosphotyrosine. Right: detection of immunoprecipitated proteins to demonstrate equivalent loading per well. Arrows, where present, indicate positions of immunoprecipitated proteins. Arrowheads indicate positions of coimmunoprecipitated proteins (see text). Lower band detected in B, right, is immunoglobulin. Molecular masses are shown in C, left. Molecular masses of proteins detected with each Ab are described in text (see MATERIALS AND METHODS). Results shown are representative of at least 5 independent experiments for each protein examined.


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Fig. 4.   Effect of apical vs. basolateral EGF (100 ng/ml) addition to postconfluent cell populations on tyrosine phosphorylation of PLC-gamma (A), SHC (B), PI3K (C), and pp60src (D) contained in Triton X-100-insoluble fraction. Left: detection of phosphotyrosine. Right: detection of immunoprecipitated proteins. Arrows, where present, indicate positions of immunoprecipitated proteins. Arrowheads indicate positions of coimmunoprecipitated proteins (see text). Lower band detected in D, right, is immunoglobulin. Molecular masses are shown in C, left. Molecular masses of proteins detected with each Ab are described in text (see MATERIALS AND METHODS). Results shown are representative of at least 5 independent experiments for each protein examined.

Tyrosine phosphorylation of the detergent-soluble fraction of all four signaling proteins paralleled the pattern observed for total cell protein (Fig. 3, left). A somewhat different pattern was observed when tyrosine phosphorylation of signaling proteins in the Triton X-100-insoluble fraction (Fig. 4, left) was examined. PLC-gamma protein in the Triton X-100-insoluble fraction exhibited a significant level of tyrosine phosphorylation in the absence of EGF that was not increased markedly by addition of EGF to either compartment. Furthermore, tyrosine-phosphorylated EGFR was not coprecipitated with PLC-gamma in this compartment (Fig. 4A, left), in contrast to the coprecipitation observed in the Triton X-100-soluble compartment (see above). Tyrosine phosphorylation of detergent-insoluble SHC protein displayed a pattern similar to that of detergent-soluble SHC (Fig. 4B, left). Although no tyrosine-phosphorylated PI3K regulatory subunit was detected in the detergent-insoluble fraction (Fig. 4C, left), minimal levels of tyrosine-phosphorylated ~120- and ~180-kDa proteins could sometimes be detected after prolonged exposure of the blots. However, the phosphotyrosine signal of these coprecipitated proteins was not increased by EGF addition to either compartment. pp60src in the detergent-insoluble fraction exhibited a basal level of tyrosine phosphorylation, but, in contrast to what was observed in the detergent-soluble fraction, this level was demonstrably increased by addition of EGF to either compartment (Fig. 4D, left).

PLC-gamma is concentrated along the lateral membrane. Because PLC-gamma , but not SHC, PI3K regulatory subunit, or pp60src, exhibited a differential accessibility to apical and basolateral EGFR, we asked whether PLC-gamma , but not the other signaling proteins, exhibited a subcellular distribution consistent with this differential accessibility. The signaling proteins were localized in postconfluent transfected cells maintained in the absence of EGF. For comparison, the localizations of a basolateral membrane marker protein, E-cadherin, and an apical membrane marker protein, gamma -glutamyl transpeptidase (2), were also determined in postconfluent cell populations. Immunofluorescence microscopy revealed that all four signaling proteins exhibited a diffuse distribution within the cytoplasm (Fig. 5). In addition to this diffuse staining, PLC-gamma protein staining was concentrated at lateral cell borders. The other signaling proteins did not exhibit this concentration at lateral cell borders. E-cadherin protein exhibited a diffuse, somewhat granular intracellular staining pattern and a sharp staining at the lateral membranes (Fig. 5j). This pattern was similar to that observed for PLC-gamma but not the other signaling proteins. Staining for gamma -glutamyl transpeptidase exhibited a punctate distribution over the entire apical cell surface overlaid on a granular intracellular staining pattern (Fig. 5i). The punctate apical staining pattern is consistent with staining of microvilli and is distinct from the staining pattern exhibited by any of the signaling proteins, suggesting that the signaling proteins examined here are not concentrated at the apical membrane.


