From Genentech, Inc., South San Francisco, California
94080 and the § Institut für Biologie III, University
of Freiburg, D-79104 Freiburg im Breisgau, Germany
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ABSTRACT |
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ErbB3 is unique among other members of the
receptor tyrosine kinase family of growth factor receptors in that its
kinase domain is enzymatically impaired. This renders it incapable of
transducing a signal in response to ligand binding. However, in
conjunction with ErbB2, ErbB3 is a potent mediator of signaling by the
growth factor heregulin. Heregulin binding to ErbB3 induces formation of a heterodimeric complex with ErbB2, and this results in
transactivation of the ErbB2 kinase. Although interaction between the
extracellular domains of these receptors is an essential part of this
process, it was not clear whether interaction between the cytoplasmic
domains is also necessary for transactivation. By examining the
abilities of a series of cytoplasmic domain mutants of ErbB3 to
activate ErbB2, we have found a discrete sequence of three amino acid
residues (LVI), located at the carboxyl-terminal end of the impaired
ErbB3 kinase region, that is obligatory for transactivation. We
conclude that formation of a functional ErbB2-ErbB3 signaling complex
requires the presence of a specific structural feature within the ErbB3 cytoplasmic domain and suggest that ErbB2 transactivation results from
a physical interaction between the cytoplasmic domains of these receptors.
Receptor tyrosine kinases play a pivotal role in the transduction
of extracellular signals into the cells. The binding of cognate growth
factors to these cell-surface receptors results in receptor
oligomerization and activation of the intrinsic kinase activity (1, 2).
This leads to receptor phosphorylation and triggers a cascade of
intracellular signaling events that ultimately elicit a variety of
cellular responses such as proliferation, differentiation, survival, or migration.
An extensively characterized subgroup of this receptor superfamily is
the ErbB group of receptors, also known as the class I receptor
tyrosine kinases. Members of this group include the epidermal growth
factor receptor (EGFR1 or
ErbB1), ErbB2 (also termed HER2 or Neu), ErbB3 (HER3), and ErbB4
(HER4). EGFR binds several distinct ligands including EGF and
transforming growth factor- The extensive interreceptor associations that occur in the ErbB family
serve to increase the repertoire of cellular responses to growth factor
stimulation and to fine-tune growth factor signaling. At least 10 different homo- and heteromeric combinations of ErbB proteins have been
reported (10, 11). However, these combinations are not equally
favorable. The interreceptor interactions are hierarchically organized,
where ErbB2 is the preferred heteromeric partner, and it favors
interaction with ErbB3 (12, 13).
Cross-talk between ErbB2 and ErbB3 is especially important as the
kinase of ErbB3 is dysfunctional. The impaired kinase activity has been
demonstrated in several systems. It was initially reported in insect
cells expressing ErbB3 (14), later by demonstrating the lack of any
biological activity in cells expressing ErbB3 alone (10, 11), and
recently by biochemical analysis of the purified kinase domain (15).
Alterations of four amino acid residues in the kinase region that are
otherwise conserved among all protein tyrosine kinases (16) may account
for the lack of catalytic activity. ErbB2, however, is characterized by
a constitutively active kinase (17). The physiological significance of
this heteromeric complex is emphasized by the fact that the presence of
ErbB2 in ErbB3-expressing cells significantly enhances the
transformation ability (18, 19). Inhibition of ErbB2 and ErbB3 complex
formation abolishes HRG-mediated signaling (9, 13, 20). Additionally, active ErbB2-ErbB3 receptor complexes have been seen in several mammary
tumor cell lines, indicating the relevance of this heteromeric receptor
aggregate in human neoplasia (18, 21).
HRG binds with low affinity to kinase-inactive ErbB3. Recruitment of
ErbB2 into the HRG-ErbB3 complex leads to the formation of a high
affinity HRG-binding receptor, which is capable of generating a
tyrosine phosphorylation signal due to the kinase activity of ErbB2
(7). Studies on the isolated extracellular domain of ErbB3 show that
ligand binding is exclusively mediated by the extracellular region of
ErbB3 (22). Furthermore, the interaction between ErbB2 and ErbB3 upon
HRG stimulation is seen with a modified version of the extracellular
domain of ErbB3 containing a glycosylphosphatidylinositol moiety
anchoring it to the plasma membrane (23). Also, receptor IgGs
consisting of the extracellular domains of ErbB3 and ErbB2 fused to an
immunoglobulin Fc domain show increased HRG binding affinity compared
with ErbB3 binding alone (24). Taken together, it appears that HRG
binding and the affinity shift for HRG binding in the presence of ErbB2
require only the extracellular domains of these receptors.
