The ErbB signaling network consists of four
transmembrane receptor tyrosine kinases and more than a dozen ligands
sharing an epidermal growth factor (EGF) motif. The multiplicity of
ErbB-specific ligands is incompletely understood in terms of signal
specificity because all ErbB molecules signal through partially
overlapping pathways. Here we addressed the action of epiregulin, a
recently isolated ligand of ErbB-1. By employing a set of
factor-dependent cell lines engineered to express
individual ErbBs or their combinations, we found that epiregulin is the
broadest specificity EGF-like ligand so far characterized: not only
does it stimulate homodimers of both ErbB-1 and ErbB-4, it also
activates all possible heterodimeric ErbB complexes. Consistent with
its relaxed selectivity, epiregulin binds the various receptor
combinations with an affinity that is approximately 100-fold lower than
the affinity of ligands with more stringent selectivity, including EGF.
Nevertheless, epiregulin's action upon most receptor combinations
transmits a more potent mitogenic signal than does EGF. This remarkable
discrepancy between binding affinity and bioactivity is permitted by a
mechanism that prevents receptor down-regulation, and results in a
weak, but prolonged, state of receptor activation.
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INTRODUCTION |
Various biological processes are controlled by intercellular
interactions that are mediated by polypeptide growth factors. Examples
include embryonic development, neuronal functions, hematopoiesis, and
pathological situations, like wound healing and malignant transformation. The mechanism transmitting extracellular signals ultimately starts with binding of the growth factor to a cell surface
receptor, that in many cases carries an intrinsic tyrosine kinase
activity (1). These receptors fall into several subgroups sharing
structural and functional characteristics. Each subgroup of receptors
specifically recognizes a family of structurally homologous growth
factors. Perhaps the most striking multiplicity of related growth
factors is exemplified by the epidermal growth factor
(EGF)1 family of
molecules (2). This six
cysteine-containing motif of 45-60 amino acids is shared by all
members of the family, and it functions as the receptor binding portion
of the molecule. Currently there are four known receptors for EGF-like
ligands, constituting the ErbB subgroup of receptor tyrosine kinases
(also known as HER, or type I receptor tyrosine kinases (3)). Whereas ErbB-1 binds many ligands, including EGF, transforming growth factor
(TGF
), and amphiregulin, both ErbB-3 and ErbB-4 bind to a family
of isoforms, collectively known as neuregulins (also called Neu
differentiation factors, heregulins, glial growth factors, and
acetylcholine receptor inducing activity) (4). A related group of
molecules, termed NRG2, binds to the same two receptors (5-7), and a
third molecule, NRG3, exclusively binds to ErbB-4 (8). Two other
ligands, betacellulin (9), and the heparin-binding EGF-like growth
factor (10, 11) bind to both ErbB-1 and ErbB-4. Interestingly, the most
oncogenic member of the ErbB family, ErbB-2, binds none of the EGF-like
ligands with high affinity. However, recent studies indicate that
ErbB-2 functions as a shared low affinity receptor that binds the
apparently bivalent EGF-like ligands with low affinity, once they are
presented by either one of the high affinity receptors (12).
Despite shared receptor specificity, it is clear that the multiple
EGF-like ligands play distinct physiological roles: gene targeting
experiments showed that loss of function of ErbB-1 (13-15) more
severely impairs embryonic development than inactivation of one of its
ligands, TGF
(16). On the other hand, targeting of either neuregulin
(17), ErbB-2 (18), or ErbB-4 (19), resulted in the same embryonic
cardiac defect, indicating that activation of an ErbB-2/ErbB-4 receptor
combination is exclusively mediated by neuregulin in the developing
heart. That ligand multiplicity related to tissue-specific expression
is suggested by distinct spatial and temporal patterns of expression of
the various ligands (reviewed in Ref. 2), and also by experiments with
transgenic mice demonstrating tissue selectivity of specific ErbB-1
ligands (20). Part of the physiological selectivity of ligands with shared receptors may be attributed to their domains that flank the
EGF-like motif, including the presence of heparin-binding sites,
sugars, and specific protein motifs.
In this study we addressed the functional identity of epiregulin, a
recently identified ligand of ErbB-1 (21, 22). Like TGF
, this ligand
was originally isolated from the medium of transformed fibroblasts, and
its transmembrane precursor carries only short sequences that flank the
EGF-like motif. Epiregulin expression is relatively restricted; except
for macrophages and placenta, other human tissues contain very low or
no epiregulin transcripts, but most types of epithelial tumors are
characterized by high expression of the growth factor (23). Although
epiregulin competed with EGF on the binding to ErbB-1, it displayed
relatively low affinity to ErbB-1-overexpressing cells (21). On the
other hand, the factor displayed dual biological function in
vitro: it stimulated proliferation of fibroblasts, smooth muscle
cells, and hepatocytes, but inhibited growth of several tumor-derived
epithelial cell lines (21). These observations, and the emerging
broader than expected specificity of EGF-like ligands to ErbB proteins
(reviewed in Ref. 24), prompted us to analyze the selectivity of
epiregulin to ErbB proteins. Here we report that epiregulin is a
pan-ErbB ligand that activates all ligand-stimulatable combinations of ErbB proteins with variable efficiency. Strikingly, in a model cellular
system, epiregulin more potently activates mitogenesis than does EGF,
although the affinity of EGF to ErbB-1 is approximately 100-fold
higher. This superiority of epiregulin is independent on the presence
of other ErbB proteins, and appears to result from a relatively
inefficient mechanism of receptor inactivation.
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EXPERIMENTAL PROCEDURES |
Materials, Buffers, and Antibodies--
A recombinant form of
NDF-
1177-246 was kindly provided by Amgen (Thousand
Oaks, CA). Human recombinant EGF and TGF
were purchased from Sigma.
