From the
Neu differentiation factor (NDF, or heregulin) and epidermal
growth factor (EGF) are structurally related proteins that bind to
distinct members of the ErbB family of receptor tyrosine kinases. Here
we show that NDF inhibits EGF binding in a cell type-specific manner.
The inhibitory effect is distinct from previously characterized
mechanisms that involve protein kinase C and receptor internalization
because it occurred at 4 °C and displayed reversibility. The extent
of inhibition correlated with both receptor saturation and affinity of
different NDF isoforms, and it was abolished upon overexpression of
either EGF receptor or ErbB-2. Binding kinetics and equilibrium
analyses indicated that NDF reduced the affinity, rather than the
number, of EGF receptors, through an acceleration of the rate of ligand
dissociation and deceleration of the association rate. On the basis of
co-immunoprecipitation of EGF and NDF receptors, we attribute the
inhibitory effect to the formation of receptor heterodimers. According
to this model, EGF binding to NDF-occupied heterodimers is partially
blocked. This model of negative trans-regulation within the ErbB family
is relevant to other subgroups of receptor tyrosine kinases and may
have physiological implications.
Many growth factors, cytokines, and neurotrophic factors
activate receptor tyrosine kinases. The activated receptors become
phosphorylated on tyrosine residues, thereby initiating intracellular
signaling, leading to cellular responses
(1) . One of the
subgroups of transmembrane tyrosine kinases includes the receptor for
EGF
Although NDF activates ErbB-2/HER-2 in mammary and neuronal cells,
no interaction occurs in ovarian and in fibroblastic cells that
overexpress this receptor
(11) . These and additional lines of
evidence raised the possibility that the interaction between NDF and
ErbB-2 involves another molecule that belongs to the family of EGF
receptor. Phosphorylation of ErbB-4 by heregulin has been reported
(12) . Moreover, by constructing soluble forms of ErbB proteins
we found that
Unlike ErbB-3 and ErbB-4 that bind
multiple isoforms of NDF ErbB-1 binds many different ligands
(5) . Binding of EGF induces receptor dimerization and increases
the intrinsic tyrosine kinase activity, which leads to receptor
autophosphorylation and tyrosine phosphorylation of various cellular
substrates
(16) . In addition to this activation pathway, EGF
receptor serves as a target for several negative regulatory mechanisms.
EGF itself is responsible for receptor down-regulation because of an
increase in the rate of receptor internalization and degradation. In
addition, several growth factors induce a rapid decrease in high
affinity EGF binding by two different pathways. The first one involves
activation of protein kinase C (PKC) and is observed after treatment
with bombesin and phorbol esters such as TPA
(17) . The second
pathway, induced by EGF, platelet-derived growth factor, fibroblast
growth factor, interleukin 1, and tumor necrosis factor
In
the present study we demonstrate a new pathway of trans-regulation of
EGF receptors. We have found that NDF can reduce the binding of
radiolabeled EGF in certain human tumor cells, but not in other cell
lines, even when the cells are maintained at 4 °C. The inhibitory
effect appears to be independent of PKC activation, it involves a
decrease in affinity, rather than number, of EGF receptors, and it
displays reversibility. On the basis of these and additional lines of
evidence, we propose a model that attributes the trans-regulatory
effect of NDF to heterodimerization of ErbB proteins.
MaterialsEGF (human, recombinant) was purchased from Boehringer Mannheim and
recombinant NDF preparations were from Amgen (Thousand Oaks, CA).
Iodogen and BS
To visualize surface-exposed EGF receptors we
employed an alternative assay that made use of covalent cross-linking
of radiolabeled EGF to its membrane receptor
(35) . Under normal
conditions this assay detected two protein bands upon resolving whole
cell lysates by gel electrophoresis (Fig. 6). Presumably, these
bands represent monomers and dimers of the ligand-receptor complexes
because they disappeared in the presence of high concentrations of
unlabeled EGF (Fig. 6), and they were immunoprecipitable by
antibodies to human EGF receptor (data not shown). However, the
identitification of the upper band as a receptor dimer remains open.
Exposure to NDF prior to the cross-linking step reduced labeling of
both receptor species, with no detectable change in their
electrophoretic mobilities (Fig. 6). Together with the data
presented in , our results implied that NDF did not affect
the number of EGF receptors, but reduced their binding affinities.
