Báo cáo Y học: Chimeric receptor analyses of the interactions of the ectodomains of ErbB-1 with epidermal growth factor and of those of ErbB-4 with neuregulin potx

Chimeric receptor analyses of the interactions of the ectodomains
of ErbB-1 with epidermal growth factor and of those of ErbB-4
with neuregulin
Jae-Hoon Kim
1,
*, Kazuki Saito
1,2
and Shigeyuki Yokoyama
1,2,3
1
Yokoyama CytoLogic Project, ERATO, Japan Science and Technology Corporation, c/o Tsukuba Research Consortium,
Tokodai, Tsukuba, Japan;
2
RIKEN Genomic Sciences Center, Suehiro-cho, Tsurumi, Yokohama, Japan;
3
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan
A series of chimeric receptors was generated between the
epidermal growth factor (EGF) receptor, ErbB-1, and its
homologue, ErbB-4, to investigate the roles of the extracel-
lular domains (I–IV) in the ligand specificities. As compared
with ErbB-1 and the chimeras with both domains I and III of
ErbB-1, the chimeras with only one of these domains
exhibited reduced binding of
125
I-labeled EGF. Particularly,
the contribution of domain III was appreciably larger than
that of domain I of ErbB-1 in
125
I-labeled EGF binding.
Nevertheless, the chimeras with domain III of ErbB-1 and
domain I of ErbB-4 were prevented from binding to

125
I-labeled EGF competitively by the ErbB-4 ligand, neu-
regulin (NRG). On the other hand, NRG did not compete
with
125
I-labeled EGF for binding to the chimeras with the
ErbB-1 domain I and the ErbB-4 domain III. Therefore,
NRG binding to ErbB-4 depends much more on domain I
than on domain III. With respect to autophosphorylation
and subsequent ERK activation, EGF activated the chi-
meras with either domain I or III of ErbB-1. In contrast,
NRG activated the chimeras with the ErbB-4 domain I and
the ErbB-1 domain III, but not those with the ErbB-1
domain I and the ErbB-4 domain III. Therefore, the relative
contributions between domains I and III of ErbB-4 in the
NRG signaling are different from those of ErbB-1 in the
EGF signaling.
Keywords: chimeric receptors; epidermal growth factor;
ErbB family; ligand recognition; neuregulin.
The ErbB-family tyrosine kinases play central roles in the
proliferation, differentiation, and development of cells [1].
The family is composed of four members, including the
ErbB-1/epidermal growth factor (EGF) receptor [2], ErbB-
2/neu [3], ErbB-3 [4], and ErbB-4 [5]. Each of these receptors
has an extracellular region, a single transmembrane region,
and a cytoplasmic sequence containing a tyrosine kinase
domain and a C-terminal tail. The extracellular region has
about 40% homology among the four family members and
can be further divided into four domains (I–IV); the
N-terminal domain I has sequence similarity to domain

III, which is flanked by two cysteine-rich domains, II and IV.
More than a dozen ligands have been found to interact
with the ErbB-family receptors [6]. These ligands have a
characteristic structure called the EGF-like motif, which is
defined by three disulfide bridges [7–9], and can be classified
into three major groups according to their receptor-binding
specificities. The first group consists of EGF, transforming
growth factor a, and amphiregulin, all of which bind
directly to ErbB-1. The isoforms of neuregulin (NRG, also
known as heregulin and neu differentiation factor) are
members of the second group, and have specific affinity for
ErbB-3 and ErbB-4. The third group is composed of the
ligands that bind to both ErbB-1 and ErbB-4, such as
betacellulin, heparin-binding EGF, and epiregulin.
The binding of ligands to the extracellular region of
receptors causes receptor dimerization and autophosphory-
lation of the C-terminal tail [10]. The phosphorylated
tyrosine residues in the tail serve as docking sites for the
proteins that possess a src homology 2 (SH2) domain [11] or
a phosphotyrosine-binding (PTB) domain [12]. All members
of the ErbB family have docking sites for growth factor
receptor-bound protein 2 (Grb2) and/or SH2-containing
polypeptide (Shc), both of which have SH2 and/or PTB
domains. Recruitment of adapter proteins by the phos-
phorylated receptors can stimulate the Ras signaling
pathway, leading to the activation of extracellular signal-
regulated protein kinases (ERKs) [13]. The Ras/ERK
pathway is one of the most important and well-studied
pathways that transduce extracellular signals into the
intranuclear activation of gene expression [14].

