Divisions of 1 Gastroenterology
and 4 Hematology/Oncology, 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,
tyrosine phosphorylation; 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.
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
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-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.
-catenin; proliferation; epidermal
growth factor receptor downregulation and endocytosis
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
· cm2 or
higher were used for experiments.
-MEM
(ICN) with 10% fetal calf serum (Hyclone), penicillin, streptomycin,
and glutamine. At 14 days in culture, the medium was changed to cold
-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).
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RESULTS |
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---|
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
· 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.
|
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.
|
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).
|
|
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.
|
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.
|
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. -Catenin, a known EGFR substrate (15), is an important component of
adherens junctions. The highly localized distributions of FAK and
-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
-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 -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).
|
In striking contrast, -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
-catenin. The lower band corresponded exactly to
tyrosine-phosphorylated
-catenin on the anti-phosphotyrosine blot
and the upper band corresponded to non-tyrosine-phosphorylated
-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
-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
-catenin.
|
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. -Catenin forms complexes with
E-cadherin at the adherens junction and in the cytoplasm (21).
Therefore,
-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
-catenin
coprecipitated with E-cadherin, regardless of the degree of
-catenin
tyrosine phosphorylation (compare Fig.
8B,
left and
right). However, tyrosine
phosphorylation of the coprecipitated
-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
-catenin. Again, only apical EGFR stimulation led to tyrosine phosphorylation of
-catenin (Fig.
8C). This further suggests that not
all
-catenin was complexed with E-cadherin in the Triton-soluble fraction.
-Catenin was immunoprecipitated from the Triton-insoluble fraction
under denaturing conditions. Again, tyrosine phosphorylation of
-catenin was greater after apical than after basolateral EGFR stimulation (Fig. 8D,
left). Furthermore, more
-catenin
protein was found in the Triton-insoluble fraction after stimulation of apical than after basolateral EGFR (Fig.
8D,
right).
In K2 cells, -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).
-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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DISCUSSION |
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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 -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
-catenin after apical than after basolateral EGF stimulation in K2
cells demonstrated compartmentalization of
-catenin.
Analysis of EGF-induced tyrosine phosphorylation of -catenin in
various cell fractions revealed that the missorted apical EGFR had much
greater access to a Triton-soluble pool of
-catenin than basolateral
EGFR. Specifically, apical EGFR had access to
-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
-catenin staining. It has been previously reported that
increased tyrosine phosphorylation of
-catenin led to dissociation
with E-cadherin at adherens junctions (4). However, we did not see any
decrease in
-catenin at the lateral cell membrane in response to
either apical or basolateral EGFR activation by
X-Z plane confocal microscopy,
and coprecipitation of
-catenin with E-cadherin occurred
independently of the level of tyrosine phosphorylation of
-catenin.
The higher degree of tyrosine phosphorylation of
-catenin by apical
EGFR did produce a greater appearance of
-catenin protein in the
Triton-insoluble cell fraction than that produced by basolateral EGFR
stimulation.
-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
-catenin in the Triton-insoluble pellet may represent nuclear
translocation, we do not have any evidence to support this.
The differential tyrosine phosphorylation of -catenin in K2 cells is
likely due to the overexpression of EGFR per se rather than to
perturbation of
-catenin localization as a result of EGFR
overexpression. This was evident by the fact that
-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 -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 |
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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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