From the Division of Cell Biology and Immunology, Department of Pathology, University of Utah, Salt Lake City, Utah 84132
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ABSTRACT |
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ErbB-2/HER2 is an important signaling partner for
the epidermal growth factor receptor (EGFR). Overexpression of erbB-2
is also associated with poor prognosis in breast cancer. To investigate how erbB-2 amplification affects its interactions with the EGFR, we
used a human mammary epithelial cell system in which erbB-2 expression
was increased 7-20-fold by gene transfection. We found that
amplification of erbB-2 caused constitutive activation of erbB-2 as
well as ligand-independent activation of the EGFR. Overexpression of
erbB-2 strongly inhibited erbB-2 down-regulation following transactivation by EGFR. Significantly, down-regulation of activated EGFR was also inhibited by erbB-2 amplification, resulting in enhanced
ligand-dependent activation of the EGFR. The rate of EGFR
endocytosis was not affected by erbB-2 overexpression, but the rate of
lysosomal targeting was significantly reduced. In addition, erbB-2
overexpression promoted rapid recycling of activated EGFR back to the
cell surface and decreased ligand dissociation from the EGFR. Our data
suggest that overexpression of erbB-2 inhibits both its down-regulation
and that of the EGFR. The net effect is increased signaling through the
EGFR system.
The epidermal growth factor
(EGF)1 family of receptors
and ligands contains four related receptor tyrosine kinases and seven related ligands (1-5). Overexpression of each of the receptors (EGFR,
erbB-2, erbB-3, and erbB-4) has been correlated with poor prognosis in
breast and other cancers (6-8). Compelling clinical studies on breast
cancer reveal that amplification of erbB-2 has a high degree of
correlation with disease recurrence and poor survival (9). Both
clinical and basic research studies indicate a role for erbB-2
amplification in initial transformation events and in progression to
metastases (10, 11). Despite the correlative evidence between erbB-2
overexpression and breast cancer, however, the mechanism by which
erbB-2 facilitates cell transformation is unknown.
The EGFR has five known ligands (EGF, transforming growth factor- The EGFR is negatively regulated by both serine and threonine
phosphorylation as well as by intracellular trafficking (23). Rapid
internalization and lysosomal degradation result in short-term as well
as long-term down-regulation of receptor activity (24). Proper
trafficking of the EGFR is important for regulation of cell growth
(25). Many signaling complexes are thought to be activated by assembly
at the cell surface, and internalization has been proposed to
negatively regulate this activity (24, 26). Once internalized, occupied
receptors are sorted in recycling endosomes and subsequently degraded
in lysosomes, effectively reducing the number of activated receptors.
Overexpression of EGFR has been shown to impair their down-regulation,
apparently because of limiting levels of regulatory molecules that
mediate rapid endocytosis and lysosomal targeting (24, 27-29).
Although activation of erbB-2, erbB-3, and erbB-4 has been well
characterized, the negative regulation of these receptors has not been
extensively studied. There are conflicting reports regarding the
trafficking of erbB-2 following transactivation with EGF. We, as well
as other investigators, have described efficient down-regulation and
lysosomal targeting of erbB-2 in three different cell types (20, 30).
In contrast, chimeric receptors composed of the EGFR extracellular
domain and the erbB-2 cytoplasmic domain, were not down-regulated
following activation with EGF (31). In another study, down-regulation
of erbB-2 was investigated in SKBR-3 cells, which have an amplified
erbB-2 gene (32). It was reported that although erbB-2 was
transactivated following EGF treatment, there was no measurable change
in erbB-2 half-life.
One way to reconcile these disparate results is to postulate that
although lysosomal targeting is normally involved in erbB-2 down-regulation, it is impaired when erbB-2 is overexpressed. To test
this hypothesis, we examined the negative regulation of erbB-2 in
mammary epithelial cells that overexpress the protein due to
introduction of a transgene. We found that overexpression of erbB-2
severely inhibits its own down-regulation. Intriguingly, we also found
that down-regulation of the EGFR was inhibited even though EGFR levels
were similar between parental and erbB-2 overexpressing cells.
Consistent with the findings of other investigators, we found that
overexpression of erbB-2 results in an elevated basal level of
activated receptors (33, 34). However, we not only observed
constitutive activation of erbB-2, but also of EGFR. These results show
that receptor cross-talk is not only important in receptor activation,
but also plays a role in negative regulatory processes.
Antibodies--
N-13 polyclonal antibody directed against the
amino-terminal 13 residues of the EGF receptor was a gift from Dr.
Debora Cadena. 1917 polyclonal antibody directed against the
carboxyl-terminal 18 residues of erbB-2 was provided by Dr. Gordon
Gill. Ab5 mouse monoclonal antibody against the extracellular domain of
erbB-2 was from Oncogene Sciences. 225 mouse monoclonal antibody
against the EGFR was purified from hybridomas obtained from the
American Type Culture Collection. RC20 anti-phosphotyrosine/horseradish peroxidase conjugate was purchased from Transduction Laboratories. Antibodies 13A9 against the EGFR and 4D5 against human erbB-2 were
gifts from Genentech. These were directly labeled with Alexa 488 and
Alexa 594 dyes, respectively, following the manufacture's protocol
(Molecular Probes, Inc.).
Cell Culture--
B82 mouse L cells transfected with the gene
for human EGF receptor were grown in Dulbecco's modified Eagle's
medium containing dialyzed 10% calf serum (HyClone) and 5 µM methotrexate. The human mammary epithelial cell lines
MTSV1-7, ce1, and ce2 have been described previously (35) and were
obtained from Dr. Joyce Taylor-Papadimitriou. They were grown in
Dulbecco's modified Eagle's medium containing 10% calf serum
(HyClone), 1 µM insulin, and 5 µM
dexamethasone. Selection for erbB-2 expression was maintained using 500 µg/ml G418.