A number of studies have described the ligand interac-
tions of the ErbB family, but most of them have dealt with
only the EGF receptor (ErbB-1). Among the four extracel-
lular domains of ErbB-1, domain III is considered to be
the major binding site for EGF. First, cross-linking of
Correspondence to K. Saito or S. Yokoyama, RIKEN Genomic
Sciences Center, Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
E-mail: or
E-mail:
Abbreviations: CHO, Chinese hamster ovary; DMEM/F-12,
Dulbecco’s modified Eagle’s medium/nutrient mixture F-12; EGF,
epidermal growth factor; ERK, extracellular signal-regulated protein
kinase; NRG, neuregulin; PTB domain, phosphotyrosine-binding
domain; SH2 domain, src homology 2 domain.
*Present address: Center for Cellular Switch Protein Structure,
Korea Research Institute of Bioscience and Biotechnology, Yusong,
Taejon, South Korea.
(Received 26 November 2001, revised 7 March 2002,
accepted 12 March 2002)
Eur. J. Biochem. 269, 2323–2329 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02877.x
125
I-labeled EGF to ErbB-1 in A431 cells resulted in a single-
labeled CNBr fragment (residues 294–543), which involves
domain III [15]. In addition, some monoclonal antibodies
that recognize epitopes in domain III competitively inhibited
the EGF binding [16]. By replacing domain III with that of
the human EGF receptor, the chicken EGF receptor gained
higher affinity for mammalian EGF, similar to that of the
entire human receptor [17,18]. The C-terminal part of EGF
was found to be located near Lys456 of domain III in the

ligand–receptor complex by a cross-linking experiment [19].
Among the ectodomain fragments, the one corresponding to
domain III showed the highest affinity for EGF [20].
Nevertheless, it has been reported that domain I as well as
domain III is involved in EGF binding, suggesting bivalent
binding of EGF to the EGF receptor. A deletion in the
N-terminal region of domain I impaired the EGF binding of
ErbB-1 [21]. The N-terminus of EGF was linked to Tyr101
in domain I by using a covalent cross-linking reagent [22].
Chimeras between the chicken and human EGF receptors
revealed that domain I contributes somewhat to the binding
of EGF, in addition to the major contribution of domain III
[18]. At present, the bivalent manner of EGF binding to the
receptor, in which both domains I and III are utilized, is
accepted within the mechanism of receptor dimerization [23].
Considering that the extracellular domains share high
sequence homology among the four ErbB members, it is
possible that the ligand binding by the ectodomains of other
family members is similar to that of ErbB-1. However, with
respect to the EGF-like motif of the ErbB-3/4 ligand, NRG,
the determinant residues for the specific binding to ErbB-3/4
are somewhat different from those of EGF [24]. The
determinants of NRG are clustered in a part that corres-
ponds to the N-terminal part of EGF, while those of EGF are
in two different parts: the central antiparallel b sheet and the
surface including Tyr13, Leu15, Arg41, and Leu47 [25–28].
In the present study, we constructed a series of chimeric
receptors between ErbB-1 and ErbB-4 (Fig. 1). These two
members of the ErbB family have different specificities for
the ligands, EGF and NRG, respectively, but are very

