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
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ABSTRACT |
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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- (PLC-
) 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-
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
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INTRODUCTION |
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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). 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-, 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.
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MATERIALS AND METHODS |
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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- (PLC-
) 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).
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 (-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.
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-
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
-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.
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RESULTS |
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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 -catenin was normal in Cl4 cells
expressing wild-type human EGFR, and this localization was not
disrupted dramatically by EGF treatment (18a).
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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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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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Basolateral but not apical EGFR interacts with PLC-.
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-
, 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-
, 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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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-
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PLC- is concentrated along the lateral membrane.
Because PLC-
, but not SHC, PI3K regulatory subunit, or
pp60src, exhibited a differential
accessibility to apical and basolateral EGFR, we asked whether PLC-
,
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,
-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-
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-
but not the other signaling
proteins. Staining for
-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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DISCUSSION |
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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- was restricted only to basolateral
receptors. EGF-induced tyrosine phosphorylation of PLC-
was
restricted to the detergent-soluble pool. Detergent-insoluble PLC-
exhibited a basal level of tyrosine phosphorylation that was not
increased by EGF addition, suggesting that this fraction of PLC-
was
not accessible to EGFR expressed at either surface. Our results suggest
that activation of EGFR at the basolateral membrane will initiate
PLC-
-mediated signaling events, whereas activation of apical EGFR
will not.
In the absence of EGF, PLC- 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-
was
detergent soluble, it is tempting to speculate that it is this lateral
membrane-associated PLC-
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-
-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- activity has been reported to be essential for cell
motility (10). It is tempting to speculate that basolateral EGFR
activation, which will activate PLC-
, 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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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