From the University of Nijmegen, Department of Cell Biology, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands
Received for publication, November 22, 2002, and in revised form, January 23, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
EGF-like growth factors activate their ErbB
receptors by promoting receptor-mediated homodimerization or,
alternatively, by the formation of heterodimers with the orphan ErbB-2
through an as yet unknown mechanism. To investigate the selectivity in
dimer formation by ligands, we have applied the phage display approach to obtain ligands with modified C-terminal residues that discriminate between ErbB-2 and ErbB-3 as dimerization partners. We used the epidermal growth factor/transforming growth factor The recent determination of the crystal structure of the
extracellular domain of ErbB-1 in complex with its ligands epidermal growth factor (EGF)1 or
transforming growth factor Dimerization of ligand-bound receptor tyrosine kinases is a mechanism
that is thought to activate the intrinsic kinase domain followed by
transphosphorylation and subsequent docking of cellular signal
transducing proteins. As a consequence, ligand binding serves as a
potential site for regulation of cell proliferation in diseases where
ErbB receptors are overexpressed, as has been observed for ErbB-1 and
ErbB-2 in multiple human cancers (3). ErbB-2 has no known ligand, but
by decelerating the ligand dissociation rate, it serves as a preferred
dimerization partner for all other ErbB members (4-6). Heterodimer
formation with ErbB-2 is especially important in the case of the
ErbB-3, which together with ErbB-4 forms the natural receptor for the
different neuregulins (NRGs). ErbB-3 contains a defective kinase and,
hence, ErbB-3 homodimers are biologically inactive (7, 8). The
ErbB-2/ErbB-3 heterodimer, however, is the most prominent and strongest
transforming signaling complex activated by NRG-1 (9-12) and provides
an attractive model system to study the mechanism of ligand-induced
ErbB heterodimerization.
EGF-like growth factors share a structurally conserved EGF motif,
characterized by three disulfide-bonded loops (the A-, B-, and C-loop),
in addition to a linear N-terminal and C-terminal region. Structural
and mutational analyses have shown that residues in the A-loop, C-loop,
and C-terminal linear region of EGF and TGF- To evaluate the contribution of residues in the linear C-terminal
region of EGF-like ligands for selective dimer formation, we applied
the phage display technique to select ligands that discriminate between
ErbB-2 and ErbB-3 as dimerization partners in ErbB-3 complexes. In
earlier work we and others showed that EGF chimeras in which the linear
N terminus was replaced by either NRG (biregulin) or TGF- Construction of T1E Mutants--
The construction of T1E has
been described previously (28). Residues in the linear C-terminal
region after the sixth cysteine in pEZZ/Fx/T1E were replaced by the
corresponding residues of TGF- Cell Lines--
Interleukin-3-dependent murine 32D
hematopoietic progenitor cells transfected with distinct human
ErbB-encoding viral vectors or plasmids were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum
(Invitrogen), 0.25 ng/ml murine interleukin-3 (Promega, Madison, WI)
and kept under continuous selection using 0.6 mg/ml G418 (Calbiochem)
or 0.4 mg/ml hygromycin B (Sigma) (12). The human mammary carcinoma
cell line MDA-MB-453 was cultured in a 1:1 mixture of Dulbecco's
modified Eagle's medium and Ham's F-12 medium supplemented with 10%
fetal calf serum. Human embryonic kidney 293 cells were cultured in
Dulbecco's modified Eagle's medium (Invitrogen) supplemented with
10% fetal calf serum.