similar in their other properties. Actually, both ErbB-1 and
ErbB-4 have a ligand-promoted tyrosine kinase activity,
whereas ErbB-2 does not have any authentic ligands and
ErbB-3 is deficient in the kinase activity. Thus, we found
that the relative contributions among the extracellular
domains to the cognate-ligand binding are different between
ErbB-1 and ErbB-4.
EXPERIMENTAL PROCEDURES
Materials
Human EGF (recombinant) and NRG1-b1 (the EGF-like
motif of neuregulin 1-b1, amino-acid residues 176–246,
recombinant) were purchased from R&D Systems Inc.
(Minneapolis, MN, USA). Murine
125
I-labeled EGF was
from NEN Life Science Products, Inc. (Boston, MA, USA).
Anti-(ErbB-1) Ig (sc-03), anti-(ErbB-4) Ig (sc-283), and anti-
ERK2 Ig (sc-153), and a monoclonal antibody to
phosphotyrosine (sc-508), were purchased from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA, USA). The anti-
[phospho-p44/42 MAP kinase (Thr202/Tyr204)] Ig was
obtained from New England Biolabs, Inc. (Beverly, MA,
USA).
Construction of expression plasmids for ErbB-1,
ErbB-4, and chimeric receptors
The mammalian expression plasmid for ErbB-1 was con-
structed as described previously [29]. The full-length cDNA
molecule encoding ErbB-4 was obtained from human brain
Quick-Clone cDNA (Clontech) by the PCR, and was
inserted into the AflII–Xba I sites of the mammalian

expression plasmid pcDNA3.1/Zeo(+) (Invitrogen). A
schematic diagram of the constructed chimeric receptors is
shown in Fig. 1A. The constructs were carefully designed to
maintain the disulfide bond connections within the domains
[30]. The chimeric receptor 1111-4 was generated by
replacing the full-length extracellular region of ErbB-4 (1–
639) with that of ErbB-1 ()24 to 614). The chimeric
receptors 1114-4, 1144-4, and 1444-4 were engineered to
Fig. 1. Schematic representation and expression of ErbB-1, ErbB-4, and
chimeric receptors. (A) EC represents the extracellular region, which
consists of domains I, II, III, and IV; TM, the transmembrane region;
TK, the tyrosine kinase domain; and CT, the C-terminal tail. Con-
structs 1111-1 and 4444-4 correspond to the human wild-type ErbB-1
and ErbB-4, respectively. (B) Whole-cell lysates of CHO cell clones
were resolved by 7.5% SDS/PAGE and were transferred to a nitro-
cellulose membrane. To confirm the expression of the receptors, the
membrane was immunoblotted with an appropriate antibody that
recognizes a region of the C-terminal tail of ErbB-1 or ErbB-4. The
positions of the molecular mass markers (kDa) are shown on the left.
2324 J H. Kim et al. (Eur. J. Biochem. 269) Ó FEBS 2002
contain N-terminal portions of the extracellular region of
ErbB-1 ()24 to 479, )24 to 311, and )24 to 163) and
C-terminal portions of ErbB-4 (500–1308, 333–1308, and
187–1308), respectively. To create chimeric receptors that
have the ErbB-1 cytoplasmic region in common (4444-1,
4441-1, 4411-1, and 4111-1), the entire extracellular region
of ErbB-1 or portions thereof ()24 to 615, )24 to 477, )24
to 311, and )24 to 163) were replaced by the corresponding
regions of ErbB-4 (1–639, 1–498, 1–332, and 1–186,
respectively). All of the chimeric receptors mentioned above

were constructed by the PCR with appropriate primers, and
were confirmed by DNA sequence analysis.
Cell lines and cell culture
Chinese hamster ovary (CHO) cells, which lack endogenous
ErbB-1 and ErbB-4, were grown in Dulbecco’s modified
Eagle’s medium/nutrient mixture F-12 (DMEM/F-12)
medium (Life Technologies, Inc) supplemented with 10%
fetal bovine serum and antibiotics. The mammalian
expression vectors, which were constructed to express
thewild-typeorchimericreceptors,wereintroducedinto
CHO cells by the LipofectAMINE method (Life
Technologies Inc). Several transfectants were selected in
complete medium containing Zeocin (0.2 mgÆmL
)1
).
Comparable expression of the receptors was confirmed by
immunoblotting of the cell lysates (Fig. 1B). As often
observed for membrane proteins, the bands were rather
broad in the blots, due to the heterogeneity of the attached
carbohydrate chains.
EGF binding assay
Confluent cells in 12-well plates were incubated in duplicate
with 10 ngÆmL
)1125
I-labeled EGF in DMEM/F-12 medium
containing 1 mgÆmL
)1
BSA at 4 °C for 2 h. The free
125
I-