Radioactive materials were purchased from Amersham (Buckinghamshire,
United Kingdom). IODO-GEN and BS3 were from Pierce. A
monoclonal antibody to the ErbB-2 protein, mAb L26 (25), was used to
stimulate ErbB-2. A monoclonal anti-phosphotyrosine antibody (PY-20,
Santa Cruz Biotechnology) was used for Western blot analysis. A mAb to
the active form of MAPK (doubly phosphorylated on both tyrosine and
threonine residues of the TEY motif) (26) was a gift from Rony Seger.
Binding buffer contained Dulbecco's modified Eagle's medium with
0.5% bovine serum albumin and 20 mM HEPES. Solubilization
buffer contained 1% Triton X-100, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1.5 mM EGTA, 1.5 mM
MgCl2, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, aprotinin (0.15 trypsin
inhibitor unit/ml), and 10 µg/ml leupeptin.
Peptide Synthesis--
Epiregulin was synthesized on an Applied
Biosystems (ABI) model 431 peptide synthesizer fortified with UV
feedback monitoring at 301 nm, and using Fmoc
(9-fluorenylethoxycarbonyl-)-Rink amide AM resin. Only the EGF-like
domain of the murine epiregulin (21) was synthesized. The conventional
ABI monitor previous peak algorithm was employed up to five times with
a cut-off of 3.5% of the first deprotection. A secondary deprotection
was performed and followed by double coupling. Acetic
anhydride/1-hydroxybenzotriazole capping was utilized at the end of
each coupling, followed by washing with 1:1
trifluoroethanol/dichloromethane. The peptide was deprotected and
removed from the resin as described (27), with the following modifications: methoxyindole (2%) was added to reagent K, and the
reaction time was changed to 3.5 h. Small quantities of the reduced peptides were purified by reverse-phase high performance liquid
chromatography and examined by matrix-assisted laser desorption ionization mass spectral analysis. The crude reduced protein was dissolved in a Tris-HCl buffer, pH 6.0, containing guanidium HCl (6 M) and diluted to a concentration of 0.06 mg/ml in
methionine-containing buffer (10 mM) that included 1.5 mM cystine, 0.75 mM cysteine, and 100 mM Tris, pH 8.0. The mixture was stirred for 48 h at
4 °C, and the oxidized protein isolated on a C-4 VYDAC 10 micron
preparative column (22 × 250 mm) using a 0.1% trifluoroacetic
acid/water/acetonitrile gradient. The oxidized protein was lyophilized
and characterized by mass spectrometry and amino acid analysis, and
shown to be homogeneous. Electrospray mass spectrometry was used to
verify the mass of the synthetic peptide.
Cell Lines--
MDA-MB453 cells were purchased from the American
Type Culture Collection (Rockville, MD). The Chinese hamster ovary
(CHO) cell lines expressing various ErbB proteins or their combinations were described previously (28). The establishment of a series of
interleukin 3 (IL-3)-dependent 32D myeloid cells expressing all combinations of ErbB-1, ErbB-2, and ErbB-3 has been described (29).
To generate an ErbB-4-overexpressing derivative of 32D cells, we used
an LTR-erbB-4 expression vector that was electroporated into
32D cells as described (30). Cell lines co-expressing ErbB-2 or ErbB-3,
together with ErbB-4, were established by transfection of the pLXSHD
reteroviral vector (31), directing ErbB-4 expression, into ErbB-2- or
ErbB-3-expressing cells (D2 and D3, respectively) by using
electroporation (Bio-Rad Genepulser, set at 400 volts and 250 millifarad). After a 24-h long recovery, cells were selected for 4-5
weeks in medium containing histidinol (0.4 mg/ml). Clones expressing
the two receptors were identified by using Western blotting, and
isolated by limiting dilution. Due to differences in promoter potency,
the selected cell line that singly expresses ErbB-4 (E4 cells)
contained approximately 10-12-fold more ErbB-4 molecules than cell
lines expressing the combinations of ErbB-4 with ErbB-2 (D24 cells) or
with ErbB-3 (D34 cells). A cell line expressing only approximately
5 × 104 ErbB-4 molecules per cell was established by
using previously described procedures (29) and denoted D4.
Radiolabeling of Ligands, Covalent Cross-linking, and Ligand
Binding Analyses--
Growth factors were labeled by using IODO-GEN as
described (32). The specific activity was approximately 5 × 105 cpm/ng. For covalent cross-linking analysis, cells
(106) were incubated on ice for 1.5 h with
125I-EGF, 125I-NDF-
1, or
125I-epiregulin (each at 100 ng/ml). The chemical
cross-linking reagent BS3 was then added (1 mM), and after 90 min on ice, cells were pelleted and
solubilized in solubilization buffer. For analyses of ligand displacement with 32D cells, 106 cells were washed once
with binding buffer, and then incubated for 2 h at 4 °C with a
radiolabeled ligand (1 ng/ml) and various concentrations of an
unlabeled ligand in a final volume of 0.2 ml. Nonspecific binding was
determined in the presence of a 100-fold molar excess of the unlabeled
ligand. To terminate ligand binding, each reaction tube was washed once
with 0.5 ml of binding buffer and loaded on top of a 0.7-ml cushion of
bovine serum. The tubes were spun (12,000 × g, 2 min)
to remove the unbound ligand. Ligand displacement from CHO cells was
analyzed with cell monolayers grown in 24-well dishes. Monolayers were
washed once with binding buffer and then incubated for 2 h at
4 °C with 1 ng/ml of the radiolabeled ligand, along with increasing
concentrations of an unlabeled growth factor. Then, cells were washed
three times with ice-cold binding buffer. Labeled cells were lysed for
15 min at 37 °C in 0.5 ml of 0.1 N NaOH solution
containing 0.1% sodium dodecyl sulfate, and the radioactivity was
determined. Nonspecific binding was calculated by subtracting the
binding of radiolabeled ligands to untransfected CHO cells, or by
performing the binding assays in the presence of a 100-fold excess of
the unlabeled ligand.