Ligand Displacement Experiments--
Recombinant human
NRG-1 Construction of the Phage T1E46-50 Library--
A
phage library of T1E randomized by mutation to NNS codons (N = G/T/A/C, S = G/C) at positions Tyr-46, Arg-47, Asp-48, Leu-49, and
Lys-50 (T1E numbering) was constructed by a PCR-based approach using
fUSE5/T1E as template (29, 31). The randomized region in the peptide
growth factor was directly followed by the SfiI restriction
site, thereby omitting the EGF residues Trp-51 to Arg-55. Because the
randomized sequence was located in close proximity of the pIII fusion
point, a flexible (Gly-Gly-Gly-Ser)2 linker sequence was
introduced in the wild-type fUSE5 vector after the second
SfiI site before to the gene encoding pIII. Wild-type fUSE5 contains an out-frame stuffer fragment between the SfiI
sites, thereby eliminating background phage (32). The PCR fragments encoding the randomized T1E gene were cloned into the fUSE5/linker phage vector using both SfiI sites. Ligation products were
processed and electroporated into Escherichia coli TG-1
cells (Stratagene) for phage production. The number of independent
transformants was determined by titration on tetracycline-containing
plates to estimate the size of the library. Randomly picked clones from the library were analyzed by cycle sequencing (PerkinElmer Life Sciences) to confirm the diversity of codon use and the expected amino
acid distribution. Phage preparations were carried out after standard
polyethylene glycol 8000/NaCl precipitation procedures. Titers of
filter-sterilized phages were estimated by both titration and
spectrophotometric determination and expressed as titraing units
(tu).
Preparation of ErbB-IgG Fusion Proteins--
Gene constructs
encoding the extracellular domain of human ErbB receptors were fused to
the hinge and Fc regions of the human IgG1 heavy chain (referred
to as ErbB-IgG (33)). Subconfluent HEK-293 cells were transfected with
the expression vector pCDM7/IgB3 or a mixture of pCDM7/IgB3 with
pCDM7/IgB2 using LipofectAMINE 2000 (Invitrogen) according to the
manufacturer's protocol. Conditioned culture supernatants containing
the soluble dimeric IgG fusion proteins were harvested 5-10 days after
transfection and purified by affinity chromatography on a 1-ml Hi-trap
protein A-Sepharose column (Amersham Biosciences). Purified IgG fusion
proteins were eluted with 0.1 M citric acid, pH 4.2, into
tubes containing 1 M Tris, pH 9.0. ErbB-IgG preparations
were quantified by Fc-ELISA using human IgG as a standard, whereas the
purity and presence of dimeric species was confirmed by
SDS-polyacrylamide electrophoresis and immunoblotting with polyclonal
antibodies directed against human Fc (Nordic, Tilburg, NL).
Phage Selection on ErbB-3-IgG Fusion Proteins--
Nunc
immunoabsorbant wells were precoated overnight at 4 °C with 0.2 µg
of goat-anti-human Fc-specific IgG (Jackson Immunoresearch Laboratories, West Grove, PA) in 100 µl of PBS (137 mM
NaCl, 2.7 mM KCl, 1 mM
Na2HPO4, 2 mM
KH2PO4). Wells were washed in PBS, 0.05% (v/v)
Tween 20 (washing buffer) and blocked for 1 h in 0.2 ml of PBS,
0.2% (w/v) BSA (blocking buffer) at room temperature. Next, wells were
coated with 100 ng of ErbB-3-IgG for 2 h in PBS, 0.2% BSA, 0.05%
Tween 20 (binding buffer). Wells were rinsed twice and incubated with
1-3 × 1010 tu T1E46-50 phages in 0.1 ml
of binding buffer. After incubation for 2 h, unbound phages were
removed by rinsing 12 times with washing buffer. Bound phages were
eluted by the addition of 0.1 ml of glycine buffer (50 mM
glycine, 150 mM NaCl, pH 2.7) for 10 min, and the eluate
was neutralized with 25 µl of 1 M Tris/HCl, pH 8.0. The eluate was used for phage titration and infection of logarithmic cultures of TG-1 cells.
Whole Cell Phage Selection--
Phage selections on MDA-MB-453
cells were carried out by incubation of 5-10 × 106
cells in suspension with 1-3 × 1010 tu
T1E46-50 phages in 3 ml of blocking buffer. In the case of
ErbB-3-IgG depletion, the phages were subjected to ErbB-3-IgG as
described above before cell selection. The unbound phages from
ErbB-3-IgG wells were subsequently transferred to 12-ml Falcon tubes,
diluted to 3 ml with binding buffer, and added to MDA-MB-453 cells.