Details of the molecular mechanism that leads to the activation of
ErbB2 kinase are unknown, and in particular, the role of the
intracellular domain of ErbB3 is uncertain. We questioned whether
structural elements in the intracellular domain of ErbB3 were needed
for the transactivation of ErbB2. In this study, we designed a series
of C-terminal deletion and substitution mutants of ErbB3 and assessed
the phosphorylation status of the receptors in the complex. We report
here that a distinct three-amino acid segment (LVI) in the
intracellular domain of ErbB3 is required for transactivation of ErbB2
and propose that this is an intermediate step between ligand-induced
receptor dimerization and kinase activation.
Materials--
Monoclonal anti-ErbB2 antibody 3E8 has been
described previously (25). Polyclonal rabbit anti-ErbB2 antibody was
acquired from Dako Corp. (Carpinteria, CA). Polyclonal rabbit
anti-ErbB3 (C-17) and rabbit anti-ErbB4 antibodies were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine
antibody conjugated to horseradish peroxidase was obtained from
Transduction Laboratories (Lexington, KY). Monoclonal antibody 5B6
raised against the herpes simplex glycoprotein D signal sequence has
been described elsewhere (26). The EGF-like domain of
HRG Cell Culture and Transient Transfections--
COS-7 cells
(American Type Culture Collection, CRL1651) and K562 cells (CCL243)
were cultured in a 50:50 mixture of Ham's F-12 medium and Dulbecco's
modified Eagle's medium supplemented with 10% heat-inactivated fetal
bovine serum, 2 mM glutamine, and 10%
penicillin/streptomycin. COS-7 cells were transfected using the
LipofectAMINETM protocol obtained from Life Technologies,
Inc. Transfections were carried out for 24-36 h, and experiments were
initiated immediately. A stable K562 cell line that expressed human
ErbB3 was generated by electroporation using a derivative of pcDNA3
(Invitrogen) containing the open reading frame of erbB3.
This cell line was transiently transfected with the appropriate ErbB2
expression plasmids using SuperFect transfection reagent purchased from
QIAGEN, Inc. Cells (1 × 107) were transfected with 30 µg of DNA and 120 µl of SuperFect reagent according to the
supplier's standard protocol and were subjected to further analysis
after 48 h.
Construction of Receptor Mutants--
Site-directed mutagenesis
was performed to obtain C-terminal ErbB3 truncation or internal
deletion/substitution mutants (28). All receptor constructs were
derived from a pRK7-based vector (29). The endogenous signal sequences
of ErbB2 and ErbB3 were replaced with the herpes simplex signal
sequence of glycoprotein D for quantitative analysis (30). The cDNA
sequence of erbB2 was subcloned into the XbaI and
ClaI sites of pBluescript SK 125I-rHRG Binding Assay--
COS-7 cells were
transfected in 15-cm plates. After 30 h, cells were removed from
dishes using 2 mM EDTA in phosphate-buffered saline. Cells
were transferred onto 96-well plates at a density of 1.8 × 105 cells/well in final volume of 250 µl of binding
buffer (Dulbecco's modified Eagle's medium, 10%
penicillin/streptomycin, and 0.2% bovine serum albumin). Cells were
incubated with varied concentrations of rHRG Tyrosine Phosphorylation Assay--
COS-7 cells were transfected
in 12-well plates. After 24-36 h, cells were washed with serum-free
Ham's F-12 medium/Dulbecco's modified Eagle's medium and
serum-starved for 2-4 h. Cells were stimulated with the indicated
concentrations of HRG. After incubation for 8 min at room temperature,
the medium was carefully aspirated, and reactions were stopped by
adding 250 µl of sample buffer (5% SDS, 1% dithiothreitol, and 25 mM Tris-HCl, pH 6.8). Each sample (20 µl) was subjected
to SDS-polyacrylamide electrophoresis using a 4-12% gradient gel
(Novex) and then electrophoretically transferred onto a nitrocellulose
membrane. Blots were probed with monoclonal anti-phosphotyrosine
antibody conjugated to horseradish peroxidase (125 ng/ml), and
immunoreactive bands were visualized with chemiluminescence detection reagent (ECL, Amersham Pharmacia Biotech).
Chemical Cross-linking and Immunoprecipitation--
Transfected
COS-7 cells were resuspended in Hanks' balanced salts containing 20 mM HEPES, pH 7.4, and incubated with radiolabeled 125I-rHRG The Intracellular Domain of ErbB3 Is Necessary for
Transactivation of the ErbB2 Kinase--
To determine whether
activation of the ErbB2 kinase by ErbB3 requires a specific interaction
with the cytoplasmic domain of ErbB3, we constructed a truncated
version of ErbB3. This mutant, designated ErbB31-665,
essentially lacks the entire cytoplasmic domain of the receptor. The
functional characteristics of ErbB31-665 were examined by
transiently expressing the truncated receptor in COS-7 cells.