Receptor Down-regulation Assay--
Ligand-induced receptor
down-regulation was measured as follows: cells grown in 24-well plates
were incubated at 37 °C for up to 90 min without or with various
ligands in binding buffer. The cells were then put on ice, rinsed twice
with binding buffer, and surface-bound ligand molecules removed by
using a 7-min long incubation in 0.5 ml of solution of 150 mM acetic acid, pH 2.7, containing 150 mM NaCl
(33). The number of ligand-binding sites that remained exposed on the
cell surface was then determined by incubating cells at 4 °C with
radiolabeled EGF (20 ng/ml) for 90 min.
Lysate Preparation and Western Blotting--
For analysis of
total cell lysates, gel sample buffer was added directly to cell
monolayers or suspensions. For other experiments, solubilization buffer
was added to cells on ice. The adherent CHO cells were scraped with a
rubber policeman into 1 ml of buffer, transferred to microtubes, mixed
harshly, and centrifuged (10,000 × g, 10 min at
4 °C). Samples were resolved by gel electrophoresis through 7.5%
acrylamide gels, and electrophoretically transferred to nitrocellulose
membranes. Membranes were blocked for 2 h in TBST buffer (0.02 Tris-HCl buffered at pH 7.5, 0.15 M NaCl, and 0.05% Tween
20) containing 1% milk, blotted for 2 h with 1 µg/ml primary
antibody, washed, and reblotted with 0.5 µg/ml secondary antibody
linked to horseradish peroxidase. Immunoreactive bands were detected
with an enhanced chemiluminescence reagent (Amersham Corp.).
Cell Proliferation Assays--
Cells were washed free of IL-3,
resuspended in RPMI 1640 medium at 5 × 105 cells/ml,
and treated without or with growth factors or IL-3 (1:1000 dilution of
medium conditioned by IL-3-producing cells). Cell survival was
determined by using the MTT assay as described previously (29). MTT
(0.1 mg/ml) was incubated for 2 h at 37 °C with the analyzed
cells. Living cells can transform the tetrazolium ring into dark blue
formazan crystals, that can be quantified by reading the optical
density at 540-630 nm after lysis of the cells with acidic isopropyl
alcohol (34).
Cellular Differentiation Assays--
MDA-MB453 human mammary
cancer cells were plated in chamber slides (Lab-Tek) and then incubated
for 4 days in the absence or presence of ligands (50 ng/ml). Cells were
stained with oil red O, to visualize neutral lipids, as described
previously (35).
 |
RESULTS |
Induction of Cellular Differentiation and Tyrosine Phosphorylation
by Epiregulin in the Absence of ErbB-1--
The duality of
epiregulin's activity, namely, mitogenicity for some normal cells and
growth inhibition of epithelial tumor cells (21), may depend on
expression patterns of ErbB proteins, and thus may be explained by
epiregulin's interaction with receptor species other than ErbB-1. As
an initial test of this paradigm we examined the biological effect of
epiregulin on MDA-MB453 mammary tumor cells, which are devoid of the
EGF-receptor (ErbB-1), but can undergo phenotypic differentiation in
response to EGF-like ligands (36). Evidently, these cells underwent
growth arrest in response to long-term incubation with epiregulin, and
displayed phenotypic differentiation that included cell flattening, and appearance of neutral lipid-containing vesicles (Fig.
1A). EGF, at 1-200 ng/ml, was
inactive in inducing cell differentiation (data not shown), whereas
similar phenotypic alterations were induced also by NDF/neuregulin, a
ligand that interacts with both ErbB-3 and ErbB-4 (37). Consistent with
their biological effects on MDA-MB453 cells, both epiregulin and NDF,
but not EGF, were able to stimulate tyrosine phosphorylation of a
180-kDa protein at concentrations higher than 10 ng/ml (Fig.
1B). In conclusion, epiregulin action on the mammary
epithelial cell line we examined is independent of ErbB-1, and is
distinct from the effect of EGF.

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Fig. 1.
Induction of cellular differentiation and
tyrosine phosphorylation by epiregulin in mammary cells lacking
ErbB-1/EGF receptor. A, MDA-MB453 human mammary cancer
cells, that express no ErbB-1, were plated in chamber slides and then
incubated for 4 days in the absence (CONTROL) or presence of
epiregulin (50 ng/ml). Cells were stained with oil red O, to visualize
neutral lipids. Note the appearance of lipid droplets
(yellow) in epiregulin-treated cells. The magnification used
was × 600. B, following an overnight starvation,
106 MDA-MB453 cells were incubated for 2 min at 37 °C
without or with EGF, NDF- 1, or epiregulin, at the indicated
concentrations. Whole cell lysates were then prepared, resolved by gel
electrophoresis, and immunoblotted with an antibody to phosphotyrosine
(PY20). Bound antibody was detected by using a chemiluminescence-based
method.
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Epiregulin Is a Relatively Potent Stimulator of ErbB-1, but It Can
Transmit Biological Signals Also through Combinations of Other
Receptors--
To directly address the specificity of epiregulin to
ErbB receptors, we employed a previously described series of cell lines derived from the IL-3-dependent 32D myeloid cell line (29). Parental 32D cells express no ErbB protein, but as a result of transfection, the derivative lines singly express ErbB-1, ErbB-2, ErbB-3, or ErbB-4 (cell lines denoted D1, D2, D3, and E4,
respectively). Likewise, co-expression of two ErbB proteins established
cell lines with various combinations. For example, D13 cells co-express ErbB-1 and ErbB-3. Analysis of cell proliferation in the absence of
IL-3, but in the presence of increasing concentrations of epiregulin, EGF, or NDF-
1, revealed several interesting characteristics of epiregulin. First, the factor was more potent than EGF on cells singly
expressing ErbB-1 (D1 cells, Fig.
2A), as well as on cells expressing combinations of ErbB-1 with either ErbB-2 (D12 cells) or
ErbB-3 (D13 panel in Fig. 2A). Not only were the
dose-response curves of epiregulin shifted to the left, but this ligand
exerted in D1 and D13 cells a higher maximal response than EGF.