Quantification of ErbB-2, EGFR, and Phosphotyrosine
Levels--
Confluent cultures were lysed in RIPA buffer (36), debris
was removed by centrifugation and samples were brought to 2% SDS, 1%
To determine the phosphotyrosine content of erbB-2 and the EGFR, cells
were lysed for 10 min at 0 °C in RIPA buffer supplemented with 100 µM Na3VO4. The lysate was
clarified by centrifugation for 10 min at 10,000 × g,
and receptors were immunoprecipitated with 5 µl of either the 1917 or
4D5 antibodies (erbB-2) or 2 µg of 225 (EGFR) and 100 µl of protein
A-Sepharose (50% slurry). The beads were washed several times in lysis
buffer and boiled in SDS sample buffer prior to gel electrophoresis and
transfer to nitrocellulose. Phosphotyrosine levels were measured by
detection with RC20 antibody and the Renaissance ECL kit from NEN Life
Science Products Inc. Blots were exposed to film, and analyzed using a Bio-Rad Imaging Densitometer and the Molecular Analyst software package.
Fluorescence Microscopy--
Distribution of both EGFR and
erbB-2 was evaluated using fixed and permeabilized cells as described
previously (20). Cells were labeled for 1 h with a mixture of 13A9
and 4D5 antibodies directly labeled with Alexa 488 and Alexa 594 dyes,
respectively, at a final concentration of 1 µg/ml. After rinsing, the
cells were mounted in Prolong anti-fade medium (Molecular Probes, Inc.) and viewed with a Nikon inverted fluorescence microscope with a ×60,
1.4 N.A. oil immersion objective. Images were acquired as described
below. Specificity of the labeling was verified by using control cells
(B82) lacking EGFR and human erbB-2.
To follow the transfer of EGFR to lysosomes, the lysosomes were first
labeled by incubating cells for 15 min at 37 °C with 5 mg/ml
fluorescein-labeled dextran (Mr 10,000, anionic,
lysine fixable; Molecular Probes, Inc.), then chased for an additional 2 h. During the chase period, cells were pulsed for 15 min with 1.5 × 10 Binding Analysis--
Number of surface-associated erbB-2
molecules was determined by steady state analysis (38). 4D5 antibody
was radioiodinated to a specific activity of 4.5 × 106 cpm/pmol (39) and cells were incubated with
concentrations from 6.7 × 10 Fractional Recycling--
Cells were grown to confluence in
35-mm dishes and switched to serum-free Dulbecco's modified Eagle's
medium containing 20 mM HEPES (pH 7.4) and no bicarbonate
(D/H/B) 12 h before experiments. The cells were incubated at
37 °C in 0.1-30 ng/ml 125I-EGF for 3 h to allow
the sorting process to reach a steady state (39, 41). Cells were then
washed with acid-strip (50 mM glycine-HCl, 100 mM NaCl, 2 mg/ml polyvinylpyrrolidone, pH 3.0) for 2 min at 0 °C to remove surface-bound ligand (39), rinsed twice with phosphate-buffered saline, and returned to 37 °C in D/H/B containing 1 µg/ml unlabeled ligand to prevent rebinding and reinternalization of recycled ligand. The medium was collected at 10 min and an aliquot
counted for total radioactivity. Cells were solubilized with 2% sodium
dodecyl sulfate and the amount of radioactivity remaining was
determined. The medium was loaded on a 15% native polyacrylamide slab
gel and the intact and degraded EGF was separated by isotachophoresis
(41). After drying the gel, the relative amount of radioactivity in the
bands corresponding to intact and degraded EGF was quantified using a
Bio-Rad G250 Molecular Imager. Cell number per plate was determined by
counting parallel plates. Fraction of intact ligand was then plotted as
a function of ligand in the cells at the start of the chase (lost into
the medium + amount remaining) as described previously (28).
ErbB-2 Half-life Measurements--
Cells were labeled to steady
state (24 h) with cysteine and methionine-free Dulbecco's modified
Eagle's medium (ICN) supplemented with 250 µCi/ml
EXPRE35S35S from NEN Life Science Products Inc.
which contains both radiolabeled methionine and cysteine. Cultures were
rinsed 6 times with normal culture medium and chased with medium with
or without 100 ng/ml EGF. At 0, 1, 3, 5, and 7 h chase, cells were
lysed in RIPA buffer and equal amounts of protein were subjected to
immunoprecipitation with 5 µl of 1917 antibody. Samples were then
separated by SDS-gel electrophoresis and the gels were then dried on
3MM paper followed by quantitation of erbB-2 bands using a Bio-Rad
Molecular Imager as described above.
Characterization of Cells Overexpressing ErbB-2--
Ligand
binding not only activates the EGFR but also initiates negative
regulatory processes. Overexpression of the EGFR, however, has been
shown to inhibit this negative regulation. Since erbB-2 acts as a
signaling partner of the EGFR, we wanted to determine whether erbB-2
overexpression affected its own negative regulation, or that of the
EGFR. We employed a human mammary epithelial cell line (MTSV) and two
derivative lines (ce-1 and ce-2) which have been transfected with the
gene for erbB-2 (35, 42). The parent cell line, derived from human
breast aspirates, was immortalized with SV40 large T antigen, but is
not tumorigenic. Transfection with the erbB-2 gene alters the growth
characteristics of the ce-1 and ce-2 cells in that the transfectants
grow in soft agar can be propagated as tumors in nude mice, and show
disorganized growth on collagen gels (35).
Western blot analysis and binding of radiolabeled anti-erbB-2 (4D5) at
37 °C was used to quantify the expression levels of erbB-2 in these
cells. This approach was taken because equilibrium binding could not be
achieved at 0 °C due to the very slow binding of 4D5. Steady state
binding at 37 °C also allowed us to estimate the relative
distribution of erB-2 between cell surface and intracellular pools
(38). Preliminary experiments showed that treating the cells overnight
with 4D5 did not appear to alter the cellular distribution of erbB-2
(data not shown).