Expression of ErbB31-665 at the cell surface was confirmed
by competitive binding analysis using 125I-rHRG
To determine whether deletion of the cytoplasmic domain interfered with
the ability of the receptor to interact with ErbB2, COS-7 cells were
co-transfected with erbB2 and either erbB3 or erbB31-665 cDNA, and chemical cross-linking
experiments were performed using 125I-rHRG
To confirm the association of ErbB31-665 with ErbB2,
cross-linked proteins were immunoprecipitated using a monoclonal
antibody (3E8) directed against ErbB2. As shown in lanes 5 and 6 in Fig. 1B, a cross-linking pattern similar
to that seen in lanes 1 and 3 was
observed. Since 125I-rHRG
We next looked at the ability of truncated ErbB3 to induce tyrosine
phosphorylation upon HRG stimulation. Since COS-7 cells naturally
express low levels of ErbB2, which form heteromeric complexes with
ectopically expressed ErbB3, co-transfection with exogenous
erbB2 was not necessary in these experiments. Cells that
expressed full-length ErbB3 showed a dose-dependent
increase in tyrosine phosphorylation after HRG stimulation (Fig.
2). The double band visible at ~185 kDa
represented phosphorylated ErbB2 and ErbB3. In contrast, although
ErbB31-665 binds HRG and associates with ErbB2, it was not
able to activate the ErbB2 kinase. The results shown in Fig. 2
suggested that a direct or indirect interaction between the
intracellular domains of ErbB3 and ErbB2 was necessary for HRG-mediated
activation of the intrinsic kinase activity of ErbB2.
Mapping the Intracellular Transactivation Domain of ErbB3--
We
questioned whether the entire intracellular domain of ErbB3 or only a
segment of it was required for ErbB2 transactivation. To address this
issue, a series of ErbB3 truncation mutants containing smaller
deletions from the C terminus were constructed. These are shown in
schematic form in Fig. 3A. The
receptor mutants were transiently expressed and subjected to
HRG-induced receptor activation analysis as described above. In these
mutants, the phosphorylation signals observed were exclusively due to
tyrosine phosphorylation of endogenous ErbB2 because the truncation
mutants lacked the tyrosine phosphorylation sites located on the C
terminus of the full-length receptor. Phosphorylation results are also
indicated in Fig. 3A. Surprisingly, ErbB31-952
did not show HRG-induced phosphorylation, whereas
ErbB31-963 showed a dose-dependent increase in
ErbB2 phosphorylation upon HRG stimulation. To further map this area,
we engineered additional truncation mutants to identify regions that
may contribute to ErbB2 transactivation. ErbB31-959 was
able to stimulate receptor phosphorylation, yet additional deletion of
Ile959 resulted in a decreased phosphorylation signal, and
deletion of Val958 in ErbB31-957 completely
abolished stimulation of tyrosine phosphorylation (Fig. 3B).
Thus, we were able to localize the region responsible for the
transactivation activity to an area N-terminal to amino acid 959. This
critical region is found at the C-terminal end of the impaired kinase
domain, as shown in Fig. 3C.
Fine Mapping of the Transactivation Area in the Context of the
Full-length Receptor by Internal Deletion and Substitution
Mutants--
Further characterization of the ErbB3 transactivation
region was carried out by constructing internal deletion and
substitution mutants in the context of the full-length receptor.
Although Tyr956 on ErbB3 has not been described as a
potential phosphorylation site, we questioned whether
Tyr956 might play a role in the regulation of
transactivation, possibly by serving as a phosphorylation site for
another kinase. We therefore replaced Tyr956 with
phenylalanine in the full-length ErbB3 receptor. Analysis of the
substitution mutant Y956F showed a strong phosphorylation signal,
indicating that Tyr956 does not play a direct role in the
transactivation process (Fig. 4A).
Fig. 4A summarizes a series of internal deletion mutants
that were designed to fine map the region responsible for ErbB2
transactivation. ErbB3
It is possible that deletion of three amino acids at positions 957-959
altered the spatial alignment of important structural features on ErbB3
relative to corresponding sites on ErbB2. The inability of ErbB3
To further characterize this area, a series of double and single
substitution mutants were designed. These mutants are outlined in
schematic form in Fig. 4A along with results of the
phosphorylation analysis for each. Interestingly, in contrast to
ErbB3 The LVI Segment Is Conserved in ErbB4 and Is Necessary for ErbB2
Transactivation--
Sequence alignment of the tyrosine kinase domains
of members of the ErbB receptor family revealed that the LVI segment is conserved in ErbB4 as well as ErbB3 and EGFR (Fig.