Most mammalian receptor tyrosine kinases belong to subgroups
of highly related transmembrane proteins. This multiplicity is not
found in insects, suggesting that it has been developed in order to
provide solutions for more complex physiological requirements in
mammals
(38) . The ErbB subgroup of receptors is one of the best
studied families, not only because it is widely expressed in
mesenchymal, neuronal, and epithelial cells, but also because members
of this family were implicated in the development of human cancer
(39) . As expected, the biological activities of ErbB proteins
display variation; whereas ErbB-1 delivers mitogenic signals, that are
strictly ligand-dependent, ErbB-2 delivers growth signals even in the
absence of a ligand
(40) , and NDF, which binds to ErbB-3 and
ErbB-4, can promote cellular differentiation
(12) . Despite this
functional heterogeneity, several lines of evidence indicate that ErbB
proteins retained an ability to cross-talk and thereby trans-modulate
incoming signals
(41) . In the present study we identified a new
trans-modulatory effect between NDF and EGF receptors and discuss the
potential biological significance of this molecular interaction.
Before dealing with the mechanism by which NDF inhibits EGF binding,
it is relevant to list the biochemical characteristics of this process.
1) Inhibition is partial and never exceeds 90% of maximal EGF
binding (Fig. 1).
2) The effect of NDF is cell type specific
and appears to be affected by the relative expression levels of ErbB
proteins (Fig. 5).
3) The extent of inhibition is proportional
to saturation of NDF receptors and correlates with ligand affinity of
different isoforms (Figs. 3 and 1 C).
4) The inhibitory
effect is independent of temperature (Figs. 1, A and
B) and requires no preincubation (data not shown).
5)
Simultaneous occupation of NDF receptors is essential for display of
reduced EGF receptor binding (Fig. 4).
6) Decreased EGF
binding is due to reduction in affinity (Fig. 7), rather than
number () of EGF receptors.
Because direct interaction
between NDF and EGF receptors was undetectable
(6, 8) the inhibitory effect of NDF cannot be caused by direct
competition on the ligand-binding site of EGF receptor. On the basis of
specific recovery of radiolabeled NDF in immunoprecipitates of EGF
receptors (Fig. 9) and the existence of heterodimeric complexes
between EGF receptors and either ErbB-2
(22, 23) or NDF
receptors
(13) , we favor the following model of
trans-regulation of ErbB-1 by NDF receptors. Depending on the relative
levels of expression of different ErbB proteins, NDF binding to its own
receptors, namely ErbB-3 and ErbB-4, induces their homodimerization, as
well as heterodimerization with ErbB-1 and ErbB-2. In the case of
ErbB-1, EGF binding to the predimerized receptor, which already exists
in a complex with a ligand-occupied NDF receptor, is partially
inhibited. Apparently, this is due to acceleration of the rate of EGF
dissociation, while the on rate of the heterodimerized receptor for the
second ligand binding event is reduced in comparison with that of
monomeric or homodimeric receptor species.
Although a direct proof
to this model cannot be obtained with the currently available methods,
it can explain all of the above described characteristics, and it is
consistent with observations that were made in related experimental
systems. For example, the proposed mechanism attributes lack of effect
of NDF on EGF binding in certain cell types to the quantitative
relationships between homo- and heterodimers of ErbB-1. Although
alternative explanations to the cell type specificity, such as
different expression levels of ErbB-3 and ErbB-4, cannot be excluded,
it is worth noting that receptor density was identified as a major
determinant of dimer formation in a theoretical model
(42) , and
in experimental systems involving homodimers of ErbB-2
(43) or
heterodimers of ErbB-1
(44) . Similarly, the model we suggest
can explain two characteristics of the inhibitory phenomenon,
sensitivity to low pH (Fig. 4) and lack of temperature dependence
(Fig. 1); both are landmarks of receptor dimerization
(45, 46) ,
The major feature of the proposed model
refers to the kinetics of binding of a second ligand to predimerized
receptors. We speculate that conformational changes that take place
within heterodimers of ErbB-1 result in partial blocking of EGF
receptors. It is interesting that according to a recent study, EGF
binding to a receptor in a dimer having one receptor already bound
occurs with lower affinity than the initial binding event
(47) .
Moreover, the lower affinity of binding to the second binding site was
proposed to be the reason for negatively curved Scatchard plots that
are often, but not always, observed in the case of EGF. Independent of
the exact molecular mechanism of inhibition of EGF binding by NDF, this
effect and especially its possible sensitivity to the expression
profile of ErbB proteins may have important physiological implications.
