(Received for publication, September 25, 1995; and in revised form, January 3, 1996)
From the
The group of subtype I transmembrane tyrosine kinases includes the epidermal growth factor (EGF) receptor (ErbB-1), an orphan receptor (ErbB-2), and two receptors for the Neu differentiation factor (NDF/heregulin), namely: ErbB-3 and ErbB-4. Here we addressed the distinct functions of the two NDF receptors by using an immunological approach. Two sets of monoclonal antibodies (mAbs) to ErbB-3 and ErbB-4 were generated through immunization with recombinant ectodomains of the corresponding receptors that were fused to immunoglobulin. We found that the shared ligand binds to highly immunogenic, but immunologically distinct sites of ErbB-3 and ErbB-4. NDF receptors differed also in their kinase activities; whereas the catalytic activity of ErbB-4 was activable by mAbs, ErbB-3 underwent no activation by mAbs in living cells. Likewise, down-regulation of ErbB-4, but not ErbB-3, was induced by certain mAbs. By using the generated mAbs, we found that the major NDF receptor on mammary epithelial cells is a heterodimer of ErbB-3 with ErbB-2, whereas an ErbB-1/ErbB-2 heterodimer, or an ErbB-1 homodimer, is the predominant species that binds EGF. Consistent with ErbB-2 being a shared receptor subunit, its tyrosine phosphorylation was increased by both heterologous ligands and it mediated a trans-inhibitory effect of NDF on EGF binding. Last, we show that the effect of NDF on differentiation of breast tumor cells can be mimicked by anti-ErbB-4 antibodies, but not by mAbs to ErbB-3. Nevertheless, an ErbB-3-specific mAb partially inhibited the effect of NDF on cellular differentiation. These results suggest that homodimers of ErbB-4 are biologically active, but heterodimerization of the kinase-defective ErbB-3, probably with ErbB-2, is essential for transmission of NDF signals through ErbB-3.
Signals for growth and differentiation are mediated by binding
of soluble growth factors to transmembrane receptors, that carry an
intrinsic tyrosine kinase activity(1) . The group of subtype I
receptor tyrosine kinases includes four members that are characterized
by ectodomains with two cysteine-rich sequences. Despite extensive
structural homology, these receptors differ in their ligand
specificities. Thus, ErbB-1 (also called HER-1) binds several distinct
ligands whose prototype is the epidermal growth factor (EGF), ()whereas ErbB-3 and ErbB-4 are the respective low and high
affinity receptors for more than dozen isoforms of the Neu
differentiation factor
(NDF/heregulin)(2, 3, 4) . The fourth member
of the family, ErbB-2/Neu remains an orphan receptor because no fully
characterized ligand of this receptor has been reported(5) .
Besides the interest in ErbB proteins as mediators of signal
transduction, these receptors attracted attention due to their
involvement in cancer development(6) . Both ErbB-1 and ErbB-2
are oncogenic when overexpressed in murine
fibroblasts(7, 8) , and their overexpression in human
adenocarcinomas is associated with poor
prognosis(9, 10) . Likewise, ErbB-3 is overexpressed
in some adenocarcinomas, but its prognostic significance is still
unclear (11, 12, 13) .
Unlike ErbB-1 and ErbB-2, whose expression patterns include many mesenchymal tissues, both ErbB-3 and ErbB-4 are not expressed in fibroblasts and their expression in epithelial cells is limited to specific organs. On the other hand, mesenchymal cells are the major producers of the ligands for ErbB-3 and ErbB-4, implying that these receptors may play a role in mesenchyme-epithelium interactions(14, 15) . However, ErbB-3 differs from ErbB-4, as well as from other receptor tyrosine kinases, in certain structural motifs of the catalytic portion(13, 16) . These differences are probably responsible for the severely impaired kinase activity of ErbB-3(17) . Nevertheless, ErbB-3 contains many tyrosine autophosphorylation sites that are potential docking residues for signaling proteins that include a phosphotyrosine-specific binding cleft, called Src homology 2 (SH-2) domain. For example, ErbB-3 appears to allow coupling of ErbB-1 to phosphatidylinositol 3`-kinase (PI3K)(18, 19) . In addition, the relatively low ligand binding affinity of ErbB-3 is augmented by co-expression of ErbB-2(20) . Consistently, prevention of ErbB-2 expression at the cell surface, by using intracellular antibodies, significantly impaired signaling by NDF(21) , due to acceleration of ligand dissociation rate(22) . These and other observations led to the possibility that ErbB-3 functions as a kinase-defective docking protein analogous to the IRS-1 substrate of insulin receptor(23) . However, experiments that made use of chimeric proteins comprised of the extracellular domain of ErbB-1 fused to the cytoplasmic portion of ErbB-3, implied that ErbB-3 is a ligand-activable kinase that transmits proliferative signals through interactions with several SH-2 proteins, including phosphatidylinositol 3-kinase and SHC(24, 25) . To which extent these signaling events are mediated by ErbB-2, which forms heterodimers with ErbB-3(26) , is currently unknown. Another open question relates to the dual effect of NDF as a mitogen (26, 27) or as a growth-arresting and differentiation-inducing factor(28, 29) . Potentially, this duality may correlate with the fact that NDF binds to two distinct receptors.
