(Received for publication, October 19, 1995; and in revised form, December 15, 1995)
From the
Numerous epidermal growth factor (EGF)-related peptide binding
members of the ErbB family of receptor tyrosine kinases have been
described. While several EGF agonists bind and activate ErbB-1/EGF
receptor, neu differentiation factor (NDF) functions as a ligand for
ErbB-3 and ErbB-4. However, it is currently unknown which specific
subsets of ErbB receptors become activated in response to each of these
ligands. The present study addresses this issue using the T47D breast
tumor cell line, which expresses moderate levels of all the presently
known ErbB receptors. We show that all the EGF agonists, but not NDF,
stimulated tyrosine phosphorylation of ErbB-1. In contrast, all the
EGF-related factors except amphiregulin were able to induce tyrosine
phosphorylation of ErbB-2. The ability to induce tyrosine
phosphorylation of ErbB-3 varied dramatically among the different
EGF-related peptides. While EGF, transforming growth factor
(TGF)-, and amphiregulin only had a moderate effect, NDF
dramatically increased the ErbB-3 phosphotyrosine content. Most
notably, heparin binding EGF-related growth factor (HB-EGF) and
betacellulin (BTC) were more effective than other EGF agonists.
Consequently, only NDF, HB-EGF, and BTC significantly stimulated
association of phosphatidylinositol kinase activity with ErbB-3. Among
the EGF agonists, HB-EGF induced a low level of ErbB-4 tyrosine
phosphorylation, while BTC was as efficient as NDF in activating
ErbB-4. The BTC activation of ErbB-4 appears to be independent of
ErbB-1, as shown by pretreatment of cells with an antibody that
inhibits binding of EGF agonists to ErbB-1. As a result of the
differential activation of ErbB receptors, most of the EGF-related
growth factors had distinguishable biological activities on cultured
mammary epithelial cell lines.
Many polypeptide growth factors exert their function by binding
cell surface receptors with intrinsic protein tyrosine kinase activity.
Ligand binding to the extracellular domain of a receptor tyrosine
kinase (RTK) ()induces dimerization, activation of the
intracellular kinase domain, and autophosphorylation by an
intermolecular mechanism(1) . Tyrosine-phosphorylated residues
serve as high affinity binding sites for SH2 or phosphotyrosine binding
domain containing proteins and allow for the modulation of
intracellular signaling pathways(2, 3) . RTKs play a
fundamental role in the regulation of cell growth and differentiation.
A large number of RTK subclasses has been described, among which the
type I/ErbB family of RTKs is of particular interest due to their
frequent involvement in human cancer. Four members of this family are
currently known: epidermal growth factor (EGF) receptor/ErbB-1, ErbB-2,
ErbB-3, and ErbB-4(4, 5, 6, 7) .
Aberrant expression of ErbB-1 has been observed in various human
tumors(8) . Overexpression of ErbB-2 in the presence or absence
of gene amplification is frequently found in tumors arising at many
sites, especially of the breast and ovary where it correlates with poor
patient prognosis(9) . High levels of ErbB-3 have been
described both in breast tumor cell lines (10) and in primary
breast cancer(11) . In order to fully understand the normal
function of ErbB receptors as well as their role in neoplastic
transformation it is crucial that their regulation by growth factors be
elucidated in detail.
Regulation of ErbB receptor activity appears
to be very complex since a large number of ErbB ligands have been
described. All of these peptides are derived from transmembrane
precursors and are cleaved to give rise to the mature, soluble ligands.
Depending on their binding specificities, they can be subdivided into
two classes. The first group of ligands binds to the EGFR and includes
EGF itself, transforming growth factor (TGF) , amphiregulin (AR),
heparin-binding EGF-like growth factor (HB-EGF), and betacellulin
(BTC)(12, 13, 14, 15, 16, 17) .