Steady state binding of anti-erbB-2 indicated an accessible pool of
9.8 × 104 and 1.5 × 106 erbB-2
molecules per cell in MTSV and ce2 cells, respectively (Fig.
1A), a 15-fold increase in the
transfected cell line. Acid stripping of the bound radiolabeled 4D5
showed that surface expression of erbB-2 was 6.3 × 104 in the case of MTSV cells and 6.4 × 105 for ce2 cells. ErbB-2 expression in the ce-1 line
varied between 2- and 6-fold higher than the parental cells (data not
shown). Western blot analysis indicated a 23-fold increase in erbB-2
mass in the ce2 versus MTSV lines, somewhat higher than the
values derived from the steady state analysis, suggesting that not all cellular erbB-2 readily exchanges with the cell surface. The affinity of the 4D5 antibody for erbB-2 was similar for both the MTSV and ce2
cells at 12 and 10 nM, respectively. The percent of 4D5
antibody found internalized at steady state was also similar at 41%
(±4%) and 55% (±14%) for MTSV and ce2 cells, respectively.
The number of EGFR in these cells was determined by Scatchard analysis
conducted at 0 °C to prevent receptor down-regulation. As shown in
Fig. 1B, both MTSV and ce2 cells displayed similar numbers
of surface EGFR (5.7 × 105 and 9.2 × 105 per cell, respectively) of predominantly a single
affinity class. The affinity of these receptors, 1.4 nM, is
similar to what has been described previously for fibroblasts (43).
Western blots of detergent extracts of MTSV, ce1, and ce2 cells confirm
that they all express a similar number of EGFR (data not shown).
Analysis of the Western blots using a molecular imager indicated that
relative to cell protein content, ce2 cells express approximately 10%
higher EGFR levels whereas the levels of EGFR in ce1 cells was
indistinguishable from the parental MTSV cells.
These data indicate that the ratio of EGFR to erbB-2 at the cell
surface of MTSV cells is approximately 9:1 whereas the ratio in ce2
cells is approximately 1:1. To characterize erbB-2 and EGFR
distribution in these cells, sparse cultures were fixed, permeabilized,
and stained using directly labeled erbB-2 and EGFR antibodies. As shown
in Fig. 2, erbB-2 was found at both the
cell surface and in a collection of intracellular vesicles. The EGFR showed a very similar distribution pattern with EGFR and erbB-2 both
colocalized at the cell surface and in intracellular vesicles (arrows in Fig. 2). These data suggest that overexpression
of erbB-2 is not accompanied by any striking alteration in either its
cellular distribution or affinity for antibodies. In addition, the
number, distribution, and affinity of EGFR do not appear to be greatly
altered as a result of erbB-2 overexpression.
Amplification of ErbB-2 Inhibits the Down-regulation of
ErbB-2--
Because the EGFR transactivates erbB-2, changing the ratio
of erbB-2 to EGFR may alter EGF-mediated erbB-2 phosphorylation. Both
parental MTSV cells and overexpressing ce2 cells were treated with EGF
for 5 and 10 min. ErbB-2 and EGFR were then immunoprecipitated followed
by Western blot analysis for phosphotyrosine (Tyr(P)), to monitor
receptor activation. As shown in Fig. 3,
addition of EGF increased Tyr(P) levels of both EGFR and erbB-2. In the
case of the parental MTSV cells, very little receptor phosphorylation was observed in the absence of EGF addition. Surprisingly, in ce2
cells, there was a substantial amount of phosphorylation of both erbB-2
and the EGFR in the absence of EGF. Although increased basal activation
of erbB-2 as a result of overexpression has been documented by other
investigators (34, 35, 44), higher basal activation of the EGFR has not
previously been reported. Addition of EGF further increased the level
of erbB-2 phosphorylation approximately 3-fold in ce2 cells as compared
with 9-fold in the MTSV cells (average of three experiments),
indicating that the EGFR was capable of transactivating erbB-2 in both
cell types.
It seemed possible that constitutive activation of both the EGFR and
erbB-2 could be due to autocrine production of EGF-like ligands. We
tested this idea by blocking EGFR activation using antagonistic EGFR
antibodies 225 and 13A9 (45, 46). Neither antibody affected the
constitutive activation of either the EGFR or erbB-2 (Fig. 3),
suggesting that overexpression of erbB-2 alone was responsible.
To determine whether overexpression of erbB-2 affects its
down-regulation, we transactivated erbB-2 by treating both MTSV and ce2
cells with 100 ng/ml EGF for 24 h. As a comparison, we also
examined a well characterized fibroblast cell line (20). Loss of erbB-2
was assessed by Western blot analysis. As shown in Fig.
4, A and B,
fibroblasts and MTSV cells showed a 80 and 72% loss of erbB-2,
respectively, by 24 h following EGF treatment. However, the ce2
cell line only displayed a 30% loss in erbB-2 mass. Calculating the
net amount of erbB-2 loss following 24 h EGF treatment showed that
the MTSV cells lost a similar amount of erbB-2 mass relative to ce2
cells (2.0 × 104 versus 2.3 × 104 arbitrary molecular imager units). Thus, erbB-2
overexpression affected the relative amount erbB-2 degraded in response
to EGF, not the absolute amount.