5). We questioned whether a similar
transactivation mechanism also occurred between ErbB2 and ErbB4. To
assess ErbB2 transactivation in an ErbB2-ErbB4 heterodimer, we
eliminated the intrinsic kinase activity of ErbB4 by constructing an
ErbB4 mutant in which Lys726 at the ATP-binding site was
replaced with methionine (ErbB4M726) (Fig.
6A). The lack of kinase
activity in this mutant was verified by phosphorylation analysis using
a human hematopoietic cell line (K562) that is devoid of all ErbB
family members (data not shown). Receptor activation analysis in COS-7
cells expressing ErbB4M726 showed a
dose-dependent phosphorylation signal upon HRG treatment (Fig. 6B). These data confirmed that a similar
transactivation of ErbB2 also occurred in ErbB2-ErbB4 heterodimers. To
investigate the importance of the LVI segment in ErbB4, a three-amino
acid deletion mutant lacking residues Leu960,
Val961, and Ile962 was constructed
in the context of ErbB4M726. COS-7 cells expressing
ErbB4M726 An Associated Cytoplasmic Kinase Is Not Involved in Receptor
Autophosphorylation upon HRG Stimulation--
It is conceivable that
ErbB2 transactivation is not the result of a direct interaction that
occurs solely between ErbB3 and ErbB2. One scenario is that a third
protein with tyrosine kinase activity is bound to the cytoplasmic
domain of ErbB3 and that this kinase phosphorylates ErbB2. This would
be similar to the transactivation mechanism observed in cytokine
receptor and T-cell antigen receptor signaling (31, 32). If the
activity of a third kinase was directly regulated by ErbB3, the kinase
activity of ErbB2 would not be required for receptor phosphorylation.
To test this possibility, we constructed a kinase-inactive ErbB2 mutant
in which Lys732 at the ATP-binding site was replaced with
methionine. Ectopic expression of this mutant was then performed in a
hematopoietic cell line that expressed only ErbB3 (K562erbB3)
(Fig. 7A). If a cytoplasmic
kinase was responsible for autophosphorylation, cells that co-express
ErbB3 and kinase-inactive ErbB2 would still show a phosphorylation
signal upon HRG treatment. Cells transfected with wild-type
erbB2, erbB2M732, or control
expression plasmids were incubated with HRG and then subjected to
immunoprecipitation with antibodies directed against ErbB2 or ErbB3.
Cells transfected with erbB2 cDNA demonstrated tyrosine
phosphorylation on ErbB2 and ErbB3 (Fig. 7B,
lanes 1-4). Interestingly, ErbB2 was
constitutively phosphorylated, whereas ErbB3 phosphorylation was
HRG-dependent. In cells transfected with either
erbB2M732 or control expression plasmids, HRG
stimulation caused no autophosphorylation on ErbB2 or ErbB3. The
expression of ErbB2 and ErbB2M732 was verified by Western
blot analysis (Fig. 7B). These data showed that deactivation
of the ErbB2 kinase completely abolished the phosphorylation signal and
confirmed that ErbB3 has no intrinsic kinase activity.
Autophosphorylation of the receptors was therefore not due to a
cytoplasmic kinase activated by binding to the transactivation segment
of ErbB3, but was the result of the ErbB2 kinase.
The allosteric oligomerization model proposed for EGFR by
Schlessinger (33, 34) predicts that ligand binding induces formation of
receptor dimers, which brings the intracellular domains into close
proximity, and causes them to phosphorylate one another in
trans. Because ErbB3 lacks intrinsic kinase activity and
ErbB2 does not bind HRG, this model does not fully explain the
phosphorylation pattern observed in the ErbB2-ErbB3 complex following
HRG stimulation. It was also previously unclear whether activation of
the intrinsic kinase required specific cytoplasmic domain interactions.
Here, we present data showing that transactivation of the ErbB2 kinase by ErbB3 requires the presence of a structural element within the ErbB3
cytoplasmic domain. Using ErbB3 deletion and substitution mutants, we
found a discrete sequence of three amino acids (LVI) at the carboxyl
terminus of the inactive kinase domain of ErbB3 that is necessary for
ErbB2 transactivation. Deletion of segments distal to this region had
no effect, indicating that they are not required for this activity. At
present, we cannot rule out the possibility that additional sequences
N-terminal to this region may also contribute to the transactivation of ErbB2.