Potentially, this mechanism may act as a filter of incoming signals; by
blocking binding of a second growth factor, a cell may avoid excessive,
or even opposing, growth regulatory signals. Furthermore, if indeed
this process is mediated by heterodimer formation, then it may shed new
light on the frequent overexpression of ErbB proteins in certain human
adenocarcinomas
(39, 48) . Overexpression of a
particular ErbB receptor is expected to force heterodimerization of the
overexpressed receptor with other ErbB molecules, and thereby favor
binding of one ligand over others. This model may be relevant to the
growth advantage of ErbB-2 overexpressing clones of human
adenocarcinomas
(48) , but it leaves many open questions. For
example, it is unclear whether heterodimer formation is a random,
rather than a hierarchial process, that prefers certain ErbB
heterodimers over others. Another open question relates to the
relationships between homodimers and heterodimers and their relative
ligand binding affinities. Lastly, it will be interesting to examine
the relevance of our observations with ErbB proteins to other
sub-groups of receptors, such as the
Confluent monolayers of T47D cells
in 24-well dishes were incubated with 25 ng/ml of the indicated
ligands. Following 2 h at 4 or at 37 °C, the monolayers were washed
and further incubated for 90 min at 4 °C with 5 µg/ml of a
monoclonal antibody to EGF receptor (R1) and subsequently washed. The
amount of bound antibody, reflecting the level of surface-exposed EGF
receptors, was determined by incubating the cells at 4 °C for 90
min with radiolabeled rabbit antibodies to mouse immunoglobulin G, and
determination of the bound radioactivity. Control monolayers were
incubated with a first antibody to Kit/stem cell factor receptor and
their background binding was subtracted. Averages of triplicate
determinations and their standard errors are shown. The experiment was
repeated three times with the same results.
We thank L. Defize, J. Mendelsohn, M. Waterfield, M.
Guthman, and J. Blechman for monoclonal antibodies and E. Peles, S.
Lavi, Daniel Schindler, and N. Ben-Baruch for help and advice.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
Discussion
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
(ErbB-1), Neu/ErbB-2 (also called HER-2),
ErbB-3/HER-3
(2, 3) , and the recently discovered
ErbB-4/HER-4
(4) . In contrast with ErbB-1, for which many
ligands have been characterized
(5) , ligands that directly
interact with other ErbB proteins were not known until recently. The
observation in different cells of a secreted activity that stimulated
phosphorylation of ErbB-2 on tyrosine residues eventually led to the
isolation of cDNAs encoding novel EGF-related proteins
(6, 7, 8) . The 44-kDa rat factor, named Neu
differentiation factor (NDF), stimulated tyrosine phosphorylation of
ErbB-2 and induced synthesis of milk components (casein and lipids) in
certain breast carcinoma cell lines
(7) . The homologous human
factors, termed heregulins, were found to be mitogenic for other
mammary tumor cells
(8) . The NDF cDNA sequence predicted a
transmembrane glycoprotein precursor (pro-NDF) containing an EGF-like
domain, an immunoglobulin homology unit, and a variable length
cytoplasmic domain. At least 10 isoforms of NDF exist and they fall
into two groups,
and
, that differ in their EGF-like domains
and in receptor binding affinity
(9) . It has been previously
suggested that NDF isoforms are generated by alternative splicing and
perform distinct tissue-specific functions
(9, 10) .
isoforms of NDF specifically bind to the ErbB-3 and
ErbB-4 receptors, but not to a soluble ErbB-2-alkaline phosphatase
fusion protein
(13) . In agreement with these in vitro studies, when ectopically expressed, the full-length ErbB-3
(13, 14, 15) and ErbB-4
(13) receptors
conferred specific binding, as well as kinase activation, by the
various isoforms of the ligand.
, appears
to be independent of PKC activation
(18) , but it nevertheless
involves serine-threonine phosphorylation of the receptor
(19) .
Another mechanism of modulation of EGF binding involves
heterodimerization with the ErbB-2 protein. Subsequent to EGF binding,
ErbB-2 undergoes increased tyrosine phosphorylation
(20, 21) that is due to complex formation of ErbB-2 with the
ligand-occupied ErbB-1
(22, 23) . Heterodimer formation
appears to be affected by receptor overexpression, and it leads to the
appearance of EGF-receptors with very high affinity
(23) .
were from Pierce. All other chemicals were
purchased from Sigma unless otherwise indicated.
Cell Culture
T47D, A-431, MCF-7, and SKBR-3 cells were
obtained from the American Type Culture Collection (Rockville, MD).
MCF-7/ErbB-2, a transfected cell line, was established as described
earlier
(11) .
Establishment of MCF-7/ErbB-1 Cells
To establish
MCF-7 cells that overexpress ErbB-1, the human EGF-receptor cDNA was
digested with the restriction enzymes EcoRV and XhoI
and inserted into the mammalian expression vector pcDNA3-NEO
(Invitrogen, San Diego, CA). MCF-7 breast cancer cells were then
transfected with the resulting pcDNA3-NEO/ErbB-1 plasmid by
electroporation. Drug-resistant colonies were selected in medium
containing 0.7 mg/ml gentamycin, grown as pools, and assayed for EGF
receptor expression by I-EGF binding and Western
blotting.
Antibodies
Polyclonal antibodies against the
C-terminal portion of Neu/HER-2 (NCT) and against the cytoplasmic
portion of the EGF receptor (RK2) were generated as described
(24, 25) . Monoclonal antibodies against the
extracellular part of EGF receptor (528, R1, and 2E9) have been
previously described
(26, 27, 28) .