The kinetics of receptor loss showed that erbB-2 loss in the parental
MTSV cells was completed within 6 h (Fig. 4C). The
level of erbB-2 in the overexpressing ce2 line was only reduced about 5% at the same time. We confirmed that the accelerated loss of erbB-2
protein was due to enhanced degradation by labeling cells with
35S-amino acids and immunoprecipitating erbB-2 following
EGF addition (data not shown). EGF decreased the half-life of erbB-2 in
MTSV cells from 6 to 1 h, whereas the same treatment decreased
erbB-2 half-life from 6 to 5 h in ce2 cells. Reverse transcription
polymerase chain reaction analysis of mRNA levels indicated that
EGF did not affect the erbB-2 mRNA level (data not shown). These
results suggest that overexpression of erbB-2 inhibits its
down-regulation by reducing the degradation rate of the transactivated
receptor pool.
ErbB-2 Overexpression Increases Signaling through the EGFR--
As
previously noted, we observed that overexpression of erbB-2 resulted in
the constitutive activation of both the EGFR as well as erbB-2 (Fig.
3). The 6-fold increase in basal EGFR activation was similar to the
7-fold increased basal activity of erbB-2 (Fig. 3). Because erbB-2 acts
as a signaling partner to the EGFR, it seemed possible that erbB-2
overexpression may drive heterodimer formation. Activation of the EGFR,
however, is usually followed by several negative regulatory processes,
such as desensitization and down-regulation (23). To explore the effect
of erbB-2 overexpression on both positive and negative regulation of
the EGFR, we examined the time course of EGF-induced EGFR activity.
MTSV and ce2 cells were treated with a high concentration of EGF for up
to 2 h. At different time intervals, cells were solubilized and
the EGFR were immunoprecipitated. Receptor levels and phosphorylation states were then determined using Western blots. As shown in Fig. 5A, there was no
phosphorylation of the EGFR in MTSV cells in the absence of EGF. In
contrast, ce2 cells displayed a high constitutive level of EGFR
phosphorylation which was not affected by the addition of an
antagonistic EGFR antibody. In the case of both MTSV cells and ce2
cells, the addition of EGF caused an increase in Tyr(P) content of the
EGFR. However, receptor Tyr(P) levels rapidly decreased in the case of
MTSV cells, but remained elevated in the ce2 cells. A similar pattern
was observed for EGFR mass (Fig. 5A). Receptor levels
decreased following EGF addition for MTSV cells, but receptor levels
remained relatively constant in ce2 cells.
We analyzed the pattern of EGF-induced EGFR phosphorylation in terms of
both Tyr(P) to EGFR ratio and in terms of phosphorylated receptors per
cell (Fig. 5, B and C, respectively). This
analysis showed that the amount of Tyr(P) as a function of receptor
mass was actually depressed following EGF addition in cells
overexpressing erbB-2. The kinetics of EGFR phosphorylation in parental
MTSV cells showed a rapid rise followed by a subsequent decline,
characteristic of receptor desensitization. However, there was no sign
of EGFR desensitization in the ce2 cells.
When analyzed in terms of total phosphorylated receptors per cell, the
MTSV cells displayed a rapid loss of activated EGFR such that by 2 h following EGF treatment, their levels were similar to the
constitutive level of EGFR activation in ce2 cells (Fig. 5C). Addition of EGF to ce2 cells caused a persistently high
level of activated EGFR, evidently due to the suppression of receptor loss. We repeated these experiments using cells that express only 6-fold higher levels of erbB-2 (ce1 cells) and found an intermediate result in that the constitutive level of EGFR activation and the degree
of receptor loss was between that observed for ce2 and MTSV cells (data
not shown).
Our results indicate that the overexpression of erbB-2 inhibits
down-regulation of the EGFR. To test this idea directly, both MTSV and
ce2 cells were treated with 100 ng/ml EGF and at various times the
cells were solubilized and the EGFR levels were determined by Western
blot analysis. As shown in Fig. 6, the
loss of EGFR mass in both MTSV and ce2 cells was biphasic. The parental
MTSV cells lost half their EGFR mass in less than 2 h, whereas the ce2 cells lost the same amount in about 10 h. Thus down-regulation of the EGFR is inhibited in cells that overexpress erbB-2.
ErbB-2 Overexpression Inhibits EGFR Down-regulation at Multiple
Levels--
Ligand-induced down-regulation of the EGFR is regulated at
three distinct levels: endocytosis, endosomal sorting, and lysosomal targeting (39, 41, 47). It has been suggested previously that erbB-2
overexpression inhibits internalization of the EGFR (48). This, in
turn, could inhibit EGFR down-regulation. To test for this possibility,
we examined the kinetics of both intracellular and cell surface
accumulation of EGF. If overexpression of erbB-2 was inhibiting EGFR
internalization, then we should observe an increased amount of EGF at
the cell surface and a corresponding decrease in intracellular ligand.
MTSV and ce2 cells were incubated with 125I-labeled EGF at
37 °C. At various times, the relative amount of ligand either inside
the cell or at the cell surface was determined. As shown in Fig.
7 (bottom panel), ce2 cells
displayed a pronounced increase of 125I-EGF binding to the
cell surface relative to MTSV cells, especially at the longer time
points (>20 min). The 3-4-fold elevation in binding could not be
explained by the relatively small differences in EGFR levels between
MTSV and ce2 cells (see Fig. 1B). Paradoxically, we also
observed an increased level of intracellular 125I-EGF in
ce2 cells, but only after about 20 min incubation with ligand (Fig. 7,
top panel). The similar amount of internalized ligand in
both MTSV and ce2 cells at early time points is inconsistent with an
inhibition of internalization. The accumulation of intracellular 125I-EGF at longer incubation times suggests an inhibition
of lysosomal degradation.