After incubation for 2 h on a rowing boat shaker, cells were
rinsed 7 times in washing buffer by spinning for 4 min at 1000 rpm
followed by resuspension and a final wash with PBS. Cells were
transferred twice to clean tubes to remove phages non-specifically
bound to the plastic. Specific elution was performed by competition
with a 1:1 mixture of the anti-ErbB-2 monoclonal antibodies L26 and L96
(Neomarkers, Fremont, CA (34)) at 50 µg/ml in binding buffer for
1 h at 4 °C on a rowing boat shaker. Cells were spun, and the
supernatant was collected as ErbB-2/ErbB-3 phage fraction. The
remaining cell-bound phages were harvested by a 10-min incubation in 1 ml of acid elution buffer followed by neutralization upon adding 0.2 ml
of 1 M Tris-HCl, pH 8.0. Cells were spun for 5 min at 1200 rpm, and the eluate was collected as ErbB-3 fraction. The distinct
eluted fractions were used for phage titration and infection of
logarithmic cultures of TG-1 cells.
Phage ELISA--
Small scale phage preparations were used for
the screening of individual clones in ELISA assays. PEG 8000 precipitates of culture broth of clones grown overnight in 2 ml 2×
TY (16 g/liter bacto-tryptone, 10 g/liter yeast, 5 g/liter NaCl)
supplemented with 12.5 µg/ml tetracycline in 12-well plates were
resuspended in 100 µl of PBS, typically resulting in phage titers of
1-5 × 1010 tu/ml. Whole cell ELISAs on 32D sublines
in suspension were performed as described (29). The procedure for
ErbB-IgG ELISA was similar as described (20), with the following
modifications; homodimeric ErbB-3-IgG fusion proteins were coated at 50 ng/well, and heterodimeric ErbB-23-IgG fusions proteins were coated at
100 ng/well in 0.1 ml of binding buffer.
Cell Proliferation Assays--
32D cells that coexpress ErbB-2
and ErbB-3 (D23 cells) were washed in RPMI 1640 medium to deprive them
of interleukin-3. Subsequently cells were seeded into 96-well tissue
culture plates at a density of 5.0 × 104 cells/well
in 0.1 ml of RPMI supplemented with 0.1% BSA together with serial
dilutions of filter-sterilized phages or recombinant growth factors.
Cell survival was determined after 24 h of incubation at 37 °C
using the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide
(MTT) assay, as previously described (29).
The Linear C-terminal Region of T1E Influences the
ErbB-2/ErbB-3 Binding Efficiency--
Previous work
showed that the chimeric EGF-like ligands biregulin and T1E bind only
weakly to ErbB-3 due to sub-optimal sequences in the N-terminal linear
region (26, 29). The observed high affinity binding of T1E and
biregulin for cells expressing both ErbB-2 and ErbB-3 thus appears to
depend critically on additional stabilization of the complex by ErbB-2.
The strong dependence on ErbB-2 as a dimerization partner makes this
type of molecules a good model system for identifying residues that
mediate the selective recruitment of ErbB-2 versus
ErbB-3 as a dimerization partner.