Buffered Solutions
Binding buffer contained
Dulbecco's modified Eagle's medium with 0.1% bovine serum
albumin. HNTG buffer contained 20 mM HEPES, pH 7.5, 150
mM NaCl, 0.1% Triton X-100, and 10% glycerol. Solubilization
buffer contained 50 mM Tris-HCl, pH 7.5, 150 mM NaCl,
1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1.5 mM EGTA, 1.5
mM MgCl, 2 mM sodium orthovanadate, 1
mM phenylmethylsulfonyl fluoride, aprotinin (0.15 trypsin
inhibitor unit/ml), and 10 µg/ml leupeptin.
Radiolabeling of Ligands
Human recombinant EGF and
human recombinant NDF-1
were labeled with
Iodogen (Pierce) as follows: 5 µg of protein in PBS was mixed in an
Iodogen-coated (1 µg of reagent) tube with Na-
I (1
mCi). Following 10 min at 23 °C, tyrosine was added to a final
concentration of 0.1 mg/ml, and the mixture was separated on a column
of Excellulose GF-5 (Pierce). The range of specific activity varied
between 2 and 5
10
counts/min/ng.
Chemical Cross-linking
Monolayers (10cells) of T47D cells were incubated on ice for 2 h with either
I-EGF (20 ng/ml) or
I-NDF-
1
(10 ng/ml). The
chemical cross-linking reagent BS
was then added (1
mM), and after 45 min at 22 °C cells were again kept on
ice and washed with PBS. Cell lysates were prepared and analyzed by gel
electrophoresis.
Lysate Preparation and Immunoprecipitation
For
analysis of total cell lysates, gel sample buffer was directly added to
cell monolayers. For other experiments, solubilization buffer was added
to the monolayer of cells on ice. The proteins in the lysate
supernatants were immunoprecipitated with aliquots of the protein
A-Sepharose-antibody complex for 1 h at 4 °C. Immunoprecipitates
were then washed three times with HNTG (1 ml each wash).
Ligand Binding Analysis
Monolayers of cells
(1-2 10
cells/well) in 24-well dishes were
washed once with binding buffer and then incubated with 5 ng/ml of
either
I-NDF-
1
or
I-EGF in the same buffer. The unlabeled ligand at
different concentrations was coincubated with a radiolabeled ligand for
2 h at 4 °C, and the cells were washed three times with ice-cold
binding buffer. Labeled cells were lysed in 0.5 ml of 0.1 N
NaOH, 0.1% SDS for 15 min at 37 °C and the radioactivity was
determined by using a
-counter. Nonspecific binding was determined
by the addition of 100-fold excess of the unlabeled ligand. Scatchard
analysis was performed using the computerized program LIGAND
(29) .
Analysis of Ligand Association
T47D cells in
24-well dishes were first preincubated for 1 h at 4 °C with
NDF-1
(25 ng/ml) or medium alone, and then
the association of radiolabeled EGF was examined at 4 °C by
incubating the cells for 0.5-10 min with 5 ng/ml of
I-EGF. The cells were rapidly washed thrice with binding
buffer, dissolved in 0.1 M NaOH, 0.1% SDS, and the
radioactivity was determined. The association data were analyzed by
determination of the ratio between the amount of ligand bound at full
saturation ( B
) and that at time t ( B
). Based on published theories of
ligand association
(30) , the term
ln
(1- B
/ B
), which is a measure
of the association rate, was calculated and presented as a function of
time.
Analysis of Ligand Dissociation
Confluent
monolayers of T47D cells in 24-well dishes were incubated for 2 h at 4
°C with 5 ng/ml I-EGF and then washed three times.
Dissociation was monitored by using two different protocols. According
to the first one, the cells were incubated in binding buffer, with or
without NDF-
1
(25 ng/ml), for various
periods of time at 4 °C, and the amounts of released and
cell-associated radioactive ligand were determined. Alternatively,
after washing unbound radiolabeled EGF, the cells were incubated for 5
min at 4 °C with high concentration of EGF (100 ng/ml), and then
dissociation was assayed in the presence or absence of NDF as described
above.
In order to investigate the functional
consequences of possible cross-talk between ErbB proteins, we used T47D
human mammary tumor cells, which express relatively high levels of
erbB-4
(4) , and analyzed binding of radiolabeled NDF and EGF.