Each of these peptides competes with EGF for receptor binding (13, 14, 16, 17) and therefore this
family of growth factors is referred to as the EGF agonists. Another
family of EGF-related peptides is composed of the neu differentiation
factors (NDFs)/heregulins(18, 19) , ligands for ErbB-3
and ErbB-4(20, 21) . There are at least 12 different
isoforms arising from a single gene by alternative splicing and
depending on the sequence of their EGF-like repeat they are classified
as either
or
isoforms(22) . However, despite the
large number of NDFs no differences in receptor binding specificities
appear to exist: ErbB-3 functions as a low affinity receptor for all
NDF isoforms while ErbB-4 serves as a high affinity
receptor(23) .
An additional level of complexity in ErbB-mediated signaling has been brought forward by the finding that binding of a specific ligand not only triggers the formation of receptor homodimers, but also heterodimers. For instance, EGF induces tyrosine phosphorylation of ErbB-2 through formation of ErbB-1/ErbB-2 dimers(24, 25) . Similarly, NDF stimulates tyrosine phosphorylation of ErbB-2, presumably through formation of ErbB-3/ErbB-2 and ErbB-4/ErbB-2 dimers(21, 26, 27) . An important consequence of heterodimerization of EGF and NDF receptors with ErbB-2 is a dramatically increased affinity for the respective ligand(26, 28, 29) . However, heterodimerization may also serve to diversify the nature of the intracellular signal elicited by a specific growth factor. The prototype example supporting this notion is the recent observation that EGF stimulates the association of phosphatidylinositol (PtdIns) 3-kinase with ErbB-3, presumably through EGFR/ErbB-3 heterodimerization followed by cross-phosphorylation(30, 31) .
In view of the large number of ErbB ligands and the extensive cross-talk occurring between ErbB receptors, the knowledge about functional differences in the signaling capacities of EGF-related peptides has remained fragmentary. Thus, the present study was designed as a comparative analysis of all the currently available EGF-related growth factors with respect to activation of ErbB receptors and biological activity.
Figure 1: Tyrosine phosphorylation of ErbB-1 (A) and ErbB-2 (B). T47D cells were starved for 24 h in serum-free medium and treated for 10 min at room temperature with 100 ng/ml of the indicated factors prior to lysis. ErbB-1 and ErbB-2 were immunoprecipitated with, respectively, a 1:1 mixture of mAb EGFR and mAb 528 or the antiserum 21N, subjected to SDS-PAGE, and analyzed by Western blotting with a phosphotyrosine-specific mAb (upper panels). Filters were stripped and reprobed with, respectively, antiserum 15E or 21N (lower panels).
Previously, NDF and several EGF agonists have been shown to induce the tyrosine phosphorylation of ErbB-2(19, 24, 36) . However, BTC- or HB-EGF-induced phosphorylation has not been reported so far. Thus, we compared all the factors with respect to activation of ErbB-2. With the exception of AR, the EGF agonists, as well as NDF, induced tyrosine phosphorylation of ErbB-2 to a similar extent, presumably due to the ability of both EGF- and NDF-receptors to heterodimerize with this receptor (Fig. 1B).
Figure 2: Tyrosine phosphorylation of ErbB-3 and association with PtdIns kinase activity. After 24 h starvation in serum-free medium, T47D cells were treated for 10 min at room temperature with 100 ng/ml of the indicated factors, lysed, and ErbB-3 was immunoprecipitated with antibody C17. A, immunoprecipitates were subjected to SDS-PAGE and analyzed by Western blotting with a phosphotyrosine-specific mAb (upper panel). Filters were stripped and reprobed with antibody C17 (lower panel). B, associated PtdIns kinase activity was determined in an immune complex kinase assay using PtdIns as a substrate. Bars show the fold stimulation of PtdIns kinase activity in comparison to untreated cells.