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Fig. 5.   Immunofluorescence detection of PLC-gamma (a and e), SHC (b and f), PI3K (c and g), and pp60src (d and h) in unextracted (a-d) and Triton X-100-extracted (e-h) populations of wild-type human EGFR-expressing cells. Immunofluorescence detection of gamma -glutamyl transpeptidase (i) and E-cadherin (j) in unextracted populations of wild-type EGFR-expressing cells is also shown. Arrowheads denote lateral concentration of PLC-gamma in both unextracted (a) and Triton X-100-extracted (e) cells and vesicular localization of SHC (f) and PI3K (g) in Triton X-100-extracted cells. Scale bar (h), 20 µm. Results are representative of at least 3 independent experiments for each protein examined.

The tyrosine-phosphorylated PLC-gamma protein was soluble in Triton X-100 (see above). Because there was not a major shift in the amount of PLC-gamma protein that was Triton X-100 soluble vs. insoluble upon EGFR activation (see above), we reasoned that the PLC-gamma protein that becomes tyrosine phosphorylated must already reside in the Triton X-100-soluble pool before EGF addition. Therefore, if the lateral PLC-gamma protein is the target for basolateral EGF-mediated tyrosine phosphorylation, this staining should be strongly diminished by extraction of cells with Triton X-100 before cell fixation. Consistent with this hypothesis, most cytoplasmic and lateral immunoreactivity for PLC-gamma protein was removed by Triton X-100 pretreatment. The remaining signal, although much weaker, displayed a sharp lateral staining pattern reminiscent of staining of cell junctions (Fig. 5e). This pattern was easily distinguished from the less sharply defined lateral PLC-gamma protein detected in whole cells, suggesting that this Triton X-100-insoluble PLC-gamma protein is a minor subfraction of the total laterally distributed PLC-gamma protein.

Triton X-100 pretreatment also removed the majority of immunoreactivity for the other three signaling proteins (Fig. 5, f-h, SHC, PI3K, and pp60src, respectively). A vesicular staining pattern was observed for the Triton X-100-insoluble fraction of these three signaling proteins (see, for example, Fig. 5, f and g), suggesting that the detergent-insoluble fractions of these proteins are confined to intracellular vesicles. The nuclear staining, but not the vesicular or lateral staining, seen in Fig. 5, e-h, was observed in Triton X-100-pretreated samples when primary antibody was omitted (data not shown), indicating that it represents nonspecific binding of secondary antibody.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The functional consequences of activation of the same receptor at the two surfaces of a polarized epithelial cell for downstream signal transduction are unknown. We investigated this question directly using a model renal epithelial cell system in which functional EGFRs are expressed at both membranes of the polarized cells. We demonstrate that EGFRs at both the apical and basolateral membranes of a polarized renal epithelial cell have access to several signaling proteins in common. However, access to PLC-gamma was restricted only to basolateral receptors. EGF-induced tyrosine phosphorylation of PLC-gamma was restricted to the detergent-soluble pool. Detergent-insoluble PLC-gamma exhibited a basal level of tyrosine phosphorylation that was not increased by EGF addition, suggesting that this fraction of PLC-gamma was not accessible to EGFR expressed at either surface. Our results suggest that activation of EGFR at the basolateral membrane will initiate PLC-gamma -mediated signaling events, whereas activation of apical EGFR will not.