Preincubation of cells with EGF at 37 °C exerted only a limited
effect on NDF- and
Isoforms of NDF Reduce EGF Binding to
Mammary Tumor Cells
1 binding that was measured at 4 °C after
removal of EGF. By contrast, the reciprocal experiment indicated that
NDF-
1
inhibited, in a
concentration-dependent manner, binding of
I-EGF
(Fig. 1A). To exclude the possibility that the observed partial
inhibitory effect was due to co-internalization of EGF receptors
together with NDF receptors, we performed the experiment also at 4
°C. The results presented in Fig. 1 B implied that
the effect of NDF on EGF binding was temperature-independent. Similar
to NDF-
1
, which contained only the
EGF-like domain, the full-length form of this ligand inhibited EGF
binding to T47D cells (Fig. 1 C). More important, an
isoform of NDF was also inhibitory, but less effectively than the
isoform (Fig. 1 C). Presumably, this difference was
due to an 8-10-fold reduced affinity of NDF-
in comparison
with
isoforms
(9) .
Figure 1:
Binding of EGF and NDF to
ligand-treated T47D cells. A, monolayers of T47D cells were
preincubated at 37 °C with the indicated concentrations of either
unlabeled EGF or unlabeled NDF-1. After 2 h the cells were washed
three times with binding buffer and incubated at 4 °C with the
radiolabeled form of the other ligand (each at 5 ng/ml). The amount of
specifically bound ligand was determined by washing the monolayers 2 h
later and subtracting the radioactivity that bound in the presence of
100-fold excess of the respective unlabeled ligand. B, T47D
cells were incubated at 4 °C with different concentrations of
either NDF-
1 or EGF. The incubation was performed for 2 h in the
presence of the other ligand in its radiolabeled form, namely
I-EGF (5 ng/ml) or
I-NDF-
1
(5 ng/ml). The
monolayers were then washed three times with binding buffer and
specific ligand binding was determined after subtraction of nonspecific
binding that was measured in the presence of 100-fold excess of the
corresponding unlabeled ligand. C, varying concentrations of
three isoforms of NDF, namely NDF-
1
( open circles), NDF-
1
( filled circles), and NDF-
1
( open squares) were individually incubated with 5 ng/ml
I-EGF for 2 h at 4 °C, and the cell-bound
radioactivity was determined as described above. Means of triplicate
determinations and the corresponding standard errors ( bars)
are shown in all panels. The binding analyses were repeated three
times.
The Inhibitory Effect of NDF Is Independent of Protein
Kinase C
Because activation of PKC by tumor promoting phorbol
diesters causes down-regulation of EGF binding
(31) , we
examined the involvement of PKC in the effect of NDF. Two lines of
evidence supported lack of mediation by PKC. First, the inhibitory
effect of TPA (approximately a 60% reduction in EGF binding after a 2-h
long incubation at 37 °C with 100 ng/ml TPA; data not shown), but
not the effect of NDF, was abolished when incubation was performed at 4
°C (Fig. 2 A). Second, depletion of PKC by performing
a long incubation with a relatively high concentration of TPA
(32) significantly reduced EGF binding, but did not abolish the
inhibitory effect of NDF (Fig. 2 B).
Figure 2:
EGF
binding after treatment with TPA or NDF. A, monolayers of T47D
cells were incubated for 2 h at 4 °C with radiolabeled EGF (5
ng/ml) in binding buffer ( CONTROL), or in buffer that
contained NDF-1
(25 ng/ml) or TPA (100
ng/ml). The cells were then washed, and the specifically bound
radioactivity was determined as described under ``Experimental
Procedures.'' Each data point represents the average and range
( bars) of a duplicate determination. B, T47D cells
were treated with TPA (600 nM) at 37 °C for 20 h in order
to down-regulate PKC. The control and TPA-treated cells were then
washed extensively and incubated for 1 h at 4 °C in the presence of
buffer alone or NDF-
1
(25 ng/ml). Specific
binding of radiolabeled EGF was then determined as described under
``Experimental Procedures.'' Averages and ranges
( bars) of duplicate determinations are shown. The experiments
were repeated twice with similar results.
In analogy with
NDF, the platelet-derived growth factor also can inhibit EGF binding in
PKC-depleted cells
(18) . However, the effect of
platelet-derived growth factor appears to be mediated by an
amplification mechanism because an almost maximal effect was observed
with platelet-derived growth factor concentrations as low as those
needed for 5% of maximal receptor occupancy
(33) . To examine
this aspect with NDF as a ligand, we compared the inhibitory effect on
EGF binding with the extent of receptor saturation by NDF. Evidently,
the concentration dependences of these two parameters displayed
parallelism at NDF concentrations below 1 nM (approximately
50% receptor saturation), but they displayed disparity at higher ligand
concentrations (Fig. 3).
Figure 3:
Concentration dependences of NDF receptor
saturation and inhibition of EGF binding. NDF displacement was measured
by a 2-h long incubation at 4 °C of T47D cells with radio-labeled
NDF-1
(5 ng/ml) in the presence of
increasing concentrations of unlabeled ligand. Likewise, binding of
radiolabeled EGF (5 ng/ml) was performed in the presence of increasing
concentrations of NDF-
1
. This was followed
by extensive washing of the cell monolayers and determination of bound
radioactivity. The results are expressed as means ± S.E. of
triplicate determinations. The experiment was repeated three times with
similar results.