Since a change at a single point in the EGFR trafficking pathway could
not explain the observed accumulation of both surface-associated and
intracellular EGF in ce2 cells, it seemed possible that erbB-2 overexpression could cause multiple alterations in EGFR trafficking. We
therefore examined the individual steps. The specific internalization rate was determined by incubating cells with radiolabeled EGF for 5 min, during which time surface-associated and internalized ligand was
measured. Fig. 8A shows that
overexpression of erbB-2 did not significantly alter the
internalization rate constant (ke) for the EGFR
(0.14 min
To assess the effect of erbB-2 overexpression on EGFR endosomal
sorting, we used a previously described technique that measures the
fraction of internalized receptors that are recycled (28). To measure
fractional recycling, cells were brought to steady state with different
concentrations of 125I-EGF. Surface-associated EGF was
removed with a mild acid strip and the relative amount of intact
versus degraded EGF which subsequently appeared in the
medium was measured. We have previously shown that the ratio of intact
versus degraded EGF indicates the fraction of internalized
ligand that is recycled versus targeted to lysosomes (28).
When we used this technique on MTSV and ce2 cells, we obtained the
results shown in Fig. 8D. The parental MTSV cells showed an
increase in fractional ligand recycling from 0.25 to 0.45 as the
intracellular ligand increased from 7 × 103 to 4 × 105 molecules per cell. This "saturation" of
endosomal sorting is very similar to what has previously been described
in fibroblasts (28). The ce2 cells displayed a very similar fractional
recycling pattern, but with a greater degree of recycling at all
intracellular ligand concentrations. This suggests that overexpression
of erbB-2 inhibits sorting of EGFR from endosomes to the lysosomes, and thus promotes recycling.
To directly test if EGFR transfer to the lysosomes was impaired by
erbB-2 overexpression, a kinetic analysis of 125I-EGF
degradation was done. Cells were incubated for 5 min with 125I-EGF followed by a chase in unlabeled medium. The
amount of intracellular 125I-EGF remaining at different
times was then determined. Under these conditions, almost all of the
125I-EGF lost from the cells is degraded ligand (49)
(results not shown). As shown in Fig.
9A, there was a lag of
approximately 15 min in MTSV cells before significant loss of
internalized ligand was observed. This lag generally corresponds to the
time necessary for internalized EGF to be transferred to lysosomes
(50). Thereafter, the 125I-EGF was lost with a
t1/2 of 32 min. In the case of ce2 cells, there was
a slightly longer lag before initiation of ligand loss, after which
125I-EGF was lost with a t1/2 of 53 min.
The ce1 cells showed an intermediate rate of ligand loss (t1/2 of 46 min, data not shown). These data
indicate that erbB-2 overexpression interferes with EGFR trafficking to
the lysosomes.
To confirm that differences in ligand degradation rates were due to
differences in intracellular trafficking, we used immunofluorescence to
follow the progression of the EGFR from the cell surface to the
lysosome. The lysosomes were labeled with a pulse of fluorescently labeled dextran followed by a 2-h chase. During this chase, the cells
were pulsed with Texas Red-labeled EGF to label the EGFR. The cells
were fixed at different time periods and colocalization of the EGF with
the lysosomes was determined using digital confocal imaging. As shown
in Fig. 9B, there was little colocalization of the EGF with
lysosomes following the initial 15-min pulse. However, there was
progressive colocalization of the two fluorescent labels during the 2-h
chase period. Colocalization reached its greatest extent in both MTSV
and ce2 cells at 60-80 min (Fig. 9B). The rate at which EGF
was transferred to lysosomes was somewhat slower in the ce2 cells as
was the extent of transfer (29 versus 35% for ce2 and MTSV
cells, respectively). Colocalization of EGF in lysosomes in both cell
types declined after 80 min and never involved the majority of the
ligand, probably because our protocol only labels a fraction of total
lysosomes. The apparent decrease in colocalization is most likely due
to EGF being transferred to newly formed lysosomes lacking
fluorescein-dextran combined with degradation of previously transferred
EGF. Nevertheless, these data confirm that transfer of EGF-containing
endosomes to lysosomal structures is slower in ce2 cells relative to
MTSV cells. Thus overexpression of erbB-2 results in an inhibition of
lysosomal trafficking of EGFR.
We previously reported that erbB-2 is down-regulated following
transactivation with EGF (20) and this was due to targeting of erbB-2
to the lysosomes. This is contrary to results obtained previously with
SKBR-3 cells where erbB-2 is not degraded in response to EGF (32).
However, SKBR-3 cells express very high erbB-2 levels which could
interfere with down-regulation. To test this hypothesis, we used a
mammary epithelial cell line that expresses similar levels of erbB-2 as
SKBR-3 cells due to the introduction of a transgene (2 × 106 molecules/cell) (35). We found that although EGF
induces erbB-2 degradation in the parental MTSV cells, it had little
effect on erbB-2 levels in the overexpressing ce2 cells. Thus
overexpression of erbB-2 inhibits erbB-2 down-regulation in a similar
fashion as EGFR overexpression inhibits EGFR down-regulation (27).
Inhibition of EGFR down-regulation is due to limiting levels of sorting
components in the endocytic pathway (27), but it is unclear whether
this is the case with erbB-2. Although expression levels of erbB-2 in
ce2 cells are high relative to the parental cell line, they are
approximately equivalent to the endogenous levels of EGFR
(approximately 6 × 105 at the cell surface for both).
Because EGF addition can reduce total EGFR levels in the parental cells
by >90%, it is unlikely that saturation of some common component of
the endocytic pathway (such as coated pits) is responsible for the lack
of erbB-2 down-regulation. EGFR-mediated transactivation also does not
appear limiting for erbB-2 down-regulation because the total number of
transactivated erbB-2 molecules in the overexpressing cells is much
greater than the parental lines. Because erbB-2 overexpression inhibits
the fraction of receptors undergoing down-regulation, but not the absolute number, it appears likely that a step downstream of
transactivation is rate-limiting for erbB-2 degradation.