The present study addressed the biological rational behind the existence of two different receptors for NDF. To approach this question, we undertook an immunological strategy and attempted to detect differences between the two NDF receptors. Monoclonal antibodies have been previously shown to be efficient research tools for the study of ErbB proteins. For example, anti-ErbB-1 mAbs enabled discrimination between two types of ligand binding sites and resolved the necessity of receptor dimerization for biological actions(30) . Likewise, certain antibodies to ErbB-2 probably mimic the putative ligand of this orphan receptor(31) , and other mAbs were able to inhibit its transforming action(32) , probably because they induce growth arrest and differentiation(33) . In the case of NDF receptors, mAbs may be especially useful, because unlike the shared ligand, they may discriminate between the two different receptors. By generating two sets of mAbs to ErbB-3 and ErbB-4, here we demonstrate that certain mAbs to ErbB-4, but not mAbs to the kinase-defective NDF receptor, namely ErbB-3, are biologically active. Nevertheless, ErbB-3 can mediate NDF signals through heterodimer formation. We show that ErbB-2 is the predominant partner of ErbB-3 in epithelial cells and this combination of receptors, like ErbB-4 alone, is able to generate differentiation signals in certain mammary cancer cells.
Figure 1:
Expression and ligand specificity of
Ig-ErbB fusion proteins. A, stable transfectants were
established by cotransfecting IgB constructs (10 µg of DNA)
together with the pSV2/Neo plasmid (0.5 µg of DNA) into HEK-293
cells and this was followed by G418 selection. The growth media of
cells that stably express human Ig-ErbB-3 or -ErbB-4 fusion proteins
(denoted IgB-3 and IgB-4, respectively) were collected. IgBs were
purified from conditioned media by using Sepharose beads coupled to
protein A. Purified proteins were resolved by SDS-PAGE (7.5%
acrylamide) in the absence or presence of the reducing agent
-mercaptoethanol (
ME), followed by immunoblotting
with anti-human IgG antibody (Fc specific), and chemiluminescence-based
detection (ECL, Amersham). The locations of molecular weight marker
proteins are indicated in kilodaltons (kDa) and the presumed monomeric (M) and dimeric (D) forms of the fusion proteins are
indicated by arrows. B, covalent cross-linking of EGF
or NDF to soluble ErbB proteins. Media of HEK-293 cells that secrete
IgB-3 or IgB-4 were reacted with protein A-Sepharose beads. After
washing, the beads were suspended in 0.1 ml of PBS that contained
BS
and 10 ng/ml of
I-NDF
1
(
I-N),
I-EGF (
I-E), or 100-fold excess of unlabeled NDF (Ex.N). Following 30 min of incubation at 22 °C, the beads
were washed, heated for 5 min at 95 °C in gel loading buffer, and
subjected to SDS-PAGE. The gel was dried and exposed to an x-ray film
for 12 h at -70 °C.
In agreement with conservation of the
functional conformation by the soluble receptors, mice that were
repeatedly immunized with the purified IgB proteins developed high
titer antisera that reacted with CHO cells expressing the transmembrane
ErbB-3 or ErbB-4, but not with untransfected CHO cells (data not
shown). The respective CHO-derived cell lines, denoted CB3 and CB4 cell
lines, expressed 0.3 and 1.0 10
receptors/cell and
will be described elsewhere. Spleens from the immunized mice were
therefore used for hybridoma generation. To select hybridomas producing
anti-ErbB-3 or anti-ErbB-4 mAbs, we screened their supernatants for the
ability to immunoprecipitate the corresponding protein from lysates of
either CB3 or CB4 cells. Detection of the immunoprecipitated ErbB-4 was
performed by an in vitro kinase assay (Fig. 2A). However, because very faint, if any, signals
were obtained in the in vitro kinase assays of ErbB-3 (data
not shown), we used [
S]methionine biosynthetic
labeling of CB3 cells in order to detect immunoprecipitated ErbB-3 (Fig. 2C). Confirmation of the results of the first two
screening assays was performed by using a second assay in which mAbs
were tested for their ability to immunoprecipitate
I-NDF
affinity-labeled ErbB-3 and ErbB-4 from lysates of either T47D breast
cancer cells or CB3 cells, respectively. The results of this assay are
shown in Fig. 2, B and D.