The results of our study suggest at least two models whereby direct
sequence-specific molecular interactions may result in receptor
transactivation. These models extend the Schlessinger hypothesis (33,
34) to the ErbB2-ErbB3 system and are shown schematically in Fig.
8. In the first model, activation of
ErbB2 results from a direct interaction between the intracellular
domains of the two receptors. In this model, HRG binding to ErbB3
results in a conformational change in the extracellular domain of ErbB3 facilitating recruitment of ErbB2 and formation of the heterodimeric complex. The interaction between the extracellular domains of the
receptors aligns their intracellular domains, bringing the LVI motif of
ErbB3 into direct contact with an as yet undefined region of ErbB2.
This, in turn, leads to the activation of the ErbB2 kinase. An
alternative model assumes the participation of a third protein.
Ligand-induced heterodimerization of ErbB3 with ErbB2 allows an adaptor
molecule with specific recognition sites for each receptor to bridge
their intracellular domains. The putative adaptor protein binds to the
LVI sequence in ErbB3 and to an unknown sequence in ErbB2. We postulate
that the adaptor stabilizes interactions between adjacent cytoplasmic
domains that lead to the activation of the ErbB2 kinase, resulting in
transphosphorylation of the receptors.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(3). ErbB3 and ErbB4 bind isoforms of
the heregulin family (also designated neuregulin or Neu differentiation
factor) (4, 5). A ligand that directly binds to ErbB2 has not been
identified. Nevertheless, ErbB2 plays an important role in signaling.
ErbB2 is transactivated by heterodimerization with ligand-occupied
EGFR, ErbB3, or ErbB4 (6-9).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1-(177-244) was expressed in Escherichia coli,
purified, and radioiodinated as described previously (7, 27). The
EGF-like domain of HRG
1-(177-244) was used in all experiments and
designated rHRG
1.
(Stratagene).
Using oligonucleotide 5'-CTCTTGCCCCTCGAGGCCGCGAGC- 3', an
XhoI site at the end of the erbB2 signal sequence
was inserted by site-directed mutagenesis. A derivative of pRK7 (pChad)
was used as an expression plasmid containing the coding sequence for amino acids 1-51 of herpes simplex glycoprotein D. The
XhoI-ClaI fragment encoding mature ErbB2 was
subcloned into the XhoI and ClaI sites of the
pChad vector, fusing the glycoprotein sequence upstream of the
erbB2 sequence. The open reading frame of erbB3 was subcloned into the KpnI site of pBluescript
SK
. A SalI site was engineered using
oligonucleotide 5'-GGAGCCCCGGTCGACGCTGAAAAG-3' at the end of the
erbB3 signal sequence. The erbB3 sequence was excised with SalI and subcloned in frame into the
XhoI site of the pChad vector. C-terminal truncation mutants
were obtained by inserting a stop codon downstream of the last desired
amino acid codon. Internal deletion and substitution mutants were
engineered using oligonucleotides that removed the amino acid codons or
replaced the designated amino acid codons with alanine codons,
respectively. Confirmation of receptor constructs was obtained by DNA
sequencing using a dideoxy method modified for double-stranded DNA (T7
SequenaseTM, Version 2.0, Amersham Pharmacia Biotech).
1 and a constant amount
(75 pM) of 125I-labeled rHRG
1. Binding was
carried out on ice for 14 h. To separate cell-bound
125I-rHRG
1 from free, cells were transferred onto a
96-well filtration plate assembly (Multiscreen assay system, Millipore
Corp.) and placed on a vacuum manifold to wash and remove unbound
label. Separation was carried out as recommended by the supplier.
Samples were counted using a 100 Series Iso Data
-counter.
1 (0.5 nM) in the presence or
absence of 200 nM unlabeled rHRG
1. Incubation was
carried out at room temperature for 20 min. Chemical cross-linking was
performed by adding bis(sulfosuccinimidyl) suberate (Pierce) to a final
concentration of 1 mM and allowing it to react for 20 min
at room temperature. Samples were run on 5% SDS-polyacrylamide gels,
and cross-linked complexes were visualized by autoradiography.
Immunoprecipitation experiments were performed on transfected COS-7
cells after chemical cross-linking or on K562erbB3 cells
transfected with vector alone, erbB2, or
erbB2M753 cDNA. K562 cells were
serum-starved for 2 h and treated with rHRG
1 (10 nM) or buffer alone for 15 min at 37 °C. K562 or COS-7 cells were lysed in lysis buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1%
CHAPS, 0.5 mM Na3VO4, 0.2 mM phenylmethylsulfonyl fluoride, 50 trypsin inhibitory
units/liter aprotinin, and 10 µM leupeptin).