labeled EGF was removed by washing three times with ice-
cold NaCl/P
i
containing 1 mgÆmL
)1
BSA. The cells were
lysedin0.5mLof0.5
M
NaOH, and the radioactivity was
measured by a gamma counter. The extent of nonspecific
binding was determined in the presence of a 200-fold excess
of unlabeled EGF.
Stimulation, lysate preparation, immunoprecipitation,
and immunoblotting
The transfected CHO cells were starved in serum-free
medium containing 1 mgÆmL
)1
BSA for 24 h, and then
human EGF or NRG1-b1 was added to stimulate the cells
for 5 min. The cells were washed with ice-cold NaCl/P
i
and
lysed in a buffer containing 30 m
M
Tris/HCl, pH 7.4,
150 m
M
NaCl, 5 m
M
EDTA, 40 m

M
2-glycerophosphate,
10% glycerol, 1% Triton X-100, 1 m
M
phenyl-
methanesulfonyl fluoride, 1 m
M
sodium orthovanadate,
10 lgÆmL
)1
aprotinin, and 10 lgÆmL
)1
leupeptin. Cell
debris was removed by microcentrifugation at 4 °Cfor
10 min, and the protein concentrations of the cell lysates
were measured with a Protein Assay Kit (Bio-Rad). The
lysates were resolved by SDS/PAGE, and then were
transferred to a nitrocellulose membrane for immunoblot-
ting with an appropriate antibody. Protein bands were
visualized by the ECL system (Amersham Pharmacia
Biotech). The ERK activation was determined by
immunoblotting with an antibody that recognizes activated
ERK specifically.
RESULTS
Ligand-binding abilities of the chimeric receptors
The ligand-binding abilities of the chimeric receptors were
measured (Fig. 2). After the incubation with
125
I-labeled
EGF, the cells expressing ErbB-1 retained strong radioac-

tivity as compared to the control cells, while those expres-
sing ErbB-4 did not show any EGF binding (closed bars).
First, by elucidating the relative contributions of the four
extracellular domains of ErbB-1 to the specific binding of
the cognate ligand, EGF, we verified the assays using the
chimeras, because the contributions of the ErbB-1 ectodo-
mains to the EGF binding had already been established by
the study with the chicken and human chimeric EGF
receptors [18]. Chimera 1111-4, in which the transmembrane
and cytoplasmic regions of ErbB-1 (1111-1) were replaced
by those of ErbB-4 (4444-4), showed an adequate affinity
for
125
I-labeled EGF. As the difference in the affinity
between 1111-1 and 1111-4 may arise from the expression
level of each transfectant, 1111-4 has a similar affinity for
125
I-labeled EGF to that of 1111-1, as reported for a similar
chimera between ErbB-1 and ErbB-2 [31]. The transmem-
brane and cytoplasmic regions of ErbB-1 are not involved in
the specific EGF binding. Similarly, 1114-4 showed nearly
the same
125
I-labeled EGF binding as those of 1111-1 and
1111-4, indicating that the extracellular domain IV is not
important for the EGF binding. Furthermore, the
125
I-
labeled EGF bindings of 1111-1, 1111-4, and 1114-4 were
reduced to the background level by the addition of an excess

amount of unlabeled EGF (open bars), but not by NRG1-
b1 (gray bars). Consequently, the three N-terminal do-
mains, I–III, of ErbB-1 possess the binding sites specific for
EGF. In contrast, the replacement of domain III and of
domains III and II, resulting in 1144-4 and 1444-4,
respectively, greatly decreased the
125
I-labeled EGF binding,
indicating that domain III of ErbB-1 is primarily important
Fig. 2. Binding of
125
I-labeled EGF to the wild-type ErbB-1, ErbB-4,
and the chimeric receptors. Monolayers of CHO cell clones expressing
the wild-type ErbB-1, ErbB-4, or the indicated chimeric receptor were
incubated with
125
I-labeled EGF at 4 °C for 2 h. After the incubation,
the cells were washed three times with ice-cold NaCl/P
i
containing
1mgÆmL
)1
BSA and were solubilized with 0.5
M
NaOH. Radioactivity
retained on the cells was then measured by a gamma counter (closed
bars). For competition assays, the cells were incubated with
125
I-
labeled EGF in the presence of a 200-fold excess amount of unlabeled