Reversibility and Cell Type Specificity of the NDF
Effect
The observed partial correlation between receptor
occupancy and inhibition of EGF binding (Fig. 3), together with
the lack of dependence on temperature (Fig. 1), raised the
possibility that the mechanism of inhibition involved reversible
interactions at the cell surface. To address this mechanism we
attempted to remove surface-bound NDF by washing with a low pH solution
(34) and then assaying EGF binding. Fig. 4presents the
results of this experiment. Evidently, washing NDF-treated cells under
low pH conditions significantly reduced the inhibitory effect.
Figure 4:
Acid sensitivity of the inhibitory effect
of NDF on EGF binding. T47D cells were incubated with 25 ng/ml
NDF-1
at 4 °C for the indicated
periods of time. The cell monolayers were then washed extensively with
binding buffer and incubated at 4 °C for 5 min with 1 ml of either
binding buffer ( open squares) or 0.1% acetic acid solution
that contained 150 mM NaCl and 0.1% bovine serum albumin
( filled squares). These solutions were removed, and the
specific binding of
I-EGF was determined at 4 °C as
described in the legend to Fig. 3. Averages of triplicate
determinations and their standard errors (bars) are shown. The
experiment was repeated twice with similar
results.
The
reversible nature of the effect of NDF implied that different cells may
display a variable response due to differences in the number and
repertoire of their surface receptors. This paradigm was examined in a
series of human tumor cell lines. Unlike T47D and MCF-7 mammary tumor
cells, A-431 cells, which overexpress the EGF receptor (ErbB-1), and
SKBR-3 cells, which overexpress ErbB-2, displayed limited or no effect
of NDF on EGF binding (Fig. 5 A). Similarly, in N87 human
gastric cells that overexpress ErbB-2, we observed no inhibitory effect
of NDF (data not shown). The presumed dependence on the relative levels
of ErbB proteins was examined by establishment of two sub-lines of
MCF-7 cells that overexpress either ErbB-2 or ErbB-1 as a result of
cDNA transfection. Transfection of these cells with the corresponding
expression vector resulted in >10-fold increase in the expression of
ErbB-1 and ErbB-2, as revealed by Western blot analysis (Fig. 5,
B and C). As predicted, overexpression of either ErbB
protein in MCF-7 cells led to significantly smaller NDF inhibitory
effect in comparison with the parental cell line (Fig. 5, B and C).
Figure 5:
Cell
type specificity of the effect of NDF on EGF binding. A,
varying concentrations of NDF-1
were
incubated with
I-EGF (5 ng/ml) for 2 h at 4 °C with
various human cancer cell lines. The specific binding of labeled EGF
was then determined as described under ``Experimental
Procedures.'' The following cell types were used for binding
analyses: A431 human epidermoid carcinoma cells ( filled
circles), and the human breast cancer cell lines SKBR-3 ( open
squares), T47D ( open circles), and MCF-7
( triangles). B and C, the above experiment
was performed with MCF-7 cells and the derivative cell lines, denoted
MCF-7/ErbB-2 and MCF-7/ErbB-1, that were selected for overexpression of
ErbB-2 or ErbB-1, respectively. Averages of triplicate determinations
are shown, and the bars represent the standard errors.
Insets show the relative levels of ErbB-2 ( B) and
ErbB-1 ( C) expression in MCF-7 cells and the derived cell
lines, as determined by Western blotting of cell lysates with
polyclonal antibodies to ErbB-2 (NCT) or ErbB-1 (RK2). The experiments
were repeated twice with similar results.
The Number of Surface-exposed EGF Receptors Undergoes No
Change by NDF
Since the inhibitory effect of NDF occurred also
at 4 °C (Fig. 1 B) it was conceivable that
endocytosis and proteolytic degradation of EGF receptors were not
involved. To confirm this proposition we determined the level of
surface-exposed EGF receptors by using a binding assay of a monoclonal
anti receptor antibody to T47D cells. As control we incubated the cells
in the presence of EGF at either 4 or 37 °C and observed extensive
receptor down-regulation only at the elevated temperature
(). By contrast, the monoclonal antibody that we used
detected no change in surface expression of EGF receptors after
treatment with NDF.
Figure 6:
NDF effect on covalent cross-linking of
I-EGF to its receptor. Monolayers (10
cells)
of T47D cells were incubated at 4 °C for 2 h with
I-EGF (20 ng/ml) in the presence or absence of
NDF-
1
or 1 µg/ml unlabeled EGF ( Ex
EGF), as indicated. Covalent cross-linking was then performed by
using the bivalent reagent BS
(1 mM), and cell
lysates were prepared and subjected to gel electrophoresis. The
resulting autoradiogram is shown, and the location of a molecular
weight standard protein (myosin) is indicated. The experiment was
repeated twice.
