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

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].

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

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

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

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

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

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

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.

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,

REFERENCES
1. Walker, R.A. (1998) The erbB/HER type 1 tyrosine kinase
receptor family. J. Pathol. 185, 234–235.
2. Ullrich, A., Coussens, L., Hayflick, J.S., Dull, T.J., Gray, A., Tam,
A.W., Lee, J., Yarden, Y., Libermann, T.A., Schlessinger, J. et al.
(1984) Human epidermal growth factor receptor cDNA sequence
and aberrant expression in A431 epidermoid carcinoma cells.
Nature 309, 418–425.
3. Yamamoto, T., Ikawa, S., Akiyama, T., Semba, K., Nomura, N.,
Miyajima, N., Saito, T. & Toyoshima, K. (1986) Similarity of
protein encoded by the human c-erb-B-2 gene to epidermal growth
factor receptor. Nature 319, 230–234.
4. Kraus, M.H., Issing, W., Miki, T., Popescu, N.C. & Aaronson,
S.A. (1989) Isolation and characterization of ERBB3,athird
member of the ERBB/epidermal growth factor receptor family:
evidence for overexpression in a subset of human mammary
tumors. Proc. Natl Acad. Sci. USA 86, 9193–9197.
5. Plowman, G.D., Green, J.M., Culouscou, J.M., Carlton, G.W.,
Rothwell, V.M. & Buckley, S. (1993) Heregulin induces tyrosine
phosphorylation of HER4/p180
erbb4
. Nature 366, 473–475.
6. Riese, D.J. II & Stern, D.F. (1998) Specificity within the EGF
family/ErbB receptor family signaling network. Bioessays 20,
41–48.
7. Hommel, U., Harvey, T.S., Driscoll, P.C. & Campbell, I.D. (1992)
Human epidermal growth factor. High resolution solution struc-
ture and comparison with human transforming growth factor a.
J. Mol. Biol. 227, 271–282.
8. Kohda, D. & Inagaki, F. (1992) Three-dimensional nuclear

15. Lax, I., Burgess, W.H., Bellot, F., Ullrich, A., Schlessinger, J. &
Givol, D. (1988) Localization of a major receptor-binding domain
for epidermal growth factor by affinity labeling. Mol. Cell. Biol. 8,
1831–1834.
16. Wu, D., Wang, L., Sato, G.H., West, K.A., Harris, W.R., Crabb,
J.W. & Sato, J.D. (1989) Human epidermal growth factor (EGF)
receptor sequence recognized by EGF competitive monoclonal
antibodies. Evidence for the localization of the EGF-binding site.
J. Biol. Chem. 264, 17469–17475.
17. Lax, I., Bellot, F., Howk, R., Ullrich, A., Givol, D. & Schlessinger,
J. (1989) Functional analysis of the ligand binding site of EGF-
receptor utilizing chimeric chicken/human receptor molecules.
EMBO J. 8, 421–427.
18. Lax,I.,Fischer,R.,Ng,C.,Segre,J.,Ullrich,A.,Givol,D.&
Schlessinger, J. (1991) Noncontiguous regions in the extracellular
domain of EGF receptor define ligand-binding specificity. Cell
Regul. 2, 337–345.
19. Summerfield, A.E., Hudnall, A.K., Lukas, T.J., Guyer, C.A. &
Staros, J.V. (1996) Identification of residues of the epidermal
growth factor receptor proximal to residue 45 of bound epidermal
growth factor. J. Biol. Chem. 271, 19656–19659.
20. Kohda, D., Odaka, M., Lax, I., Kawasaki, H., Suzuki, K., Ullrich,
A., Schlessinger, J. & Inagaki, F. (1993) A 40-kDa epidermal
growth factor/transforming growth factor a-binding domain
produced by limited proteolysis of the extracellular domain of the
epidermal growth factor receptor. J. Biol. Chem. 268, 1976–1981.
21. Lax, I., Bellot, F., Honegger, A.M., Schmidt, A., Ullrich, A.,
Givol, D. & Schlessinger, J. (1990) Domain deletion in the extra-
cellular portion of the EGF-receptor reduces ligand binding and
impairs cell surface expression. Cell Regul. 1, 173–188.

the atypical Asn-X-Cys sequence of recombinant human epi-
dermal growth factor receptor. J. Biochem. 127, 65–72.
30.Abe,Y.,Odaka,M.,Inagaki,F.,Lax,I.,Schlessinger,J.&
Kohda, D. (1998) Disulfide bond structure of human epidermal
growth factor receptor. J. Biol. Chem. 273, 11150–11157.
31. Lotti, L.V., Lanfrancone, L., Migliaccio, E., Zompetta, C., Pelicci,
G., Salcini, A.E., Falini, B., Pelicci, P.G. & Torrisi, M.R. (1996)
Shc proteins are located on endoplasmic reticulum membranes
and are redistributed after tyrosine kinase receptor activation.
Mol. Cell. Biol. 16, 1946–1954.
32. Harte, M.T. & Gentry, L.E. (1995) Mutations within subdomain
II of the extracellular region of epidermal growth factor receptor
selectively alter TGFa binding. Arch. Biochem. Biophys. 322,
378–389.
33. Garrett, T.P.J., McKern, N.M., Lou, M., Frenkel, M.J.,
Bentley, J.D., Lovrecz, G.O., Elleman, T.C., Cosgrove, L.J. &
Ward, C.W. (1998) Crystal structure of the first three domains
of the type-1 insulin-like growth factor receptor. Nature 394,
395–399.
34. Jorissen,R.N.,Epa,V.C.,Treutlein,H.R.,Garrett,T.P.J.,Ward,
C.W. & Burgess, A.W. (2000) Characterization of a comparative
model of the extracellular domain of the epidermal growth factor
receptor. Protein Sci. 9, 310–324.
35. Elleman, T.C., Domagala, T., McKern, N.M., Nerrie, M.,
Lo
¨
nnqvist, B., Adams, T.E., Lewis, J., Lovrecz, G.O., Hoyne,
P.A., Richards, K.M. et al. (2001) Identification of a determinant
of epidermal growth factor receptor ligand-binding specificity
using a truncated, high-affinity form of the ectodomain. Bio-