Figure 2:
Screening of hybridoma conditioned media
and selection of mAbs. A, in vitro kinase assay.
Whole cell lysates were prepared from cultures of CB4 cells and
subjected to immunoprecipitation with supernatants (100 µl) of the
indicated hybridomas of anti-ErbB-4 antibodies. The washed immune
complexes were incubated at 22 °C with
[-
P]ATP (5 µCi) and MnCl
(10 mM). Following 20 min of incubation, the
immunoprecipitates were resolved by gel electrophoresis and
autoradiography. B, immunoprecipitation of covalent NDF-ErbB-4
complexes with mAbs. Radiolabeled NDF-
1
(5
10
cpm/ng; 10 ng/ml) was incubated at 4 °C with
monolayers of CB4 cells. Following 45 min of incubation, the chemical
cross-linking reagent BS
was added (1 mM final
concentration) and the monolayers incubated for an additional 30 min.
Whole cell lysates were prepared and subjected to immunoprecipitation
with the indicated hybridoma supernatants. For control, a mAb to
c-Kit/stem cell factor receptor was used for immunoprecipitation. C, immunoprecipitation of metabolically labeled ErbB-3.
[
S]Methionine-labeled CB3 cell lysates were
subjected to immunoprecipitation with the indicated hybridoma
supernatants (100 µl) and the precipitated complexes subjected to
SDS-PAGE. For control we used a polyclonal mouse anti-ErbB-3 antibody
(labeled PC) and an anti-c-Kit mAb (labeled c-Kit). D, immunoprecipitation of affinity-labeled ErbB-3. CB3 cells
were treated with radiolabeled NDF and their lysates subjected to
immunoprecipitation with supernatants of anti-ErbB-3 hybridomas as
described in B. For control we used a polyclonal antibody to
ErbB-3 (PC) and an anti-c-Kit mAb (labeled c-Kit).
Importantly, one
out of three anti-ErbB-3 mAbs and four out of 11 anti-ErbB-4 mAbs could
not immunoprecipitate the I-NDF affinity-labeled antigen,
although they reproducibly immunoprecipitated the
P- or
S-labeled receptor. This suggested that the corresponding
mAbs were directed to the NDF binding sites, and therefore the
antibodies are expected to inhibit ligand binding to cells. To examine
this possibility, two of the suspected antibodies, namely Ab105 to
ErbB-3 and Ab7 to ErbB-4, were incubated with CB3 or CB4 cells in the
presence of radiolabeled NDF and the specific binding of the ligand
analyzed by the method of Scatchard (41) . Evidently, both mAbs
inhibited binding of NDF to cultured cells (Fig. 3). NDF binding
to ErbB-4-expressing cells displayed two populations of ligand binding
sites whose numbers, but not affinities, were significantly reduced by
Ab7. By contrast, only one population of NDF binding sites was
exhibited by ErbB-3-expressing cells, but this was significantly
reduced, with no change in receptor number, in the presence of Ab105.
We attribute the detectability of the high affinity population of NDF
binding sites in CB4 cells to the higher receptor numbers expressed by
these cells, as compared with CB3 cells.
Figure 3:
Inhibition of NDF binding by mAbs to
ErbB-3 and ErbB-4. Monolayers of CHO cells (2 10
cells/well) overexpressing either ErbB-4 (CB4 cells, panel
A) or ErbB-3 (CB3 cells, panel B) were incubated for 2 h
at 4 °C with different concentrations of
I-NDF-
1
in the presence of
different mAbs to ErbB-4 or to ErbB-3. Nonspecific binding was
determined by the addition of 100-fold excess of the unlabeled ligand
and it was subtracted from the total amount of bound radioactivity.