It is currently unclear whether transactivation causes erbB-2
internalization. Several groups have analyzed erbB-2 endocytosis by
using labeled antibodies or EGFR extracellular domain chimeras, and
have come to different conclusions regarding whether erbB-2 undergoes
activation-induced internalization (31, 51-54). We have measured the
specific internalization rate of erbB-2 using a radiolabeled antibody
and have not observed any effect of EGF addition (0.043 min We also found that erbB-2 overexpression had a striking effect on
regulation of the EGFR. Not only did high levels of erbB-2 induce
constitutive activation of the EGFR, but they also inhibited EGF-induced down-regulation of the EGFR. Although constitutive activation of erbB-2 has previously been described as an effect of
erbB-2 overexpression, a corresponding effect on EGFR phosphorylation has not been previously reported. The high number of EGFR found in
human mammary epithelial cells and the consequent increased sensitivity
of our assays could account for our ability to observe this effect. The
constitutive phosphorylation of EGFR was quite significant, and on a
per cell basis, was higher than that observed following chronic
treatment with EGF (Fig. 5C). Monoclonal antibodies that
blocked either ligand binding (45) or receptor homodimerization (46)
had no effect on this constitutive phosphorylation, indicating that it
was probably due to expression-driven heterodimerization with erbB-2.
The biological effects of this constitutive EGFR activation are
currently unknown.
An unexpected finding was that erbB-2 overexpression also inhibited
down-regulation of activated EGFR. This resulted in prolonged signaling
through the EGFR. The mechanism by which erbB-2 inhibits EGFR
down-regulation is complex. We have found a decrease in EGF dissociation, an increase in recycling fraction, as well as an inhibition of lysosomal targeting. The overall effect of these changes
was to greatly enhance ligand-receptor stability, thus increasing the
number of activated receptors per cell. These changes are diagrammed in
Fig. 10. To determine which specific
changes were most important in increasing activated EGFR levels, we
calculated their relative contributions using the quantitative model
previously described (38). The 7-fold decrease in ligand dissociation
rates we observed would increase cell surface levels of activated EGFR only about 1.5-fold. This is because activated receptors are primarily lost by internalization, not dissociation (i.e.
ke > kd). The increase in the
time necessary to reach the lysosomes (from 15 to 20 min) would
increase intracellular pools of receptors 1.33-fold, but in combination
with the reduction in degradation rate constant (from 0.021 to 0.13 min
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
heparin binding EGF-like growth factor, amphiregulin, and betacellulin)
that directly bind and activate the receptor (12). Three other ligands,
heregulin, heregulin-2, and heregulin-3, bind to erbB-3 and/or erbB-4
(3, 5, 13). No ligand has been found that binds erbB-2, although both
EGF and heregulin can activate erbB-2 in-trans through ligand-induced
heterodimerization and subsequent tyrosine phosphorylation of erbB-2
(14-16). ErbB-2 can be directly phosphorylated by the activated
primary receptor (e.g. EGFR or erbB-4) (17). Alternately,
formation of the heterodimer induces a conformation change that
activates the intrinsic tyrosine kinase domain of erbB-2 (18-20).
Presumably, extensive interactions between EGF receptor family members
allows for diversification of signaling cascades (21, 22).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol and heated to 100 °C for 10 min. Equal amounts of protein from each sample was separated on a 5-7.5% polyacrylamide gradient gels and transferred to nitrocellulose. EGFR and erbB-2 were
detected by N-13 and 1917 polyclonal sera, respectively, using
125I-labeled protein A as described (20). The
concentrations and incubation times with 125I-labeled
protein A were in the linear range of the protein load of the gels. The
blots were analyzed by storage phosphor plates using the Bio-Rad G250
Molecular Imager. The Bio-Rad Molecular Analyst software package was
used to quantify the amount of radioactivity associated with each band.
8 M EGF-Texas Red-streptavidin
complex (Molecular Probes, Inc.) and chased for up to 105 min. Total
chase time following the initial fluorescein-labeled dextran treatment
was 2 h for all samples. Cells were fixed with 3.6%
paraformaldehyde and mounted in Prolong. Coverslips were viewed with a
Nikon inverted fluorescence microscope with a ×60, 1.4 N.A. oil
immersion objective. Sets of 3 images at 3 different focal planes
spaced 0.5 µm apart centered on the perinuclear endosomes were
acquired at 520 and 615 nm (for fluorescein and Texas Red,
respectively). The images (12 bit, 656 × 517) were acquired using
a Princeton Instruments cooled CCD camera attached to a Macintosh
workstation running Openlab software (Improvision, Inc). The image
triplets were deconvolved using nearest-neighbor subtraction (37). The
deconvolved image of the lysosomes (fluorescein) was then used to
generate a binary mask using grayscale values between 700 and 4095. This mask was then applied to the deconvolved image of the EGF (Texas
Red) to identify all lysosomal "objects" that contained EGF. The
integrated intensity of all of these objects was then taken as the
amount of EGF within lysosomal structures. A mask of the EGF image was
generated in the same way and applied to the EGF image to determine the
total integrated intensity of all EGF-containing objects within the
cell. The fraction of all EGF colocalized in lysosomes was then
calculated. At each time point, four random fields of cells were
analyzed which contained between 100 and 200 vesicles per field.
11 to 2 × 10
8 M for 3 h at 37 °C. The relative
amount of antibody associated with the cell surface was determined by
acid stripping (40) and the data was analyzed as described previously
(38). Scatchard analysis of EGF binding to cells used
125I-EGF at a specific activity of 1.6 × 106 cpm/pmol and an incubation period of 4.5 h at
0 °C using ligand concentrations from 1.7 × 10
11
to 1.7 × 10
8 M as described (40).
Specific internalization rates for the EGFR were determined as
described (40). Measurements were made using a ligand concentration of
10 ng/ml, and each rate constant determination was derived from a 5-min
incubation period with ligand. Specific internalization rates were
determined by plotting the integral surface-associated ligand against
the amount internalized, and the slopes were determined by linear
regression (40).