Consistent with the catalytic inactivity of ErbB-3 (38), and the
inability of ErbB-2 to bind any of the ErbB ligands with high affinity
(12), cells singly expressing ErbB-3 or ErbB-2 (D3 and D2 cell lines, respectively) did not respond to epiregulin (Fig. 2A). For
control, we verified that D2 cells are stimulatable by a mAb to ErbB-2 (25) (Fig. 2A), and D3 cells retained response to IL-3 (Fig. 3). Surprisingly, E4 cells that highly
overexpress ErbB-4 exhibited mitogenic response to both epiregulin and
EGF at concentrations above 5 ng/ml (Fig. 2A). In fact, the
response to EGF was reproducibly slightly higher than the mitogenic
effect of epiregulin on these cells. Due to the use of different
promoters, ErbB-4 expression in the E4 cell line was more than 10-fold
higher than that of ErbB-1 in D1 cells (see "Experimental
Procedures"). To address the possibility that epiregulin and EGF act
through ErbB-4 only when this receptor is overexpressed, we analyzed a
second cell line, D4, whose ErbB-4 expression is comparable with the
level of ErbB-1 expression in D1 cells. When tested on D4 cells, both ligands displayed mitogenic activity (Fig. 2B), along with
an ability to stimulate tyrosine phosphorylation (Fig. 2C).
Nevertheless, in terms of the maximal response to IL-3, both epiregulin
and EGF were more active on the ErbB-4-overexpressing cell line than on
the low expressor D4 cells, implying that the level of expression of
ErbB-4 affects the level of cell proliferation, but not ligand specificity.

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Fig. 2.
Proliferative responses of ErbB-expressing
derivatives of 32D myeloid cells to epiregulin and other ligands.
A, the indicated sublines of 32D cells were tested for cell
proliferation by using the MTT assay. D1, D2, D3, and E4 cells express
ErbB-1, ErbB-2, ErbB-3, and ErbB-4, respectively, whereas the other
cell lines co-express the corresponding two ErbB proteins. Cells were
deprived of IL-3 and plated at a density of 5 × 105
cells/ml in media containing serial dilutions of EGF (closed
squares), epiregulin (open squares), NDF- 1
(closed circles), or a monoclonal antibody to ErbB-2 (mAb
L26, open circles). The MTT assay was performed 24 h
later. Results are presented as fold induction over the control
untreated cells, and are the mean ± S.D. of four determinations.
Each experiment was repeated at least twice. Cells singly expressing
ErbB-3 (D3 cells) responded to none of the ligands we tested, but these
cells retained response to IL-3. B, D4 cells were tested for
cell proliferation by using the MTT assay as described above, except
that the indicated ligands were used at 100 ng/ml. For control, cells
were incubated in the absence of IL-3 or ligands. C,
ligand-induced tyrosine phosphorylation was analyzed in D4 cells by
incubating 106 cells without or with the indicated ligands
(each at 100 ng/ml). Following 2 min at 37 °C, whole cell lysates
were prepared and analyzed by immunoblotting with a mAb to
phosphotyrosine. Antibody detection was performed with a
chemiluminescence kit. Only the 180-kDa region of the blot is
shown.
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Fig. 3.
Ligand-dependent survival of
ErbB-expressing 32D cells in the absence of IL-3. The indicated
sublines of 32D cells were incubated for various time intervals at a
density of 5 × 105 cells/ml in the presence
(closed triangles) or absence of IL-3 (open
triangles), or with one of the following ligands, each at a
concentration of 100 ng/ml (except for D23 cells, that were treated
with EGF at 500 ng/ml to reflect the residual activity of this ligand
through the ErbB-2/ErbB-3 heterodimer (40, 41)): EGF (closed
squares), epiregulin (open squares), NDF- 1
(closed circles), and an antibody to ErbB-2 (mAb L26,
open circles). Cell survival was determined daily by using
the colorimetric MTT assay. The data presented are the mean ± S.D. of six determinations. Each experiment was repeated at least
twice.
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Although the effect of epiregulin on cells coexpressing ErbB-3 with
ErbB-1 (D13 cells) was higher than that of EGF, the response to NDF was
much higher, presumably because NDF better recruits ErbB-3 into
heterodimers (29, 39). Nevertheless, it is clear that also epiregulin
can recruit ErbB-3 into heterodimers, as reflected by its activity on
cells coexpressing a combination of ErbB-3 with either ErbB-2 (D23
cells, Fig. 2A) or ErbB-4 (D34 cells, Fig. 2A).
This ability of epiregulin distinguishes it from EGF, whose signaling
through the ErbB-2/ErbB-3 heterodimer occurs only at extremely high
concentrations (Fig. 2A and Ref. 40 and 41), and is
completely inactive in stimulating an ErbB-3/ErbB-4 heterodimer (Fig.
2A). Moreover, although EGF is slightly more potent than
epiregulin on ErbB-4-expressing cells (E4 panels in Figs.
2A and 3), epiregulin is superior when ErbB-2 is coexpressed with ErbB-4 (D24 panels in Figs. 2A and 3),
suggesting that this ligand is a better stimulator of the ErbB-2/ErbB-4
heterodimer. Taken together, the results presented in Fig. 2 imply that
relative to EGF, epiregulin is a better agonist of ErbB-1-containing
homo- and heterodimers. In addition, recruitment of ErbB-2, ErbB-3, and
ErbB-4 into heterodimers is more efficient in the case of epiregulin.
However, homodimers of ErbB-4 are better activated by EGF, and neither
homodimers of ErbB-2 nor ErbB-3·ErbB-3 complexes are stimulatable by
the two ligands.
These conclusions were further supported by long-term survival
experiments that are presented in Fig. 3. In this type of analysis cells are maintained in the absence of IL-3, but in the presence of
epiregulin (or other ligands) for several days, and their survival determined by using the MTT assay. Consistent with the dose curves of
the short-term mitogenic assay, at a saturating concentration epiregulin acted as a slightly better survival factor than EGF for
cells expressing ErbB-1, either alone or in combination with ErbB-3
(Fig. 3). Also consistent with the data of Fig. 2 was the observation
that EGF exerted a better survival activity on ErbB-4-overexpressing cells (E4 panel in Fig. 3). Interestingly, the presence of
ErbB-2, together with either ErbB-4 or ErbB-3, enabled epiregulin to
become a potent stimulator of cell proliferation, whereas EGF acted
primarily as a survival factor under these circumstances
(D23 and D24 panels in Fig. 3). Although survival
of cells coexpressing ErbB-3 and ErbB-4 was only slightly extended by
epiregulin (D34 panel in Fig. 3), this effect was higher
than that of EGF, reinforcing the relative preference of epiregulin for
heterodimeric receptor combinations.