EGF (open bars) or NRG1-b1(grey bars).
Ó FEBS 2002 Ligand specificities of ErbB-1/ErbB-4 chimeras (Eur. J. Biochem. 269) 2325
for the EGF binding. This agrees with previous reports that
domain III of ErbB-1 is the major binding site for EGF,
even in the bivalent manner of EGF binding. On the other
hand, chimeras 4111-1 and 4411-1, generated by the
replacement of domain I and of domains I and II,
respectively, of ErbB-1 by the corresponding domain(s) of
ErbB-4, retained significant
125
I-labeled EGF binding.
However, the replacement of domain III and of domains
III and IV, which resulted in 4441-1 and 4444-1, respect-
ively, completely abolished the
125
I-labeled EGF binding.
These results confirm that domain III of ErbB-1 plays an
important role in the cognate–ligand interaction of ErbB-1.
Nevertheless, the
125
I-labeled EGF bindings of 4111-1
and 4411-1 were appreciably smaller than those of 1111-1,
1111-4, and 1114-4, indicating that domain I of ErbB-1
participates somewhat in the EGF binding, in addition to
domain III. Chimeras 1144-4 and 1444-4, which contain
domain I but lack domain III of ErbB-1, showed weak, but
detectable,
125
I-labeled EGF binding, because the bindings
were obviously reduced by unlabeled EGF in the assays

using the same transfectant cells. This also indicates that the
ErbB-1 domain I is involved in EGF binding, but the
contribution of domain I is smaller than that of domain III.
This agrees with the previously reported conclusions, that
EGF binding to the receptor depends mainly on domain III
and less on domain I in the bivalent binding [18]. Therefore,
binding assays using the chimeras between ErbB-1 and
ErbB-4 properly evaluate the relative contributions of the
extracellular domains to the cognate-ligand bindings.
Thus, the relative contributions of the four extracellular
domains of ErbB-4 to the specific binding for the cognate
ligand, NRG1-b1, were then examined on the basis of the
competition of unlabeled NRG1-b1with
125
I-labeled EGF
for the chimera binding (grey bars in Fig. 2). Even though
4111-1 has the ErbB-1 domain III, which binds EGF most
strongly among the ectodomains of ErbB-1, the
125
I-labeled
EGF binding to 4111-1 was competitively reduced to the
background level by the addition of unlabeled NRG1-b1,
whereas those of 1114-4, 1111-4, and 1111-1 were not
affected. This suggests that domain I of ErbB-4 greatly
contributes to the cognate-NRG binding of the receptor.
Similarly, the
125
I-labeled EGF binding to 4411-1, which
also has the ErbB-4 domain I and the ErbB-1 domain III,
was reduced by the addition of unlabeled NRG1-b1. In

addition, unlabeled NRG1-b1 hardly reduced the
125
I-
labeled EGF binding of either 1444-4 or 1144-4, even
though these chimeras lack the ErbB-1 domain III, and
therefore exhibit only weak
125
I-labeled EGF binding. These
results confirm the conclusion that domain I contributes the
most to the cognate-NRG binding of ErbB-4.
Autophosphorylation of the chimeric receptors
ErbB-1, ErbB-4, and the chimeric receptors were tested for
autophosphorylation (Fig. 3). In response to the ligand
binding to the ectodomains, the dimerized receptors phos-
phorylate their own cytoplasmic C-terminal tails. After the
cells expressing ErbB-1 were stimulated with EGF or
NRG1-b1, ErbB-1 was immunoprecipitated with an anti-
(ErbB-1) Ig, and the phosphorylation of the receptor was
visualized by immunoblotting with an anti-phosphotyrosine
Ig. ErbB-1 was phosphorylated in response to EGF, but not
to NRG1-b1. Among the chimeric receptors constructed
here, 1111-4, 1114-4, 1144-4, 1444-4, 4111-1, and 4411-1
were phosphorylated by stimulation with EGF. The extents
of the autophosphorylation of 1111-1, 1111-4, 1114-4, 4111-
1, and 4411-1, which all have the ErbB-1 domain III, were
stronger than those of 1144-4 and 1444-4 without the ErbB-
1 domain III. In the
125
I-labeled EGF binding assay (closed
bars in Fig. 2), the former five receptors bound larger