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression levels of erbB-2 and EGFR in MTSV
versus ce2 cells. A, MTSV and ce2
cells were brought to steady state with varying concentrations of
125I-labeled 4D5 antibody as described under
"Experimental Procedures." The amount of surface ( ) or total
(
) cell associated antibody associated with either ce2 (main
panel) or MTSV cells (inset) was determined following
acid stripping and is presented as a steady state plot (38).
B, binding of 125I-EGF to ce2 (
) or MTSV
(
) cells. Equilibrium binding at 0 °C was done as described under
"Experimental Procedures" and is plotted as a Scatchard plot. Lines
were generated by nonlinear regression.
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Fig. 2.
Distribution of erbB-2 and the EGFR at the
cell surface is unaffected by erbB-2 overexpression. Cells were
fixed and permeabilized and incubated with directly labeled erbB-2 or
EGFR antibodies. Images were then separately acquired in the
fluorescein isothiocyanate channel (left panels)
corresponding to the EGFR and the Texas Red channel (right
panels) corresponding to erbB-2. Arrows indicate
corresponding areas of the paired images.
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Fig. 3.
Activation of erbB-2 and EGFR in MTSV
versus ce2 cells. Cells were treated with or
without EGF for 5 and 10 min. Cells were also treated with 10 µg/ml
either monoclonal antibody 225 or 13A9 for 18 h. ErbB-2 was
immunoprecipitated from the cell extracts (top panel)
followed immunoprecipitation of the EGFR (bottom panel).
Phosphotyrosine (PY) levels were then determined by Western
blot analysis. Shown is a scan from a Bio-Rad Molecular Imager.
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Fig. 4.
ErbB-2 down-regulation is inhibited by erbB-2
overexpression. Levels of erbB-2 were assessed by Western blot
analysis of extracts of the indicated cell types treated with 17 nM EGF for 24 h (panel A). The Western blot
bands were quantified by molecular imager analysis (panel
B). The percentage of erbB-2 remaining after the 24-h EGF
treatment is shown as the average with standard deviation from three to
six separate experiments. Down-regulation kinetics of erbB-2 was also
measured over a shorter time course (panel C). Shown is the
average percentage of control erbB-2 levels that remain after EGF
treatment from three separate experiments.
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Fig. 5.
Kinetics of EGFR activation in MTSV and ce2
cells. A, cells were treated with 100 ng/ml EGF for the
indicated time or were treated with 10 µg/ml 225 monoclonal antibody
for 18 h. The immunoprecipitated EGFR was then separated by
electrophoresis, transferred to nitrocellulose, and probed with
anti-Tyr(P) (top panel). After visualization of the bands,
the blots were stripped and reprobed with EGFR antibodies (bottom
panels). B, the density of the bands shown in
panel A was determined by densitometry and the ratio of the
Tyr(P) to EGFR bands was then plotted as a function of EGF treatment
time for ce2 ( ) and MTSV (
) cells. C, the density of
the Tyr(P) bands shown in panel A as a function of EGF
treatment time is shown for ce2 (
) and MTSV (
) cells.
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Fig. 6.
EGFR down-regulation is inhibited by erbB-2
overexpression. EGFR receptor mass was assessed by Western blot
analysis of cells treated with EGF for varying times up to 24 h.
Western blot bands were quantified by using a Molecular Imager and
averages from three experiments are plotted as a percent of receptor
mass in untreated cells.
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Fig. 7.
Approach of EGF to steady state binding.
Cells were incubated with 1.7 nM 125I-EGF for
the time indicated and the relative amount of ligand either inside the
cell (top panel) or at the cell surface (bottom
panel) was determined for ce2 ( ) and MTSV (
) cells by acid
stripping.
1 ± 0.01 versus 0.12 min
1 ± 0.03 for MTSV and ce2 cells, respectively). The
kinetics of initial 125I-EGF binding to both MTSV and ce2
cells was also similar (Fig. 8B), which indicates that the
forward rate constant (ka) was the same. To
determine the dissociation rate constant (kd), the
cells were incubated with EGF for 5, 10, and 15 min followed by a chase
in a large excess of unlabeled EGF (to prevent rebinding of dissociated
125I-EGF). The amount of EGF lost from the surface is a
combination of ligand internalization and dissociation from the
receptor. Thus, the rate of 125I-EGF loss from the cell
surface is equal to kd + ke. Because ke is the same in the two cell types (Fig.
7A), differences in loss will reflect kd.
As shown in Fig. 8C, loss of EGF was substantially slower
from the surface of ce2 cells as compared with MTSV cells (0.15 min
1 versus 0.23 min
1,
respectively). This did not change appreciably as a function of
incubation time. Subtracting the value of ke
measured in parallel experiments (0.135 min
1;
dashed line in Fig. 8C) yielded a value of
kd of 0.013 min
1 in ce2 cells and 0.10 min
1 in MTSV cells. Thus overexpression of erbB-2 appears
to cause a 7-fold reduction in the EGF dissociation rate constant,
which could partially explain the increased levels of EGF at the cell surface.
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Fig. 8.
Kinetics of EGF binding and recycling in ce2
and MTSV cells. A, internalization plot analysis of ce2
( ) and MTSV (
) cells was done over a 5-min period using 1.7 nM 125I-EGF as described under "Experimental
Procedures." Shown are the average of four experiments ± S.D.