Scatchard analysis was performed by using the computerized program
LIGAND. The data are presented also as saturation curves (insets). The following symbols were used: Panel A: closed squares (control), closed triangles (Ab7), open triangles (Ab94), and open circles (Ab43). Panel B: closed squares (control), closed
triangles (Ab105), and open triangles (Ab252). Each data
point represents the average of a duplicate determination and each
experiment was repeated twice.
Because ErbB-3 and ErbB-4
share ligand specificity, we examined the possibility that the ligand
binding sites of these receptors are immunologically related to each
other. However, immunoprecipitation analyses indicated that several
anti-ErbB-3 antibodies, including Ab105, were unable to recognize the
biosynthetically-labeled ErbB-4 ( Fig. 4and data not shown).
Likewise, none of our anti-ErbB-4 mAbs, including the ligand-inhibitory
Ab7, was able to immunoprecipitate ErbB-3 ( Fig. 4and data not
shown). The analysis of ErbB specificity was extended to include also
ErbB-1 and ErbB-2 by using the corresponding derivatives of CHO cells
that, respectively, express 2.0 10
and
approximately 0.8
10
molecules of the corresponding
ErbB protein. Evidently, no cross-reactivity of antibodies to NDF
receptors with either ErbB-1 or ErbB-2 was observed ( Fig. 4and
data not shown). In conclusion, despite shared ligand specificity and
homologous structures, ErbB-3 and ErbB-4 are immunologically distinct
from each other and from other members of the ErbB family.
Figure 4:
Lack of cross-reactivity of
anti-NDF-receptor mAbs. CB1, CB2, CB3, and CB4 cells were metabolically
labeled with [S]methionine, and the cell lysates
were separately subjected to an immunoprecipitation assay with either
mAbs to ErbB-4 (antibodies 7 and 72) or to ErbB-3 (antibodies 90 and
105). Proteins were separated by SDS-PAGE (7.5% polyacrylamide). An
autoradiogram of the dried gel is shown and the locations of molecular
weight marker proteins are indicated.
Figure 5: mAb-induced tyrosine phosphorylation of ErbB-4 but not ErbB-3. Monolayers of CB4 or CB23 cells were incubated for 10 min at 37 °C with the indicated antibodies at 10 µg/ml or with NDF (10 ng/ml). Cell lysates were prepared and subjected to immunoprecipitation (I.P.) with mAbs to either ErbB-4 (A), ErbB-3, and ErbB-2 (B). After gel electrophoresis, the immunoprecipitated proteins were electrophoretically transferred onto nitrocellulose and immunoblotted (I.B.) with a mAb to phosphotyrosine (P-TYR). The results of chemiluminescence-based detection are shown, along with the locations of molecular weight standard marker proteins.
The second assay examined the ability of the various mAbs to induce
accelerated degradation of NDF receptors. Transfected CHO cells were
prelabeled with [S]methionine and then chased
for 8 h in the absence or presence of either mAbs or NDF.
Interestingly, whereas NDF was unable to down-regulate ErbB-4, certain
mAbs significantly accelerated disappearance of the receptor. It is
worth noting that Abs 77 and 50 were the most active antibodies in both
receptor down-regulation and kinase stimulation, implying that these
two activities are functionally coupled. In line with this conclusion,
the rate of ErbB-3 degradation was not significantly affected by two
mAbs, and a third mAb, namely Ab252, like NDF, decelerated receptor
degradation (Fig. 6B). The observation that NDF is
unable to down-regulate its own receptors, either ErbB-3 or ErbB-4, in
CHO cells is interesting as it differs from the effect of ligands to
ErbB-1. Indeed, in experiments that are not presented we found that the
rates of NDF and EGF internalization remarkably differ in CHO and in
myeloid cells that ectopically express ErbB proteins.
Figure 6:
Effects of mAbs on receptor turnover.
Monolayers (2 10
cells per lane) of either CB4
cells or CB3 cells were biosynthetically labeled with
[
S]methionine. After a brief wash, cells were
chased for 8 h with unlabeled methionine-containing medium, in the
presence of the indicated mAbs (at 10 µg/ml) to ErbB-4 (A), or to ErbB-3 (B) or with NDF (50 ng/ml). For
control, cells were incubated with medium alone (lanes labeled None). The cells were then washed, lysed, and cell lysates
were subjected to immunoprecipitation with mAbs to either ErbB-4 (A) or to ErbB-3 (B). The resulting autoradiograms
(16 h exposure with an intensifying screen) are
shown.