ErbB-3 is unique among subclass I RTKs due to the presence of several Tyr-X-X-Met motifs in its intracellular domain, the consensus binding site for the p85 subunit of PtdIns 3-kinase(37) . Consequently, both EGF and NDF induce the association of PtdIns 3-kinase activity with ErbB-3(30, 31) . Given the differential effects of EGF agonists on ErbB-3 phosphorylation, we analyzed their ability to induce association of PtdIns-kinase activity with this receptor. T47D cells were stimulated with the various peptides, ErbB-3 was immunoprecipitated, and the associated PtdIns kinase activity was measured in an in vitro kinase assay using PtdIns as a substrate (Fig. 2B). In good correlation with the effects on tyrosine phosphorylation of ErbB-3, NDF dramatically induced ErbB-3 association of PtdIns kinase activity, while the EGF agonists were generally less active. Significantly, HB-EGF and BTC stimulated PtdIns kinase activity more strongly than the other EGF agonists. Thus, by differentially inducing tyrosine phosphorylation of ErbB-3, EGF-related growth factors have different abilities to couple to ErbB-3-specific pathways.
Figure 3:
A, tyrosine phosphorylation of ErbB-4.
T47D cells were starved for 24 h in serum-free medium and treated for
10 min at room temperature with 100 ng/ml of the indicated factors
prior to lysis. ErbB-4 was immunoprecipitated with antibody C18,
subjected to SDS-PAGE, and analyzed by Western blotting with a
phosphotyrosine-specific mAb (upper panel). Filter was
stripped and reprobed with antibody C18 (lower panel). B, competition of mAb 225 with EGF and BTC. T47D cells were
starved for 24 h in serum-free medium and either left untreated or
pretreated with 10 µg/ml mAb 225 (mAb) for 10 min
at room temperature prior to the addition of 1 nM EGF or BTC.
ErbB-4 (left panel) and ErbB-1 (right panel) were
immunoprecipitated with, respectively, antibody C18 or a 1:1 mixture of
mAb EGFR1 and mAb 528, subjected to SDS-PAGE, and analyzed by Western
blotting with a phosphotyrosine-specific
antibody.
There are at least two possibilities for the strong BTC-induced increase in tyrosine phosphorylation of ErbB-4. Either BTC directly binds and activates ErbB-4, or BTC binds to ErbB-1 and induces the formation of ErbB-1/ErbB-4 heterodimers followed by cross-phosphorylation. To test these possibilities, T47D cells were pretreated with 10 µg/ml mAb 225, which competes with EGF for binding to ErbB-1, prior to stimulation with EGF or BTC (Fig. 3B). While mAb 225 readily abolished both EGF- and BTC-induced tyrosine phosphorylation of ErbB-1, it only partially inhibited BTC-induced phosphorylation of ErbB-4. Thus, BTC appears to directly bind and activate ErbB-4, although the availability of ErbB-1 seems to enhance the activation of ErbB-4, presumably through ErbB-1/ErbB-4 heterodimerization.
Figure 4:
Effect of EGF-related peptides on the
growth of cultured mammary epithelial cells. A, growth
stimulation of MCF10A cells. B, growth stimulation of MDA MB
453 cells. Cells were plated in triplicate in the presence or absence
of the indicated factors and growth was monitored after 3 days (MCF10A
cells) or 4 days (MDA MB 453 cells) using the Cell Titer AQ kit (Promega). Bars show growth stimulation in
comparison to untreated cells
(±S.D.).
We have
previously shown that NDF, but not EGF, stimulates the
anchorage-independent growth of T47D cells(25) . To further
characterize the biological activities of the EGF-related peptides, we
tested their ability to stimulate T47D colony formation under
anchorage-independent conditions (Table 1). While TGF and
AR, like EGF, had no significant effect on growth, NDF stimulated
colony formation by almost 6-fold. Similarly, HB-EGF and BTC led to a
2-3-fold increase in the number of colonies. Thus, EGF-related
factors show distinguishable biological activities, most likely
depending on the subsets of ErbB receptors that become activated.