Immunoprecipitations were performed with anti-ErbB2 (3E8), anti-ErbB2,
or anti-ErbB3 antibody. Immune complexes were purified by absorption on
immobilized protein A/G (Ultralink immobilized protein A/G, Pierce),
and samples were subjected to SDS-polyacrylamide gel electrophoresis.
In the case of K562 samples, Western blot analysis with
anti-phosphotyrosine antibody was accomplished as described above.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1.
Untransfected COS-7 cells do not bind 125I-rHRG
1 because
they lack endogenous ErbB3 and ErbB4. As expected, ErbB31-665-expressing cells displayed an rHRG
1-binding
site (4.3 × 104 ± 7.0 × 103
sites/cell) with a dissociation constant of 1.56 ± 0.22 nM. This was comparable to cells that were transfected with
full-length erbB3 (2.7 × 104 ± 2.8 × 103 sites/cell) and displayed a binding affinity of
1.1 ± 0.34 nM (Fig.
1A) (7).
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Fig. 1.
Binding analysis of
125I-rHRG 1 to COS-7 cells expressing
ErbB31-665. A, displacement of
125I-rHRG
1 binding to COS-7 cell transfectants by
unlabeled rHRG
1. Transiently transfected cells expressing
ErbB31-665 (
) or ErbB3 (
) were incubated with
radiolabeled rHRG
1 and the indicated amounts of unlabeled rHRG
1.
Incubation was carried out on ice overnight, and cell-bound
radiolabeled ligand was separated from unbound. The results are shown
as displacement curves or Scatchard plot (inset).
B, chemical cross-linking of 125I-HRG
1 to
cell-surface receptors on COS-7 cell transfectants and subsequent
immunoprecipitation with anti-ErbB2 antibody. Cells were co-transfected
with full-length erbB2 and full-length erbB3 or
with full-length erbB2 and
erbB31-665 expression plasmids, respectively.
Cells were detached and, prior to cross-linking, were incubated with
0.5 nM 125I-rHRG
1 in the presence (+) or
absence (
) of 200 nM unlabeled rHRG
1.
Bis(sulfosuccinimidyl) suberate was added to the cell suspension,
and incubations were continued. Cells were washed and resuspended
in SDS sample buffer. For lanes 5 and 6,
following cross-linking, cell lysates were prepared, and
immunoprecipitation (IP) was performed with anti-ErbB2
antibody 3E8. Samples were run on a 5% SDS-polyacrylamide gel, and
radioactive cross-linked complexes were visualized by autoradiography.
xsHRG, cross-linked HRG; IP,
immunoprecipitation.
1 (Fig.
1B). A cross-linked product of 100 kDa was observed in cells
expressing ErbB31-665 (Fig. 1B, lane
3). The mobility of this band was consistent with the predicted
size of the mutated receptor. In addition, a higher molecular mass
complex was also present. The mobility of this higher molecular mass
complex was slightly faster than that observed when wild-type ErbB3 was
co-expressed with ErbB2 (Fig. 1B, lane 1).
1 does not become directly
cross-linked to ErbB2, these data indicated that the receptor complexes
in both the ErbB3- and ErbB31-665-transfected cells
contained ErbB2. Therefore, the high molecular mass complex represents
cross-linked receptors and cross-linked radiolabeled HRG.
Interestingly, these complexes migrated slower than expected for an
ErbB2-ErbB3 receptor dimer, suggesting that a higher order complex was
formed instead. The bands at 190 and 100 kDa in lanes
5 and 6 represented ErbB3 and
ErbB31-665, respectively, which were associated with
ErbB2, but not covalently cross-linked to it. Furthermore, these data
suggested that the interaction of ErbB31-665 with ErbB2
was similar to that of the full-length receptor. The results presented
in Fig. 1 show that deletion of the entire cytoplasmic region of ErbB3
does not interfere with either its ligand binding characteristics or
its ability to form heteromeric complexes with ErbB2.
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Fig. 2.
HRG-stimulated receptor
autophosphorylation. COS-7 cells were transfected in 12-well
plates with erbB3 or erbB31-665
expression plasmids. Cells were treated 28-32 h post-transfection with
the indicated amounts of rHRG 1. Cell lysates were subjected to SDS
gel electrophoresis and then transferred to nitrocellulose. For the
upper panels, blots were probed with anti-phosphotyrosine
antibody (
P-Tyr). The lower panels demonstrate
ErbB3 expression. Blots were probed with monoclonal antibody 5B6, which
recognizes glycoprotein D (gD)-tagged ErbB3 and
ErbB31-665.