Figure 7:
Covalent cross-linking of radiolabeled NDF
to the surface of various human tumor cell lines. I-Labeled NDF-
1
(10 ng/ml)
was incubated for 2 h at 4 °C with 5
10
cells
of the following human tumor cell lines: SKBR-3 breast cancer, MCF-7
breast cancer, CACO
colon cancer, and the PLC/PRF/5
hepatoma line. The cell monolayers were washed with PBS and then
cross-linked for 30 min with BS
(1 mM, Pierce)
followed by cell lysis. After clearance of cell debris, the
detergent-solubilized lysates were subjected to separate
immunoprecipitation reactions with antibodies to the indicated four
ErbB proteins. Immune complexes were resolved by gel electrophoresis
and autoradiography.
In order to analyze the functional consequences of the extensive interaction between ErbB-3 and ErbB-2, we examined tyrosine phosphorylation of the latter protein in SKBR-3 breast cancer cells, that overexpress ErbB-2. The results of this analysis are shown in Fig. 8. Evidently, both NDF and EGF caused phosphorylation of their direct receptors, namely ErbB-3 and ErbB-1, respectively, but these ligands also elevated tyrosine phosphorylation of ErbB-2. However, no evidence for trans-phosphorylation between ErbB-1 and ErbB-3 was obtained. It is worth noting that heterodimers containing ErbB-1 and ErbB-2 can be induced by EGF(36, 44) , in analogy to ErbB-3/ErbB-2 heterodimers that are stabilized by NDF ( Fig. 7and 9). Therefore, it is conceivable that tyrosine phosphorylation of ErbB-2 is activated in trans, either by NDF binding to ErbB-3 or by EGF binding to ErbB-1.
Figure 8:
Ligand-induced tyrosine phosphorylation of
ErbB proteins. Confluent monolayers of SKBR-3 human breast cancer cells
were incubated in serum-free medium for 12 h and then incubated for 10
min at 37 °C in PBS in the presence or absence of ligands (either
NDF-1 or EGF, each at 20 ng/ml), as indicated. The corresponding
ErbB proteins were immunoprecipitated (IP) from whole cell
lysates with specific antibodies, resolved by SDS-PAGE, and their
tyrosine phosphorylation detected by immunoblotting (IB) with
an antibody to phosphotyrosine (P-TYR). Control
immunoprecipitation was performed with a nonrelevant antibody (lanes
labeled c-Ab)
The observation that ErbB-2 is a common phosphorylation partner of NDF and EGF receptors, together with reports on the ability of ErbB-2 to enhance binding affinities of NDF and EGF(20, 22, 44) , imply that NDF receptors and EGF receptors may compete for interaction with ErbB-2. To test this prediction we performed affinity labeling experiments with radiolabeled NDF or EGF and analyzed reciprocal binding effects of the two ligands. Fig. 9depicts the results of this experiment, that was performed on T47D breast cancer cells. In agreement with the observed trans-phosphorylation between ErbB-1 and ErbB-2, it appeared that the major species that binds EGF in T47D cells was a heterodimer of ErbB-1 with ErbB-2, whereas the major NDF receptor was a heterodimer of ErbB-3 with ErbB-2. In addition, comparison of the affinity labeling patterns reflected the apparent exclusive nature of these inter-receptor interactions, as no ErbB-1/ErbB-3 heterodimers were observed. Remarkably, when the affinity labeling of EGF receptors was performed in the presence of NDF, a significant reduction in both ErbB-1 and ErbB-2 labeling was observed. This implied that NDF can inhibit EGF binding to ErbB-1 and that ErbB-2 is involved in this trans-regulatory effect. The reciprocal experiment, that examined the effect of unlabeled EGF on binding of radiolabeled NDF, revealed a lower trans-inhibitory effect. These results are consistent with our previous report that NDF can accelerate the rate of EGF release from ErbB-1 (45) , and they attribute the effect to a competition between ligand-bound ErbB-3 and ErbB-1 for the available ErbB-2. Presumably, ErbB-3/ErbB-2 heterodimers are favored over ErbB-1/ErbB-2 heterodimers, and therefore the trans-inhibitory effect of NDF is stronger than that of EGF.
Figure 9:
Trans-inhibitory effects of ErbB ligands.