NDF Reduces Binding Affinity of EGF Receptors by
Accelerating Ligand Dissociation Rate and Decelerating Its
Association
In order to further investigate the mechanism by
which NDF affected EGF receptors, we performed equilibrium
I-EGF binding assays in the presence or absence of NDF.
Fig. 7
shows that T47D cells exhibited saturable binding that
corresponded to a single population of ligand-binding sites, as
reflected in a linear Scatchard curve
(36) . Co-incubation with
NDF-
1 resulted in reduction in EGF binding, due to an increase in
the apparent dissociation constant with little effect on the number of
EGF receptors. Thus, the calculated number of receptors in the absence
of NDF was 5
10
molecules/cell, and they displayed
a dissociation constant ( K
) of 1.8
nM, whereas in the presence of NDF these parameters were 5.2
10
and 5.0, respectively.
Figure 7:
Scatchard analysis of I-EGF
binding to NDF-treated T47D cells. Monolayers of T47D cells were
incubated for 2 h at 4 °C with different concentrations of
I-EGF in the presence ( filled symbols) or
absence ( open symbols) of NDF-
1
(25 ng/ml). Binding results were analyzed by the Scatchard method
and also by plotting saturation curves ( inset). Triplicate
determinations of total binding were performed, and their means are
given. Nonspecific binding that was determined in the presence of 1
µg/ml of unlabeled ligand was subtracted. Similar results were
obtained in three other experiments.
Affinity constants
obtained under equilibrium conditions reflect both the rates of ligand
association ( k) and dissociation
( k
). It was, therefore, necessary to determine
if NDF preferentially affected one of these processes. The kinetics of
EGF association with T47D cells in the presence or absence of
NDF-
1 (25 ng/ml) were determined at 4 °C in order to minimize
involvement of enzymatic processes. Short incubation periods with
radiolabeled EGF, in the presence or absence of NDF, revealed that the
rate of EGF binding was decreased in the presence of NDF
(Fig. 8 A). Ligand dissociation analyses were also
performed at 4 °C. The cells were first saturated with the
radiolabeled ligand, then unbound EGF was removed and a short
incubation step with unlabeled EGF was performed in order to prevent
ligand reassociation. Ligand release was then followed in the presence
or absence of NDF. The dissociation data obtained were analyzed by
plotting the natural logarithm of fractional receptor occupancy,
B
/ B
, as a function
of time, where B
is the amount of ligand
bound at time t and B
is the amount of
ligand bound before starting dissociation. The negative value of the
slope of such plots should indicate the dissociation constant. This
experiment revealed that NDF slightly accelerated the rate of EGF
release (Fig. 8 B). However, in both NDF-treated and
-untreated cells the plots displayed biphasic dissociation with an
initial rapid release followed by a major component with slower
dissociation rate (Fig. 8 B). Apparently, NDF affected
only the major component by increasing the off rate ( k
= 0.22
10
s
in the absence of NDF and 0.31
10
s
in its presence). Taken together, NDF
appeared to reduce EGF binding affinity through a dual effect on both
on and off rates. This possibility was examined by using a modified
protocol of the dissociation experiment, in which we omitted the
incubation step with unlabeled EGF, in order to allow reassociation of
the radiolabeled ligand. The results of this experiment are presented
in Fig. 8 C. Comparison with the plots shown in
Fig. 8B revealed that NDF induced a remarkable effect on
the combined kinetics of EGF dissociation and reassociation.
Figure 8:
Kinetics of EGF binding in the presence of
NDF. Association ( A) and dissociation ( B and
C) kinetics were analyzed on confluent monolayers of T47D
cells in 24-well dishes. A, the cells were preincubated with
binding buffer ( closed circles) or
NDF-1
(25 ng/ml) ( open circles)
at 4 °C for 1 h, and the incubation was continued for the indicated
periods of time at 4 °C after adding
I-EGF (5 ng/ml).
The amount of EGF that was specifically bound at each time point
( B) was determined after a brief washing step. Maximal EGF
binding ( B
) was separately determined by
incubating the cells for 2 h with
I-EGF at 4 °C. Each
point represents the mean of a triplicate determination ± S.E.
B, the cells were first incubated with 5 ng/ml
I-EGF for 2 h at 4 °C and then washed three times.
Ligand reassociation was prevented by incubating the cells for 5 min
with unlabeled EGF (100 ng/ml), and this was followed by ligand
dissociation in the presence ( open circles) or absence
( closed circles) of NDF-
1
(25
ng/ml). The radioactivity released into the medium, as well as
cell-associated
I-EGF, was determined and analyzed by
plotting the natural logarithm of B/ B
versus time. ( B is the concentration of ligand
bound at time t and B
is the
concentration of ligand bound at the starting time of dissociation).