In the absence of EGF, PLC-gamma protein was concentrated at the lateral borders of cells, similar to the lateral localization of the basolateral membrane marker protein, E-cadherin, in addition to the diffuse cytoplasmic localization exhibited by all four signaling proteins. Because a substantial portion of this lateral PLC-gamma was detergent soluble, it is tempting to speculate that it is this lateral membrane-associated PLC-gamma that becomes tyrosine phosphorylated upon EGFR activation. The lateral localization of this protein would provide the basis for its restricted access to EGFR. Extrapolating from the results of Paradiso et al. (24), our results suggest that PLC-gamma -mediated signaling events will occur only in proximity to the basolateral membrane. The concept of spatially constrained signaling responses, with their potential implications for regulating intracellular events at a localized, subcellular level, may have important and far-reaching consequences for regulating epithelial cell behavior.

It was interesting that pp60src also exhibited different patterns of EGF-induced tyrosine phosphorylation for the detergent-soluble and -insoluble fractions. This difference may be merely quantitative, since the large amount of tyrosine-phosphorylated, detergent-soluble src kinase may make detection of an EGF-induced phosphorylation difficult. Alternatively, it may indicate that detergent-soluble pp60src is tyrosine phosphorylated by multiple tyrosine kinases, whereas detergent-insoluble pp60src is accessible to only a limited group of tyrosine kinases, including EGFR at either surface.

Our results indicate that membrane receptor location in a polarized epithelial cell defines, at least in part, the signaling proteins with which the receptor interacts. These results suggest that, in polarized epithelial cells expressing the same receptor at both membranes, activation of apical and basolateral receptors will initiate distinct subsets of downstream signaling pathways. If intracellular signaling events are spatially constrained, as suggested by the results of Paradiso et al. (24), activation of receptors at the two surfaces would likely produce different cellular responses. Furthermore, in diseases in which receptors are mislocalized, e.g., EGFR and c-Met in polycystic kidney disease (13, 23, 35, 36), activation of receptors at the inappropriate surface could lead to activation of inappropriate signaling pathways or lack of activation of appropriate signaling pathways. Because cellular responses result from the coordinated activation of multiple signaling pathways, activation of an abnormal complement of signaling pathways could lead to initiation of abnormal cellular responses. Activation of a signaling event at an inappropriate location within the cell could also produce abnormal cellular responses.

We have also demonstrated that receptor location affects receptor turnover (18a), which can also modulate cell behavior (34). Therefore, receptor location may be a critical parameter in determining the receptor behavior in polarized epithelial cells. EGF addition to either compartment induced tyrosine phosphorylation of both SHC and PI3K proteins (see above) and cell proliferation (18a). Activation of both proteins has been implicated in stimulation of cell proliferation (14, 25, 29). PLC-gamma activity has been reported to be essential for cell motility (10). It is tempting to speculate that basolateral EGFR activation, which will activate PLC-gamma , but not apical EGFR activation will stimulate motility in these polarized epithelial cells. Although our experimental setup does not permit analysis of cell motility under conditions of differential activation of apical and basolateral EGFR, we have confirmed that PLC activity is required for migration of these cells into a wound (data not shown). This could have important implications for receptor regulation of cell behavior under normal and pathophysiological conditions.

    ACKNOWLEDGEMENTS

We thank Dr. Kirk A. Lund (Rockwood Clinic, Spokane, WA) for assistance in developing the renal epithelial cell model system and for numerous scientific insights. The discussions, in particular, were invaluable in developing and refining the focus for this study. We also thank Dr. G. Gill (University of California, San Diego) for the gift of wild-type human EGFR cDNA construct, Dr. S. Wiley (University of Utah School of Medicine) for the gift of human EGFR-specific monoclonal antibody (no. 225), and Dr. R. Campos-Gonzalez (Transduction Laboratories, Lexington, KY) for the gift of SHC monoclonal antibody. We thank Dr. D. A. Winkelmann for providing access to and assistance with the fluorescence microscope.

    FOOTNOTES

This work was supported by grants from the American Heart Association (National Affiliate and New Jersey Affiliate) and from the Charles E. Culpeper Foundation.

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: K. Amsler, Dept. of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Ln., Piscataway, NJ 08854-5635.

Received 29 May 1998; accepted in final form 24 September 1998.

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Results
Discussion
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