Receptor Phosphorylation and MAP Kinase Activation by
Epiregulin--
Signaling by all EGF-like ligands is mediated by rapid
tyrosine phosphorylation of the respective receptors, and is ultimately funneled to the mitogen-activated protein kinase (MAP-kinase/Erk) pathway (42). The biological differences we observed between epiregulin, EGF, and NDF in subsets of 32D cells suggested that these
ligands may differ in signaling potency, and especially in their
ability to recruit the MAPK pathway. To analyze receptor phosphorylation and MAPK activation we probed blots of whole extracts, prepared from ligand-stimulated cells, with antibodies to
phosphotyrosine, or with a murine mAb that specifically recognizes the
active, doubly phosphorylated form of the ERK1 and ERK2 MAPKs (26). Surprisingly, the more mitogenic ligand of ErbB-1, epiregulin, exhibited weaker, but not less sustained, tyrosine phosphorylation of
proteins at the 180-kDa range corresponding to ErbB-1 in D1 cells (Fig.
4A). Although both EGF and
epiregulin stimulated MAPK phosphorylation in these cells, the patterns
of activation differed: a comparable increase in the activity of both
forms of the kinase was induced by epiregulin, whereas primarily the
lower form was activated after stimulation with EGF. Importantly,
although stimulation by EGF was more uniform at intermediate time
intervals (10-20 min), it completely disappeared after 30-60 min, at
which time the effect of epiregulin was still detectable. By contrast,
ErbB-4 phosphorylation was stronger with epiregulin than with EGF
(E4 panel in Fig. 4A), although the latter is a
slightly more efficient mitogen for the ErbB-4-overexpressing E4 cells
(Figs. 2A and 3). These differences between ErbB-1 and
ErbB-4 phosphorylation are cell-type independent, because they were
reproduced in a series of CHO cells expressing ErbB-1 (CB1 cells) or
ErbB-4 (CB4 cells), on a low background of the endogenous hamster
ErbB-2 (Fig. 4B). Analysis of 32D cells expressing a
combination of ErbB-2 with ErbB-3 (D23 cells) revealed that both forms
of MAPK were rapidly stimulated by epiregulin, but phosphorylation of
both ErbBs and MAPKs by EGF occurred only at very high ligand
concentrations, in agreement with recent reports (40, 41). The maximal
activation of MAPK in these cells was observed upon stimulation with
NDF, a ligand whose mitogenic effect was almost equivalent to that of
IL-3 (Fig. 3). A relatively sustained stimulation, and appearance of an
activated Erk-2, were observed upon activation of both D23 and D24
cells by their most potent ligand, namely, NDF, implying that these
features may characterize the stronger mitogenic signals. In
conclusion, the relative strength of mitogenic signals of EGF-like ligands better correlates with the duration of MAPK activation (especially the modification of Erk-2) than with the intensity of ErbB
phosphorylation.

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Fig. 4.
Kinetics of receptor phosphorylation and MAP
kinase activation by epiregulin and other ligands. The following
derivatives of 32D cells (D or E series of cell lines, panel
A), or CHO cells (CB series of cell lines, panel B),
were incubated at 37 °C for various time intervals (indicated in
minutes) with epiregulin (100 ng/ml), EGF (100 ng/ml, except for D23
cells that were treated with 500 ng/ml), or NDF- 1 (100 ng/ml): D1,
CB1, E4, and CB4 cells that singly express ErbB-1 or ErbB-4,
respectively, whereas D23, D24, and CB14 cells co-express a combination
of the corresponding two receptors. In the end of the incubation
period, whole cell lysates were prepared, cleared from debris and
nuclei, resolved by gel electrophoresis, and subjected to
immunoblotting with either an antibody to phosphotyrosine
(P-TYR), or with an antibody specific to the active doubly
phosphorylated form of MAPK, as indicated. Derivatives of CHO cells
were analyzed only with antibodies to phosphotyrosine. Signal detection
was performed by using a chemiluminescence kit.
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Low Affinity Interaction of Epiregulin with ErbB-1 and Other ErbB
Proteins--
The relatively weak stimulation of ErbB-1
phosphorylation by epiregulin (D1 panel in Fig. 4) suggested
low affinity interaction of epiregulin with ErbB-1 on D1 cells. This
possibility was addressed by employing two assays: covalent
cross-linking of a radiolabeled epiregulin to the surface of
ErbB-expressing 32D cell derivatives (Fig.
5), and ligand displacement analyses that
were performed with both 32D- and CHO-derived cell lines (Fig.
6). Epiregulin was radiolabeled with
125I and covalently cross-linked to the surface of 32D
cells by using the BS3 covalent cross-linking reagent. The
specificity of labeling by epiregulin was evident by the absence of
covalent cross-linking to ErbB-2 and ErbB-3, when these receptors were
singly expressed (D2 and D3 cells, respectively,
Fig. 5), and by displacement of radioactive epiregulin by a large
excess of the unlabeled ligand (data not shown). Interestingly, only a
very weak signal was observed when radiolabeled epiregulin was
covalently cross-linked to cells singly expressing ErbB-1, although
these cells displayed a strong cross-linking signal with
125I-EGF, whose specific radioactivity was comparable to
that of 125I-epiregulin (Fig. 5). A slightly stronger
signal was observed when cells coexpressing ErbB-1 and ErbB-2 were
analyzed, implying that the corresponding heterodimer cooperatively
interacts with epiregulin. The combination of ErbB-1 with ErbB-3 was
less efficient than that of ErbB-1 with ErbB-2, although the numbers of
ErbB-1 molecules on D1, D12, and D13 cells were similar. By contrast with ErbB-1, affinity labeling of ErbB-4 in the overexpressing E4 cell
line was very efficient in the case of both epiregulin and NDF, but
relatively weak labeling was observed with EGF (Fig. 5), in accordance
with receptor phosphorylation signals (Fig. 4A). Similar
observations were made with the D4 and CB4 cell lines (data not shown).