Averages of triplicate determinations and their standard errors
( bars) are shown. C, the experiment was performed as
in B, except that no unlabeled EGF was added to the
dissociation medium. Each of the three experiments was repeated
twice.
EGF and NDF Receptors Form Complexes in Living
Cells
The characteristics of NDF-induced inhibition of EGF
binding raised the possibility that the inhibitory effect was mediated
by direct interactions between EGF and NDF receptors. In an attempt to
demonstrate the existence of such complexes, we labeled NDF receptors
by covalent cross-linking of the radioactive ligand and tried to
immunoprecipitate the presumed complex with EGF receptor by using
various antibodies to the latter protein. When whole lysates of T47D
cells were electrophoresed after cross-linking of I-NDF
two labeled protein bands, that presumably correspond to monomers
(major species) and dimers of receptors, were observed in
polyacrylamide gels, and they both disappeared in the presence of high
concentration of unlabeled ligand (Fig. 9, upper left
panel). Immunoprecipitation of EGF receptors from such lysates
recovered both monomers and dimers of NDF receptors (Fig. 9).
Control immunoprecipitation analyses that were performed with either a
preimmune antiserum (PI), an unrelated monoclonal antibody (G63), or a
polyclonal antibody to Kit/stem cell factor receptor (Ab 212) did not
recover the labeled proteins. In addition, similar experiments, that
were performed with radiolabeled stem cell factor, showed that
co-immunoprecipitation by the RK2 antibody was specific to
surface-linked
I-NDF. Replacement of the EGF
receptor-specific antibodies with a monoclonal antibody to human
ErbB-2, namely antibody N24
(37) , also resulted in
co-immunoprecipitation signals of both monomers and dimers, which were
abolished in the presence of unlabeled NDF (Fig. 9, lower
panel). One possible interpretation of these results is that
ligand-occupied NDF receptors form heterodimers with both EGF receptors
and ErbB-2, in analogy to EGF-induced heterodimers between ErbB-1 and
ErbB-2
(22, 23) . According to this explanation,
antibodies that are directed to either ErbB-1 or ErbB-2 preferentially
co-precipitate the heterodimeric form of NDF receptors and, therefore,
this molecular species is more abundant in immunoprecipitates than it
is in whole cell lysates (compare the ratio between monomers and dimers
in the upper panels of Fig. 9). When combined with other
biochemical characteristics of the inhibitory effect of NDF, the
existence of molecular complexes between NDF and EGF receptors may
provide a structural basis for the observed receptor cross-talk.
Figure 9:
Co-immunoprecipitation of surface-linked
NDF with EGF receptors. Radiolabeled NDF-1
(10 ng/ml) or human stem cell factor (SCF, 20 ng/ml) were
incubated for 2 h at 4 °C with 10
T47D cells. After a
brief wash, BS
was added to a final concentration of 1
mM in PBS, and incubation was continued at 22 °C for 45
min. Cells were lysed at 4 °C, and the lysates were either
electrophoresed directly ( upper left panel) or first subjected
to immunoprecipitation with the indicated antibodies, and then the
immunoprecipitates were resolved by SDS-polyacrylamide gel
electrophoresis. Note that the gels shown in the upper two panels were
run under slightly different conditions, so that they differ in
separation of the two labeled bands. The following antibodies were
used: anti-EGF receptor antibodies: RK2, a rabbit antiserum directed to
a synthetic peptide derived from the cytoplasmic portion of the
receptor, and monoclonal antibodies 528 and 2E9 that recognize the
extracellular domain, the N24 anti-ErbB-2 monoclonal antibody, and
three control antibodies: PI, a preimmune rabbit antiserum, Ab212, a
rabbit antibody to the stem cell factor receptor, and G63, a monoclonal
antibody to a rat mast cell antigen. Immunocomplexes were separated on
SDS-polyacrylamide gel electrophoresis (7.5% acrylamide), dried, and
exposed to an x-ray film for 72 h at
70 °C. The resulting
autoradiogram is shown, and the location of the uppermost standard
marker protein is indicated. The lower right panel shows a
control immunoprecipitation that was performed after cells were
incubated with both
I-NDF and 1 µg/ml unlabeled NDF
(labeled Ex cold). The experiments were repeated three times
with similar results.
and
types of the
platelet-derived growth factor receptor or the different Trk receptors
for neurotrophic factors, that may be functionally interlinked by
trans-modulation of heterologous ligand binding.
Table:
Changes in the level of EGF receptor following
treatment with either NDF or EGF
, bis(sulfosuccinimidyl) suberate; NDF,
Neu differentiation factor; PBS, phosphate-buffered saline; PKC,
protein kinase C; TPA, 12- O-tetradecanoyl phorbol 13-acetate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.