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Fig. 3.
Receptor phosphorylation analysis of ErbB3
truncation mutants. A, schematic representation of
C-terminal deletion mutants and their ability to induce phosphorylation
in COS-7 cells. The portion of the extracellular domain is indicated as
ECD; the transmembrane domains (TM) are
represented as hatched boxes; and the tyrosine kinase
domains (TK) or portions of them are represented as
black boxes. The mature receptor is indicated as ErbB3,
whereas the various C-terminal truncation mutants are designated
according to their remaining amino acid residues. C-terminal mutants
were analyzed in phosphorylation assays as described in the legend to
Fig. 2, and their ability (+) or inability ( ) to induce receptor
phosphorylation is indicated on the right. B,
phosphorylation analysis of ErbB31-959,
ErbB31-958, and ErbB31-957. COS-7 cells were
transfected with the corresponding expression plasmids, and tyrosine
phosphorylation assay was performed as described in the legend to Fig.
2. C, partial amino acid sequence of ErbB3 that spans the
junction between the kinase domain and the C-terminal tail. Amino acid
residues of the kinase domain are represented in
boldface. The number beneath the amino acid isoleucine
indicates its position in the mature receptor.
P-Tyr,
anti-phosphotyrosine antibody;
gD, anti-glycoprotein D
antibody.
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Fig. 4.
Tyrosine phosphorylation analysis of internal
and substitution mutants. A, schematic diagram of
receptor mutants and their ability to induce receptor
autophosphorylation. Receptors and receptor domains are depicted as
described in the legend to Fig. 3. Internal deletion areas are shown as
gaps. Substituted amino acid residues are indicated beneath
the receptors. Receptor mutants underwent phosphorylation analysis as
described in the legend to Fig. 2, and their ability (+) or inability
( ) to induce transphosphorylation is indicated on the right.
B, phosphorylation analysis of ErbB3 mutants ErbB3
LVI and
L957A/V958A/I959A. COS-7 cells were transfected with corresponding
expression plasmids and analyzed for receptor phosphorylation as
described in the legend to Fig. 2.
P-Tyr,
anti-phosphotyrosine antibody;
gD, anti-glycoprotein D
antibody.
955-962, which lacked the eight amino acids
that span the junction between the kinase domain and the C-terminal
tail, was devoid of receptor phosphorylation activity. Additional
constructs with smaller internal deletions were engineered and tested
for transactivation activity. Deletion of three amino acid residues, Leu957, Val958, and Ile959
(ErbB3
LVI), was sufficient to abolish phosphorylation following HRG
stimulation (Fig. 4B). In contrast, deletion of two amino acid residues, Leu957 and Val958 (ErbB3
LV)
and Val958 and Ile959 (ErbB3
VI), did not
affect the transactivation potential.
LVI
to activate the ErbB2 kinase might have resulted from a gross
positional change in the ErbB3 polypeptide backbone relative to ErbB2,
rather than deletion of a specific activation motif. To address this
possibility, we replaced Leu957, Val958, and
Ile959 with alanine residues. This removes hydrophobic side
chains available for potential intermolecular interactions, but
maintains the length of the polypeptide chain. A phosphorylation signal
was not observed in cells expressing L957A/V958A/I959A (Fig.
4B), indicating that the sequence LVI is required for transactivation.
LV, cells expressing L957A/V958A did not transactivate ErbB2
upon HRG stimulation. One explanation for this finding is that, in
ErbB3
LV, isoleucine mimics leucine at position 957. But in
L957A/V958A, the substituted alanine residue was not able to replace
Leu957. This possibility was further explored with mutant
L957A. The replacement of leucine with an alanine residue resulted in a
strong decrease in the phosphorylation signal, further indicating that Leu957 played a major role in the transactivation. From
these data, we conclude that the minimal region necessary for full
transactivation of the ErbB2 kinase upon HRG stimulation is the LVI
sequence beginning at position 957. Moreover, Leu957
appears to play a key role in this hydrophobic motif. However, we
cannot rule out the possibility that areas N-terminal to this motif may
also contribute to transactivation of ErbB2.
LVI revealed no increase in tyrosine
phosphorylation signal upon HRG treatment (Fig. 6B). Thus,
analogous to the ErbB3 situation, activation of ErbB2 kinase also
requires the LVI motif in ErbB2-ErbB4 heterodimers.
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Fig. 5.
Sequence alignment of the C-terminal ends of
the ErbB tyrosine kinase domains. Sequences are displayed in a
single letter code; identical residues are denoted as dots.