Interestingly, we were unable to detect covalent cross-linking of
epiregulin to cells coexpressing ErbB-3 with either ErbB-2 or ErbB-4
(D23 and D34 lanes in Fig. 5, note that ErbB-4
expression in D24 and D34 cells is approximately 10-fold lower than in
E4 cells), although these combinations reacted with NDF. By contrast,
the ErbB-2/ErbB-4 combination displayed a clearly detectable signal
with 125I-epiregulin, reflecting the relatively high
mitogenic response of D24 cells to epiregulin (Figs. 2A and
3).

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Fig. 5.
Covalent cross-linking of radiolabeled
epiregulin and other ligands to ErbB-expressing cells. The
indicated derivatives of 32D myeloid cells (106 cells per
lane) were incubated at 4 °C with 125I-EGF,
125I-epiregulin, or 125I-NDF- 1, each at 100 ng/ml. Following 90 min of incubation, the covalent cross-linking
reagent BS3 was added (1 mM final
concentration), and cell lysates prepared after an additional 1.5 h of incubation. Lysates were resolved by gel electrophoresis and
autoradiography. The location of a 180-kDa molecular weight marker is
indicated.
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Fig. 6.
Binding of epiregulin to cell lines
expressing specific ErbB proteins and their combinations. Ligand
displacement analyses were performed with derivatives of 32D myeloid
cells (D series of cell lines, panel A), or with CHO cells
expressing ErbB-1, ErbB-4, or their combinations (CB series of cell
lines, panel B). Either radiolabeled EGF (D1,
CB1, and the left-hand CB14 panel) or radioactive
NDF- 1 (D3, D4, D23, D24, D34, CB4, and the
right-hand CB14 panel) were used. Cells (106)
were incubated for 2 h at 4 °C with the radiolabeled ligand (1 ng/ml) in the presence of increasing concentrations of an unlabeled
epiregulin (open squares), EGF (closed squares),
or NDF- 1 (closed circles). Each data point represents the
mean (less than 10% variation) of two determinations.
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We then compared the capacity of epiregulin, as opposed to EGF, to
displace a cell-bound radioactive EGF from the surface of 32D or CHO
cells singly expressing ErbB-1 (D1 and CB1 cells, respectively). In
contrast with the mitogenic superiority of epiregulin for
ErbB-1-expressing 32D cells, the apparent binding affinity of
epiregulin, as reflected by the competition curves, was 2 orders of
magnitude lower than that of EGF (D1 and CB1
panels in Fig. 6, A and B). The presence of
ErbB-4 together with ErbB-1 did not significantly alter the ability of
epiregulin to displace EGF from the surface of CB14 cells (Fig.
6B), although epiregulin was able to displace, albeit with
low efficiency, a surface-bound 125I-NDF from
ErbB-4-expressing cells (D4 or CB4 cells, Fig. 6,
A and B). The results of ligand displacement
experiments that were performed with E4 cells were qualitatively
similar (data not shown). NDF displacement by epiregulin, or EGF, was
relatively efficient in D24 cells, but only weak competition was
detectable in D34 cells, consistent with the relative mitogenic potency
of epiregulin for D24 and D34 cells (Figs. 2A and 3). Thus,
affinity labeling (Fig. 5) and ligand competition analyses (Fig. 6)
imply that epiregulin binds cooperatively to the combination of ErbB-2
with ErbB-4. By contrast, only very weak competition between epiregulin
and NDF was observed in cells expressing ErbB-3, either alone or in combination with ErbB-2 or ErbB-4 (Fig. 6A), implying that
ErbB-3, unlike ErbB-4, does not cooperate with ErbB-2 in epiregulin
binding. This conclusion is consistent with the absence of a detectable cross-linking signal in D3, D13, and D23 cells (Fig. 5). In light of
this inference the results obtained with D23 cells are interesting because epiregulin displayed only a slightly better ability than EGF to
displace NDF from these cells, but its mitogenic activity was much
stronger than that of EGF (Figs. 2A and 3). In conclusion, receptor binding analyses indicated direct interaction between epiregulin and two receptors, ErbB-1 and ErbB-4. Although neither ErbB-3 nor ErbB-2 directly interact with epiregulin, the latter protein
significantly cooperates with both direct receptors of epiregulin.
Epiregulin-induced Down-regulation of ErbB-1 Is Defective--
The
superior mitogenic activity of epiregulin is analogous to that of
TGF
. This latter ligand of ErbB-1 is a better agonist than EGF when
tested in vitro in mitogenic, angiogenic, and motogenic assays (43, 44). Apparently, the relatively potent activity of TGF
,
whose binding affinity is almost identical to that of EGF, is due to
the absence of receptor down-regulation, which allows sustained
cellular activation (45). To examine the possibility that epiregulin's
superiority is due to a defective receptor inactivation process, we
exposed CB1 cells to epiregulin, EGF, or TGF
, and determined the
extent of disappearance of ErbB-1 from the cell surface. Evidently,
whereas EGF induced gradual disappearance of the surface-exposed
ErbB-1, neither epiregulin nor TGF
led to a significant change in
the level of surface ErbB-1 (Fig. 7), although at the concentrations we used both ligands were more mitogenic
than EGF (Fig. 2A, and data not shown). In experiments that
are not presented we found that the difference in receptor down-regulation was not due to defective endocytosis of epiregulin, whose rate of internalization was comparable to that of EGF and TGF
.