Figure 5:
Cooperation of EGF-related growth factors
with insulin and EGF for the stimulation of HC11 cell growth. HC11
cells were plated in triplicate under serum-restricted conditions in
the presence of either insulin (A) or EGF (B) plus
the indicated factors. Growth was monitored after 3 days using the Cell
Titer AQ kit (Promega). Bars show growth
stimulation in comparison to cells grown in insulin or EGF alone
(±S.D.).
In this report we demonstrate that EGF-related growth factors differ in their ability to induce tyrosine phosphorylation of the various ErbB receptors in T47D breast tumor cells (see Table 2for a summary). Most notable are the different abilities of EGF agonists to induce ErbB-3 tyrosine phosphorylation, as well as the ability of BTC to efficiently activate ErbB-4 in an apparently ErbB-1-independent manner. We show that in line with their effect on ErbB-3 tyrosine phosphorylation, only NDF, BTC, and HB-EGF significantly stimulated the recruitment of PtdIns kinase activity to this receptor. Furthermore, we demonstrate that the activation of different receptor subsets by EGF-related peptides results in distinct biological activities on cultured mammary epithelial cell lines.
While most of the EGF agonists were similarly effective in inducing
tyrosine phosphorylation of ErbB-1 in T47D cells and stimulating growth
of MCF10A cells, AR was strikingly less active. This is in agreement
with the observation that AR has a significantly lower affinity for
ErbB-1 than EGF(14) . The lower affinity of AR appears to be
due to the fact that the mature form (either AR or
AR
, due to alternate N-terminal cleavage) is
truncated at its C terminus and lacks a leucine residue which is highly
conserved among EGF agonists. This leucine residue appears to be
important for high affinity binding of EGF to ErbB-1(41) . In
fact, it has been shown that an artificially C terminally extended
recombinant AR incorporating 6 additional amino acid residues predicted
from its coding sequence, as well as a methionine to leucine
substitution at position 86 (AR
) had a
50-100-fold higher activity than wild-type AR and was comparable
to EGF(42) . In line with its low affinity for ErbB-1, AR was
the only EGF agonist that failed to induce tyrosine phosphorylation of
ErbB-2 in T47D cells. This is in contrast to a previous publication
showing AR-induced phosphorylation of ErbB-2 in a number of cell lines
with relatively high ErbB-1 levels(36) . This discrepancy most
likely is due the low levels of ErbB-1 expressed in T47D
cells(27) .
Despite quite high ErbB-3 and ErbB-4 expression levels in T47D cells (27) , we failed to observe any NDF-induced increase in ErbB-1 tyrosine phosphorylation. This is in contrast to a recent publication which shows that coexpression of ErbB-1 with either ErbB-3 or ErbB-4 allows NDF to regulate ErbB-1 tyrosine phosphorylation(43) . These results strongly suggest that ErbB receptor heterodimer formation follows a strict hierarchy. If only ErbB-1 is available, ErbB-3 and ErbB-4 may be able to heterodimerize with ErbB-1 in response to NDF. However, if all the four ErbB receptors are present, other heterodimers seem to be preferentially formed (i.e. ErbB-3/ErbB-2, ErbB-4/ErbB-2, or ErbB-3/ErbB-4).
EGF has been found to induce ErbB-3 tyrosine
phosphorylation and association with PtdIns 3-kinase in ErbB-1
overexpressing A431 and MDA MB 468 cells, presumably through
ErbB-1/ErbB-3 heterodimerization(30, 31) . However,
while NDF strongly induced ErbB-3 phosphorylation and association with
PtdIns kinase activity in T47D cells, EGF only had a mild effect,
probably due to the low abundance of ErbB-1 in these cells (27) . Thus, although in ErbB-1 overexpressing tumor cells
ErbB-1/ErbB-3 cross-talk may contribute to the transformed phenotype,
our observations raise potential doubts about the relevance of this
cross-talk at physiological ErbB-1 expression levels. HB-EGF and BTC
were significantly more efficacious in inducing ErbB-3 phosphorylation
and association with PtdIns kinase activity than the other agonists.