B, surface binding of EGF to ce2 (
) and MTSV (
) cells
using 10 ng/ml 125I-EGF. Shown is the average of four
experiments ± standard deviation. C, loss of
125I-EGF from the surface of ce2 (
) and MTSV (
)
cells. The cells were incubated with 1.7 nM EGF for 5, 10, and 15 min. Cells were rinsed and incubated with 1.7 µM
unlabeled EGF to prevent rebinding of dissociated ligand. The percent
of initially bound ligand remaining on the cell surface at the
indicated times was determined by acid stripping. Shown are the average
results of all three data sets ± S.D. The solid lines
were generated by nonlinear regression. The dashed line is
loss predicted from the effects of endocytosis alone. D,
fractional recycling of 125I-EGF from ce2 (
) and MTSV
(
) cells. Cells were brought to steady state with varying
concentrations of 125I-EGF. After removal of surface
ligand, the fraction of internalized ligand which recycled back into
the medium intact was measured as described under "Experimental
Procedures." Shown is the fraction of recycled ligand plotted against
the amount of internalized ligand at the beginning of the chase
period.
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Fig. 9.
Transport of EGF to lysosomes in MTSV
versus ce2 cells. A, either ce2 ( )
or MTSV (
) cells were pulsed for 5 min with 8 nM
125I-EGF and then chased with 170 nM unlabeled
EGF for the indicated times. The amount of intact ligand remaining in
the cells was determined following acid stripping. B, cells
were pulsed for 15 min with fluorescein-dextran to label the lysosomes.
The cells were then incubated with Texas Red-labeled EGF for 15 min and
chased for the indicated time. Total chase time for the
fluorescein-dextran was 120 min for all cells. The amount of EGF
colocalized with the dextran-labeled lysosomes was determined by image
analysis as described under "Experimental Procedures."
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and 0.045 min
1 with or without EGF,
respectively).2 However,
antibodies may not accurately reflect any EGF-stimulated change in
erbB-2 internalization. Rapid endocytosis is not necessary for
down-regulation, however, because constitutive internalization rates
are sufficiently quick. For example, if constitutively internalized erbB-2 are directed entirely to the lysosomes for degradation instead
of being recycled, their half-life would be approximately 15 min (38).
The absence of a major effect of EGF on erbB-2 internalization does
suggest that heterodimers between erbB-2 and the EGFR are not
sufficiently stable to be internalized as a complex.
1), intracellular pools would increase 2-fold. This is
in good agreement with our observed doubling of intracellular ligand
pools (Fig. 7). Fractional recycling from this pool increases from
approximately 30 to 45% in the erbB-2 overexpressing cells (Fig.
8D). In combination with the expanded intracellular pools,
this translates to a 3-fold higher net recycling rate. Because of the
much larger intracellular pool of EGFR relative to the cell surface,
this would raise surface-associated EGF about 3-fold. The lowered
dissociation rate would increase this further, to the 4-5-fold range
observed in our experiments. Thus altered postendocytic receptor
trafficking is mainly responsible for the enhanced levels of activated
EGFR.
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Fig. 10.
Model of the influence of erbB-2
overexpression on EGFR trafficking. Shown are the individual
trafficking steps analyzed and the fold effect of erbB-2 overexpression
(~2 × 106 molecules/cell). Of the 5 steps shown, 2 are inhibited (lysosomal fusion and receptor dissociation) and one is
stimulated (recycling). Ligand association and endocytosis are not
affected.
Yarden and colleagues (14, 48) have recently examined the effect of erbB-2 expression on EGFR behavior. They likewise found that erbB-2 overexpression decreased the rate of EGF dissociation from EGFR and delayed their inactivation (14, 48). Although our results are in general agreement with their findings, we did observe some differences. For example, we could find no evidence for an inhibition of EGFR internalization due to erbB-2 overexpression (Fig. 8A). We also found no evidence that erbB-2 overexpression alters EGFR affinity at 0 °C (Fig. 1B) or potentiates signaling through the EGFR (Fig. 5B). The different results are probably due to cell-specific factors and our use of more specific receptor trafficking assays. Nevertheless, we confirm that erbB-2 overexpression increases signaling through the EGFR primarily by delaying receptor inactivation.
The effect of erbB-2 overexpression is to inhibit specific sorting steps in the endocytic pathway. Mechanistically, the simplest explanation for this would be competition between erbB-2 and the EGFR for limiting sorting components in the endocytic pathway. There is strong sequence similarity between erbB-2 and the EGFR in the regions that have been identified as involved in postendocytic trafficking (29, 47). It has also been proposed that heterodimer formation with erbB-2 could alter the trafficking of EGFR (48). However, the fraction of EGFR involved in heterodimer formation is unknown. It is also uncertain whether the stability of the heterodimers is sufficient to affect receptor trafficking. Experiments are currently in progress to evaluate the role of heterodimer formation in EGFR trafficking.
Although the effects of erbB-2 overexpression on EGFR basal activation
and down-regulation were unexpected, they are not inconsistent with the
emerging view of the erbB family as a highly interactive group of
receptors that coordinately regulate signal transduction (2, 55, 56).
Our findings add another layer of complexity to the role of erbB-2
overexpression in breast cancer. Reciprocally, overexpression of the
EGFR may alter the normal regulation of erbB-2. It thus appears that
regulation of signaling from a single erbB family receptor cannot be
completely understood without considering its role as a member a highly
interactive signaling group.
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ACKNOWLEDGEMENT |
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We thank Joyce Taylor-Papadimitriou for the MTSV, ce1, and ce2 cells.
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FOOTNOTES |
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* This work was supported in part by U. S. Army Breast Cancer Research Program Grant DAMD17-94-J-444 and National Science Foundation Biotechnology Program Grant BES-9421773.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. Section 1734 solely to indicate this fact.
Recipient of a predoctoral fellowship from the U. S. Army
Breast Cancer Research Program.
§ To whom all correspondence should be addressed: Dept. of Pathology, University of Utah, Salt Lake City, UT 84132. Tel.: 801-581-5967; Fax: 801-581-4517; E-mail: Wiley{at}path.med.utah.edu.
2 R. Worthylake, L, K. Opresko, and H. S. Wiley, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: EGF, epidermal growth factor; Tyr(P), phosphotyrosine; EGFR, EGF receptor.
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