amounts of
125
I-labeled EGF than the latter two. The
receptors with neither domain III nor domain I of ErbB-1,
4444-4, 4444-1, and 4441-1, showed negligible autophosph-
orylation (Fig. 3) and no
125
I-labeled EGF binding (Fig. 2).
Therefore, the contributions of domains III and I in the
EGF-induced autophosphorylation of ErbB-1 were fully
consistent with those in the EGF binding.
Furthermore, the extent of the EGF-induced auto-
phosphorylation of 1444-4 was slightly lower than that of
1144-4, while that of 4111-1 was higher than that of 4411-1
Fig. 3. Ligand-induced tyrosine autophosphorylation of ErbB-1, ErbB-4, and the chimeric receptors. CHO cell clones expressing ErbB-1, ErbB-4, or
each of the chimeric receptors were serum-starved for 24 h in serum-free medium containing 1 mgÆmL
)1
BSA. After the starvation, the cells were
treated with the indicated ligands (20 ngÆmL
)1
) for 5 min or left untreated (–). Lysates were subjected to immunoprecipitation with anti-(ErbB-1) Ig
or anti-(ErbB-4) Ig. Immunoprecipitates were resolved by 7.5% SDS/PAGE, transferred to a nitrocellulose membrane, and visualized by
immunoblotting with an anti-phosphotyrosine Ig. Membranes were stripped and reprobed with the corresponding anti-ErbB Ig to control for
protein loading.
2326 J H. Kim et al. (Eur. J. Biochem. 269) Ó FEBS 2002
(Fig. 3). This indicates that domain II may also contribute
to the ligand interaction. In this context, 4411-1 bound less
125
I-labeled EGF than 4111-1 (closed bars in Fig. 2). In the
case of transforming growth factor a, which belongs to the

same ligand group for the ErbB members as EGF, domain
II of ErbB-1 was found to be involved in the ligand
interaction by insertion mutagenesis [32]. Although three-
dimensional structures are not yet available for the ErbB
members, a comparative model of ErbB-1, based on the
structure of the type-1 insulin-like growth factor receptor
[33], shows that the N-terminal part of domain II is involved
in a lobe of domain I [34]. Domain II of ErbB-1 might
participate in the ligand interaction as a part of ÔstructuralÕ
domain I, or it may support the relative positions between
the ligand-binding domains, I and III. Cells transfected with
either 4414-4 or 1414-4 did not show any response to the
ligands (data not shown). In chimeras with such complica-
ted constructions, misfolding of the receptors might prevent
domains I and III from assuming their correct positions.
On the other hand, ErbB-4 was phosphorylated only by
NRG1-b1, but hardly by EGF. The chimeras with domain I
of ErbB-4, such as 4444-1, 4441-1, 4411-1, and 4111-1,
showed sufficient autophosphorylation in response to
NRG1-b1, while those without domain I of ErbB-4 did
not exhibit any NRG1-b1-induced autophosphorylation. In
contrast, domain III contributes much less than domain I to
NRG binding by ErbB-4, considering the loaded amounts
of the chimeras, shown in a control strip of Fig. 3, and thus
the NRG1-b1-induced autophosphorylations of 4444-1 and
4441-1 were just slightly stronger than those of 4411-1 and
4111–1. Even if the binding of NRG1-b1toErbB-4is
bivalent, like that of EGF to ErbB-1, domain I is
predominant in the specific interaction of ErbB-4 with the
cognate ligand, NRG1-b1.