This observation raised the possibility that unlike EGF, which directs
ErbB-1 to degradation in lysosomes, epiregulin binding to ErbB-1 is
followed by receptor recycling, a route taken by the TGF
-driven
ErbB-1 (45, 46). This notion was supported by an experiment that tested
the effect of monensin, a well characterized inhibitor of receptor
recycling (47), on down-regulation of ErbB-1. In the presence of the
carboxylic ionophore both epiregulin and TGF
induced significant
down-regulation of ErbB-1, but this compound was ineffective on the
extensive down-regulation that was induced by EGF (Fig. 7, and data not
shown). In conclusion, the relatively strong biological action of
epiregulin through ErbB-1 may be due to continuous recycling of ErbB-1
back to the cell surface, thus allowing prolongation of epiregulin
signaling.

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Fig. 7.
Epiregulin-induced down-regulation of
ErbB-1. CB1 cells were grown to 80% confluence in 24-well plates,
rinsed with binding buffer, and incubated at 37 °C for the indicated
time intervals with one of the following ligands (each at 1 ng/ml):
epiregulin (closed squares), EGF (circles), or
TGF- (triangles). Sister epiregulin-treated cells were
similarly incubated, except that monensin (0.1 mM) was
added to the medium (open squares). Thereafter, monolayers
were rinsed twice with binding buffer, followed by a 7-min long
incubation with a low pH stripping buffer that removes surface-bound
ligands. The level of surface receptors, relative to the number of
ligand-binding sites present before down-regulation, was determined by
incubating cells for 1.5 h at 4 °C with radiolabeled EGF. The
results are expressed as the average fraction and range
(bars) of the original binding sites that remained on the
cell surface after exposure to the non-labeled ligand at
37 °C.
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DISCUSSION |
The evolutionary pathway of the ErbB signaling module, from worms
(48) and flies (49) to mammals, indicates that duplication of genes
encoding EGF-like ligands preceded multiplication of receptor-encoding
genes. Despite multiplicity of ligands and receptors, it is clear that
the downstream signaling mechanisms, namely a linear cascade leading to
MAPK activation, has been conserved. Thus, to gain functional
diversity, variations on the common theme of ligand-ErbB-MAPK evolved
throughout evolution. Examination of the interactions between one of
the mammalian ErbB ligands, epiregulin, and various combinations of the
four ErbB proteins uncovered two novel features of the evolved module,
that are schematically presented in Fig.
8. First, epiregulin is a
broad-specificity ligand that activates all eight ligand-stimulatable
combinations of ErbBs. Second, despite its extremely low affinity,
signaling by epiregulin is more potent than the bioactivity of a high
affinity ligand, namely, EGF. The mechanisms underlying these two
features, and their functional implications, are discussed below.

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Fig. 8.
Summary of epiregulin-receptor
interactions. The horizontal gray bar represents the
plasma membrane, and the 10 possible receptor dimers are shown
schematically as double circular structures. Specific ErbB proteins are
identified by their numbers. Two ErbB ligands, epiregulin
and EGF, are compared and their relative strength of signaling, as
revealed by using an IL-3-dependent series of cell lines,
are represented by the thickness of the corresponding arrows.
Broken arrows indicate very low bioactivity. For simplicity, the
ability of EGF to stimulate an ErbB-2/ErbB-3 heterodimer at very high
ligand concentrations is not represented. Note that no ligand binds
with high affinity to ErbB-2 homodimers. Because no 32D cell derivative
co-expressing ErbB-1 and ErbB-4 has been established, the data related
to this heterodimeric combination was inferred from experiments with
transfected CHO cells. All other receptor combinations were examined in
32D cell derivatives.
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Pan-ErbB Specificity of Epiregulin--
The four mammalian ErbB
proteins can form 10 homo- and heterodimeric complexes, including an
ErbB-3 homodimer, which is biologically inactive (29), and an ErbB-2
homodimer whose formation may be driven by receptor overexpression
(50), or by a transmembrane oncogenic mutation (51). Epiregulin can
signal through all but these two homodimeric combinations of ErbBs
(Fig. 8). This broad specificity is unique; no other EGF-like ligand
has such a wide selection of receptors. However, due to its broad
selectivity, none of the receptors of epiregulin binds it with high
affinity (Figs. 5 and 6).
One of the most surprising observations made in the course of the
present study is the ability of both epiregulin and EGF to activate
ErbB-4 when this receptor is singly expressed. This observation is
reminiscent of several recent reports that identified betacellulin (9)
and heparin-binding EGF (10, 11) as ligands of ErbB-4. Conceivably,
ErbB-1 and ErbB-4 share some structural features at their
ligand-binding sites, thus defining a subgroup of direct ErbB-1
ligands, including EGF, betacellulin, and heparin-binding EGF, but
excluding TGF
and amphiregulin, as ligands with dual receptor
specificity. Nevertheless, like all other interactions of epiregulin,
binding to ErbB-4 is characterized by very low affinity; the
corresponding dissociation constant is estimated to be in the
micromolar range (D4 and CB4 panels in Fig. 6).
The affinity of the other direct receptor of epiregulin, ErbB-1, is only 10-fold better, much higher than the nanomolar or lower apparent Kd of EGF or NDF binding to their direct receptors
(Fig. 6A). However, receptor combinations containing ErbB-1
and ErbB-4 are not the only receptors for epiregulin; although this
ligand does not interact with isolated components of the ErbB-2/ErbB-3 heterodimer, it can efficiently stimulate the respective receptor combination (D23 panels in Figs. 2A and 3). This
is probably mediated by an extremely low affinity of epiregulin to
ErbB-3 (D3 panel in Fig. 6), and by a cooperative effect of
the coexpressed ErbB-2. This effect of the ligand-less ErbB-2 is
extended to heterodimers containing the direct epiregulin receptors,
namely ErbB-1 and ErbB-4; cooperativity is exemplified by the
relatively strong binding of epiregulin to cells coexpressing ErbB-1
and ErbB-2 (but not to cells co-expressing ErbB-1 and ErbB-3, Fig. 5),
and by the ability of ErbB-2 to augment epiregulin binding to ErbB-4 (compare D4 and D24 panels in Fig. 6). This
binding effect is translated to enhanced signaling by the ErbB-2/ErbB-4
heterodimer relative to the ErbB-4 homodimer, and is apparently more
relevant to epiregulin than to EGF (compare E4 and D24
panels in Fig. 2A). The mechanism underlying signal
amplification by ErbB-2, a process that is significant to tumors
overexpressing this receptor, has been previously attributed to its
ability to decelerate dissociation of NDF and EGF from
ErbB-2-containing heterodimers (25, 52). The present study apparently
extends this mechanism to epiregulin.