Amino acid sequences are numbered according to the mature proteins. The
LVI segment is boxed. The C-terminal ends
(C-term) of the tyrosine kinase (TK) domains were
determined as described by Plowman et al. (5).
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Fig. 6.
Receptor phosphorylation analysis of ErbB4
mutants. A, ErbB4 mutants shown as described in the
legend to Fig. 3. The ErbB4 receptor construct with Lys726
replaced by Met is designated ErbB4M726.
ErbB4M726 LVI lacked, in addition to the amino acid
substitution, Leu960, Val961, and
Ile962. B, phosphorylation analysis of ErbB4
mutants. COS-7 cells were transfected with the corresponding expression
plasmids, and cells were subjected to phosphorylation analysis as
described in the legend to Fig. 2. ErbB4 expression was verified using
a polyclonal anti-ErbB4 antibody.
P-Tyr,
anti-phosphotyrosine antibody.
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Fig. 7.
An associated cytoplasmic kinase is not
involved in receptor autophosphorylation. A, ErbB3
expression in stable K562erbB3 transfectants. Anti-ErbB3
antibody was used to detect ErbB3 expression in the cell lysates of the
transfectants. B, phosphorylation analysis in
K562erbB3 cells transiently transfected with erbB2
or kinase-inactive erbB2 (erbB2M732).
Incubations with or without rHRG 1 (10 nM) were performed
for 10 min at room temperature, 48 h post-transfection. Polyclonal
antibodies to ErbB2 or ErbB3 were used for immunoprecipitations
(IP). Immune complexes were subjected to SDS-polyacrylamide
gel electrophoresis and then transferred to nitrocellulose. Receptor
phosphorylation was determined using anti-phosphotyrosine antibody
(
P-Tyr). ErbB2 expression was verified using monoclonal
antibody 5B6, which recognizes glycoprotein D(gD)-tagged
ErbB2 and ErbB2M732. WB, Western blot.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 8.
Transactivation models. The schematic
depicts two models of HRG-mediated transactivation of ErbB3. ErbB3 and
ErbB2 are designated H3 and H2, respectively. The
extracellular and intracellular domains are shown as distinct
barrels. The LVI segment in ErbB3 is symbolized as a
black half-circle; a so far unidentified interaction region
in ErbB2 is shown as a hatched box.
Sequence alignment of the tyrosine kinase domains of members of the ErbB receptor family revealed that the LVI segment is conserved in EGFR as well as ErbB3 and ErbB4 (Fig. 5). Interestingly, in ErbB2, the motif is conservatively changed, having leucine replaced by valine. The conservation of LVI in the ErbB receptors suggests that the transactivation mechanism seen in the ErbB2-ErbB3 and ErbB2-ErbB4 dimers is not restricted to these combinations, but may be a widespread mechanism of transactivation in ErbB2-containing complexes. Consistent with a more general mechanism are data from down-regulation studies of ErbB2-EGFR heterodimers (35). ErbB2 down-regulation is correlated with the transactivation of ErbB2 upon EGF binding and occurs only if sequences between amino acids 899 and 958, containing the LVI motif, are present in a kinase-defective EGFR mutant. It is noteworthy that the LVI sequence in EGFR has been identified as part of the lysosomal targeting sequence (YLVI) that contributes to the down-regulation of EGFR (36). This segment has also been mapped as part of the binding site of a newly isolated sorting nexin protein (37).
Uncontrolled signaling from ErbB family members is observed in various
cancers (38). Overexpression of EGFR or ErbB2 occurs with high
frequency and often correlates with poor patient survival (39-41).
Therefore, blocking the ErbB signaling pathway may be an ideal target
for an antiproliferative agent. To date, several approaches have been
undertaken to intercept the signal generation (42). For example,
antibodies that directly bind to the extracellular domain of ErbB2 or
EGFR have been very efficient as antiproliferative reagents (43-45).
The selective inhibition of EGFR tyrosine kinase with various small
molecules also results in antitumor activity (46). The identification
of the transactivation sequence in ErbB3 suggests another way to
block signaling in a heteromeric complex. A compound that directly
interacts with the LVI segment in ErbB3 could inhibit specific
transactivation, receptor phosphorylation, and consequently all
downstream signaling pathways.
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ACKNOWLEDGEMENT |
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We thank the DNA synthesis/purification group at Genentech, Inc. for supplying oligonucleotides.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Genentech, Inc., Mail Stop 63, South San Francisco, CA 94080. Tel.: 650-225-1247; Fax: 650-225-5945; E-mail: marks{at}gene.com.
The abbreviations used are: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; HRG, heregulin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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REFERENCES |
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