Activation of downstream ERK by the chimeric receptors
To determine whether the receptor phosphorylation triggers
the activation of downstream cellular pathways, the
chimeric receptors were tested for ligand-induced ERK
activation (Fig. 4). In control cells, the ERK activity was
not affected by either EGF or NRG1-b1. The cells
expressing ErbB-1 activated ERK when treated with
EGF, but not with NRG1-b1. Chimeras 1111-4, 1114-4,
1144-4, 1444-4, 4111-1 and 4411-1, which were all auto-
phosphorylated in response to EGF, induced obvious ERK
activation upon stimulation with EGF. Although transient
overexpression of the receptors in cells may cause non-
physiological autophosphorylation, these observations
show that the phosphorylation of the receptors certainly
transmits the external EGF signal to the downstream
enzymes. However, as compared with the
125
I-labeled EGF
binding and autophosphorylation assays, it is more difficult
to see the contribution of domain III of ErbB-1 to the ERK
activation, for example, from the difference between 1114-4
and 1144-4. This may be due to the saturation of ERK
activation resulting from the amplifying effect of the
signaling cascade.
DISCUSSION
In the present study, by using chimeric receptors, the
contributions of the extracellular domains of ErbB-1 and
ErbB-4 to ligand-specific signaling were examined. In the
case of EGF signaling by ErbB-1, the receptor has two
major functional binding sites, domains I and III, as

suggested by the bivalent binding of EGF. From many
studies using individual mutations of ligand residues, two
contact sites have been mapped on EGF for the interaction
with the receptor: the central antiparallel b sheet and the
surface including Tyr13, Leu15, Arg41, and Leu47 [25–28].
Affinity labeling between ErbB-1 and EGF, using a
heterobifunctional reagent, showed that the N- and
C-terminal parts of the ligand are cross-linked to domains
I and III, respectively, of the receptor [19,22]. The b sheet of
EGF may bind to domain I of ErbB-1, and the other surface
of the ligand binds to domain III of the receptor. Recently,
Gly441 of the ErbB-1 domain III was proposed to be
involved in the binding site that recognizes Arg45 of human
EGF [35].
In contrast, domain I is predominant in the NRG
signaling by ErbB-4. In the case of NRG, although cross-
linking experiments have not been applied to the complex
with ErbB-4, several residues in the N- and C-terminal parts
Fig. 4. Ligand-induced ERK phosphorylation in the cell clones expressing ErbB-1, ErbB-4, or the chimeric receptors. CHO cell clones expressing
ErbB-1, ErbB-4, or each of the chimeric receptors were serum-starved for 24 h in serum-free medium containing 1 mgÆmL
)1
BSA. After the
starvation, the cells were treated with the indicated ligands (20 ngÆmL
)1
) for 5 min or left untreated (–). Whole-cell lysates were resolved by 10%
SDS/PAGE and were immunoblotted with an antibody specific to the active, doubly phosphorylated form of ERK. Membranes were stripped and
reprobed with an anti-ERK Ig to control for protein loading.
Ó FEBS 2002 Ligand specificities of ErbB-1/ErbB-4 chimeras (Eur. J. Biochem. 269) 2327
of the EGF-like motif of the ligand have already been
characterized as high and low affinity sites for the receptor,

respectively [36]. Considering the results of this study, the
N-terminal high affinity site of NRG1-b1 binds to the
dominant ligand-binding site, domain I, of ErbB-4.
Recently, domain I of ErbB-3 was also found to have a
binding site for NRG1-b1 [37].
Thus, our results suggest that both EGF and NRG1-b1
have a similar orientation in the complex with the cognate
receptors, which suggests a common mechanism for both
homodimerization and heterodimerization of the ErbB-
family receptors. Nevertheless, the interaction of the
ligand with the receptor domain I may be somewhat
different between EGF and NRG1-b1. Domain I of
ErbB-1 binds the central b sheet of EGF, while that of
ErbB-4 recognizes several N-terminal residues of NRG1-
b1. Although the b sheet of the ligands is located adjacent
to the N-terminus in the deduced three dimensional
structures, domain I of the receptors may bind a different
part of the cognate ligands, because an artificial ligand,
ÔbiregulinÕ, which was made by the substitution of a
partial NRG sequence for the N-terminus of EGF, bound
with high affinity to both ErbB-1 and ErbB-4 [24]. When
bound to the receptors, this artificial molecule uses the
N-terminal and b sheet parts to bind with domains I of
ErbB-4 and ErbB-1, respectively.
In conclusion, we have demonstrated differences in the
relative contributions of the domains of ErbB-1 to the EGF
signaling and those of ErbB-4 to the NRG signaling.
Analyses using chimeric receptors are very useful to
elucidate the relative contributions among the domains of
the receptors in living cells.

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