How does epiregulin recognize all six heterodimeric complexes of ErbBs?
According to a ligand bivalence model (12), a notion supported by
recent affinity labeling studies (53), and by measurements of the
stoichiometry of ligand-receptor interactions in solution (54),
epiregulin carries a high affinity binding site whose specificity is
limited to ErbB-1 and ErbB-4. Another site that is structurally
distinct and may localize to the C-terminal half of the ligand, binds
with broad specificity but low affinity to other ErbB proteins,
including ErbB-1 and ErbB-4 (thus allowing homodimer formation), as
well as to ErbB-2 and ErbB-3, to confer heterodimer formation.
Nevertheless, as is the case with EGF and NDF, the putative
"low-affinity/broad-specificity" site of epiregulin apparently
prefers ErbB-2 over other receptors. This model explains how ErbB-2
augments epiregulin signaling through the ErbB-2/ErbB-3 and
ErbB-2/ErbB-4 heterodimers.
Mechanism of Signaling Superiority of Low Affinity Ligand-ErbB
Interactions--
In their original analysis of epiregulin
interactions with various cell types, Toyoda and collaborators (21)
found that this ligand was more mitogenic than EGF for several types of
normal cells, although epiregulin binding to cells of another type (the A-431 epidermoid carcinoma line) displayed a 10-fold lower affinity. Potentially, this discrepancy could be due to the different repertoires of ErbB proteins expressed on the surface of the different lines of
cultured cells that these authors examined. However, our studies with
engineered myeloid cells excluded this possibility, because epiregulin's superiority was retained also by cells singly expressing ErbB-1. In fact, our results extend the discrepancy between binding affinity and bioactivity to signaling through ErbB-4. Thus, epiregulin is a relatively potent stimulator of mitogenesis through both ErbB-1
and ErbB-4, despite being a very low affinity ligand of these two
receptors (D1 and E4 panels in Figs.
2A and 6A). The observation that ErbB-1
phosphorylation by epiregulin is weaker than the effect of EGF (Fig.
4A), implies that receptor activation is not the sole
determinant of signaling potency. Instead, differences in the
inactivation process may be critical: apart from differential recruitment of both tyrosine-specific phosphatases (55) and the
negative regulator c-Cbl (56), endocytosis of ligand-receptor complexes
is a major process that leads to inactivation of growth factor
signaling (reviewed in Ref. 57). Our initial studies of this aspect of
epiregulin's action indicated that this ligand, unlike EGF, mediates
limited, if any, down-regulation of ErbB-1 (Fig. 7). Additional
analyses raised the possibility that epiregulin undergoes
internalization, but its receptor rapidly recycles to the cell surface
(Fig. 7). Presumably, the very low affinity of epiregulin to ErbB-1 is
insufficient to direct this receptor to lysosomal degradation, either
because phosphorylation on tyrosine residues, which is essential for
rapid internalization (58), is relatively inefficient, or because the
ligand dissociates very rapidly. It is relevant that mutations of
another receptor, that stabilize ligand-receptor interactions at the
moderately acidic conditions of early endosomes, accelerate receptor
degradation and prevent recycling (59, 60), indicating that the
strength of ligand binding is critical for receptor routing. This
mechanism of epiregulin/ErbB-1 interactions is expected to promote a
relatively weak level of receptor activation, but due to receptor
recycling, repeated association-dissociation cycles may result in
prolongation of signaling. In support of this model, we observed an
overall lower activation of MAPK by epiregulin, but this was more
prolonged than in the case of EGF (D1 panel in Fig.
4A). Variations of the proposed mechanism have previously
been reported: in the case of TGF
, whose binding affinity is
comparable to that of EGF, the more rapid dissociation of the
ligand-receptor complex in an acidic endosomal compartment drives
ErbB-1 to recycling (45). This is contrasted with the lysosomal
destination taken by an EGF-bound ErbB-1. As a result, signaling by
TGF
is often more potent than that of EGF. An even closer example is
provided by a mutant of EGF that was engineered to enhance the
mitogenic potency of the growth factor for biotechnological
applications (61). This mutant achieved mitogenic superiority through a
combination of a 50-fold lower affinity, longer retention in culture
supernatants, and a very limited receptor down-regulation.
In addition to the question how wide is the relevance of our findings
to other growth factors whose binding affinities are very low, several
other interesting questions are left open. The exceptionally broad
specificity of epiregulin joins other observations that collectively
imply non-redundancy of the multiple EGF-like ligands (reviewed in Ref.
62). Evidently, each ligand differs from other members of its family by
a unique preference for certain ErbB proteins. This, however, does not
explain how different ligands mediate mitogenesis on some cells, but
differentiation (37), survival (63), or cell motility (10) on other
types of cells, although in all cases the MAPK pathway is recruited.
Even more difficult to reason is the inhibitory activity of epiregulin
on certain epithelial cell lines (21), because all of its receptors turned out to be stimulatory for myeloid cells (Figs. 2A and
3). Perhaps a cell type-specific component lying downstream of ErbBs determines the nature of cellular response. Another puzzling issue is
the contrast between the broad selectivity of epiregulin for ErbBs, and
its very limited pattern of expression (23). This and other questions
will require in vivo studies of epiregulin's physiological
role.
We thank Barry Ratzkin (Amgen, Thousand Oaks,
CA) for the recombinant NDF preparation, Rony Seger for monoclonal
antibodies to activated MAP kinase, and Leah Klapper for the L26
antibody to ErbB-2.