Although the mechanism of the enhanced ErbB-1/ErbB-3 cross-talk in
response to HB-EGF and BTC is unclear, it seems possible that different
ErbB-1 binding peptides preferentially promote the formation of
specific heterodimers. In fact, it appears that AR also preferentially
induces ErbB-1/ErbB-3 cross-talk, since it induced a low level of
ErbB-3 phosphorylation without having an effect on ErbB-2. EGF
agonistic growth factors can be subdivided into heparin binding (AR,
HB-EGF, and BTC) and non-heparin binding (EGF and TGF) peptides.
The presence of heparan sulfate-containing proteoglycans on the cell
surface and/or in the extracellular matrix has been shown to be
essential for activity of several heparin binding growth factors,
including AR(44, 45) . It is likely that the binding
of AR, HB-EGF, and BTC to heparan sulfate-containing proteoglycans
influences receptor-ligand interactions, and it may be possible that it
facilitates the binding of specific receptor dimers, e.g. ErbB-1/ErbB-3 heterodimers. Although the precise mechanism remains
to be elucidated, the different effect of EGF-related peptides on
ErbB-3 phosphorylation seems relevant, since it directly correlates
with their effect on the soft agar growth of T47D cells.
BTC was shown to bind ErbB-1 with an affinity similar to that of EGF (46) . However, BTC-induced tyrosine phosphorylation of ErbB receptors has not been analyzed previously. Here we show that BTC is unique among the EGF-related peptides, since it has the ability to efficiently activate all the four members of the ErbB receptor family. Most importantly, it was as effective as NDF in inducing ErbB-4 phosphorylation. Binding of a specific EGF agonist to ErbB-1 per se may induce ErbB-4 phosphorylation through the formation of ErbB-1/ErbB-4 heterodimers. In fact, this mechanism may account for the slight increase in ErbB-4 tyrosine phosphorylation we observed in response to HB-EGF. However, BTC appears to directly interact with ErbB-4. First, BTC was similar to NDF in that it was more effective than other EGF agonists in stimulating the growth of MDA MB 453 cells, which do not contain detectable amounts of ErbB-1(39) . Second, pretreatment of T47D cells with mAb 225, which competes with EGF for binding to ErbB-1(32) , completely abolished BTC-induced phosphorylation of ErbB-1 while only partially inhibiting the phosphorylation of ErbB-4. In line with these observations, a recent report shows BTC-induced phosphorylation of ErbB-4 ectopically expressed in Ba/F3 cells, which lack detectable levels of endogeneous ErbB receptors(47) . However, our results also point to a role for ErbB-1/ErbB-4 heterodimers in BTC-induced signaling, since ErbB-1 appears to be required for full activation of ErbB-4.
The requirements of HC11 mouse mammary epithelial cells for both EGF and insulin for growth under serum-restricted conditions allowed another comparison of the biological activities of the peptides. While all the EGF agonists and NDF were able to replace EGF in the growth medium, none of the EGF agonists was able to replace insulin. Only NDF could partially replace insulin, thereby cooperating with EGF for the growth stimulation of HC11 cells. NDF has a similar activity on the MCF10A human mammary epithelial cells(48) . Thus, it seems that NDF has a dual EGF- and insulin-like activity enabling it to stimulate signaling pathways that normally require two separate growth factors. Activated insulin and insulin-like growth factor-1 receptors induce the rapid tyrosine phosphorylation of insulin receptor substrate-1, which contains multiple binding sites for the p85 subunit of PtdIns 3-kinase (49) . Similarly, NDF is the best activator of ErbB-3, the only ErbB receptor involved in efficient recruitment of p85(30, 37) . Thus, it is possible that the ability to efficiently activate PtdIns 3-kinase is responsible for NDFs insulin-like activity.
In conclusion, this study represents the first comprehensive analysis of all the presently available EGF-related growth factors with respect to the activation of ErbB receptors. The distinct biological activities described in this report provide a rational for the existence of the great variety of EGF-related peptides.