Monolayers of 5 10
T47D human breast cancer cells
were incubated with radiolabeled EGF or NDF (each at 10 ng/ml) in the
presence or absence of the other ligand in its unlabeled form, as
indicated. Covalent cross-linking of the radiolabeled ligands to cell
surfaces was performed as described in the legend to Fig. 7, and
this was followed by immunoprecipitation of individual ErbB proteins
and SDS-PAGE (6.5% acrylamide) of the precipitated complexes. The
autoradiograms show monomers and dimers of the affinity-labeled
receptors. Note that NDF decreased labeling of both ErbB-1 and ErbB-2
by EGF, but the latter only slightly reduced binding of NDF to ErbB-3
and ErbB-2.
Figure 10: An anti-ErbB-4 mAb induces differentiation of breast cancer cells and up-regulates the WAF-1 protein. MCF-7 cells were incubated for 4 days in the absence or presence of 10 µg/ml mAb36 to human ErbB-4. The cell monolayers were then processed for staining of neutral lipids with Oil Red O (red perinuclear droplets) or immunohistochemical localization of WAF-1 (brown nuclear staining).
In order to examine the role of ErbB-3 in transmission of the differentiation effect of NDF, we tested the ability of anti-ErbB-3 mAbs to mimic the action of NDF on two breast cancer cell lines. Unlike most anti-ErbB-4 mAbs, that were capable of inducing some cellular differentiation, none of three mAbs to ErbB-3 was effective (Table 1, and data not shown). This result is consistent with the inability of the mAbs to stimulate tyrosine phosphorylation and down-regulation of ErbB-3 (Fig. 5B and Fig. 6B). We next analyzed the ability of Ab105, which antagonizes NDF binding (Fig. 3B), to inhibit the induction of cellular differentiation by this ligand. Indeed, like in the case of a ligand-inhibitory mAb to ErbB-4, Ab105 reduced the effect of NDF on lipids and ICAM-1 (Table 1). Although the inhibitory effect of mAb105 was incomplete, it was reproducibly larger than the effect of an ErbB-4-blocking antibody. Remarkably, even at over-saturating concentrations, that completely abolish ligand binding to CB3 cells, Ab105 was unable to completely abolish the effect of NDF. Therefore, we concluded that ErbB-3 mediates the differentiation action of NDF in a non-exclusive manner. Thus, although ErbB-3 is biologically inactive, this receptor mediates part of the effect of NDF, conceivably by heterodimer formation with ErbB-2. This proposition is supported by the finding that certain anti-ErbB-2 mAbs are effective inducers of differentiation(33) .
Most mammalian receptor tyrosine kinases belong to small groups of 2-9 highly related proteins that bind to homologous growth factors. Examples include the Trk family of receptors for neurotrophic factors and the relatively large family of Eph-like receptors(48) . Because in insect cells each subgroup is represented by a single receptor, it is reasonable that receptor multiplicity evolved in order to provide physiological answers. The group of type I receptor tyrosine kinases is unique in that it contains a receptor with no known ligand, namely ErbB-2, and another receptor, ErbB-3, whose tyrosine kinase domain includes several unusual sequence motifs. In this respect ErbB-3 resembles several other receptor-like tyrosine kinases, such as Klg (49) and Vik/Ryk(50) , whose biological and biochemical functions are unknown. The present study addressed the multiplicity of type I receptor tyrosine kinases, and especially the two distinct receptors for NDF. By expressing recombinant forms of the extracellular domains of ErbB-3 and ErbB-4 in their native forms, we were able to obtain a relatively large repertoire of mAbs to these NDF receptors. On the basis of experiments that were performed with the new mAbs, we reached three major conclusions that are summarized below.
Taken together, our results are consistent with the notion that the multiplicity of ErbB proteins confers diversification of signal transduction by the corresponding ligands(5, 23) . According to the emerging model, the two NDF receptors differ in a major aspect: ErbB-4 can generate biological signals upon homodimerization, but ErbB-3 homodimers are signaling-defective. Heterodimerization apparently reconstitutes signaling by ErbB-3, and the preferred partner of this major NDF receptor is ErbB-2. However, the latter protein forms heterodimers also with ErbB-1, so that it probably functions as a common signaling subunit of NDF and EGF receptors. This proposition is consistent with the observation that abolishment of ErbB-2 expression severely impairs signaling by both growth factors(21, 22) , and it may imply that ErbB-2 can function without a ligand of its own. The existence and role of ErbB-4/ErbB-2 heterodimers remain unclear, but ErbB-4 appears to be the minor NDF receptor, at least in epithelial cells. Presumably, besides reconstitution of ErbB-3 activity, the process of receptor heterodimerization confers additional levels of complexity and regulation to the mechanism of signaling by growth factors.