In this study we have examined whether sequences in the C-terminal
linear region of T1E play a direct role in the formation of ErbB-3
homodimeric and ErbB-2/ErbB-3 heterodimeric complexes, as suggested by
a comparison of the different NRG isoforms. Thereto we initially
exchanged the C-terminal linear tail of T1E, composed of EGF sequences,
for the corresponding sequences of TGF-
The biological activity of the various T1E mutants was assessed by
competitive binding analysis on stable transfectants of 32D cells that
express defined ErbB combinations (12). Fig. 1B shows clear
differences in the ability of the T1E mutants tested to displace
radiolabeled T1E from 32D cells coexpressing ErbB-2 and ErbB-3 (D23
cells). The IC50 of both T1E6N and T1E6T (50-60 ng/ml) was
increased compared with T1E itself (6.7 ng/ml), whereas T1E6Nopt bound D23 cells with enhanced affinity (3 ng/ml)
comparable with wild-type NRG-1
To further evaluate the role of the linear C terminus in binding to
ErbB-3, the binding affinity of the T1E mutants was assessed by
125I-labeled NRG-1 Design of the T1E46-50 Phage Library and Selection
Strategy--
To assess the precise contribution of individual
residues in T1E to the recruitment of ErbB-2 or ErbB-3 into dimeric
complexes, we randomly mutated five positions in the linear C terminus
of T1E that correspond to the positions in the NRG isoforms implicated in differential ErbB binding using a phage display approach. By a
combination of positive and negative selection strategies and specific
elution methods we subsequently selected variants that discriminated
between binding to ErbB-3 homodimers and to ErbB-2/ErbB-3 heterodimers.
To this end we used homodimeric ErbB-3-IgG fusion proteins in addition
to MDA-MB-453 cells, human breast carcinoma cells with overexpression
of ErbB-2 and ErbB-3, which we have previously employed for affinity
optimization of EGF variants to ErbB-2/ErbB-3 heterodimers (29).
Fig. 1A shows the sequence of the phage library of T1E
variants. The Gln-45 residue next to Cys-44 is conserved between EGF and NRG-1 Sequences of T1E46-50 Clones Isolated for Preferential
ErbB-3 Homodimer Formation--
Phage T1E variants that preferentially
bind to ErbB-3 homodimers but not ErbB-2/ErbB-3 heterodimers were
isolated using alternating selection rounds on homodimeric ErbB-3-IgG
fusion proteins and whole MDA-MB-453 cells. During the selection on
cells the ErbB-2-dependent phage clones were depleted from
the cell surface by competitive elution with anti-ErbB-2 antibodies
that are known to impair ligand binding (34). Subsequently the
remaining phage clones bound to ErbB-3 receptors on the cell surface
were harvested by acid elution. The alternation between exposure to
soluble and cell-bound ErbB-3 receptors reduced nonspecific background
binding of phages and excluded a possible selection advantage offered
by the preformed dimeric ErbB-3-IgG compared with the monomeric
cell-expressed receptors.
Individual phage clones were randomly picked after four selection
rounds and subsequently analyzed for binding to ErbB-3
ectodomains in phage ELISA. Table I gives
a survey of the sequences of 16 T1E46-50 clones selected
for preferential binding to ErbB-3 homodimers (ErbB-3 selectants) with
their corresponding binding properties. Binding ability of the clones
to ErbB-2/ErbB-3 heterodimers was determined in ELISAs on intact cells
(D23 versus parental 32D cells) and on heterodimeric
ErbB-23-IgG fusions. Because these heterodimeric IgG fusion proteins
were generated by cotransfection of the expression vectors encoding the
respective receptors, experiments were carried out on a mixture of
ErbB-23-IgG heterodimers and ErbB-2-IgG and ErbB-3-IgG homodimers. As a
positive control EGF/WV phage was used, which is a previously
characterized EGF variant with high affinity for ErbB-3
homodimers and ErbB-2/ErbB-3 heterodimers (29). Wild-type T1E phage and
EGF phage were used as additional controls. Table I indicates that all
ErbB-3 selectants displayed strong binding to ErbB-3-IgG, with
concomitant strong binding capacity to both ErbB-23-IgG ectodomains and
D23 cells. Thus, despite the negative selection for ErbB-2 dimers, the
T1E clones selected by this approach had not lost their ability to bind
to ErbB-2/ErbB-3 heterodimers. In other words, the ErbB-3 selectants are unable to discriminate between ErbB-2 and ErbB-3 as dimerization partners.
The most striking result in the sequences obtained was the abundant
selection of Asp-48 in 81% of the ErbB-3 selectants, which is the
corresponding residue in both EGF and TGF- Sequences of T1E46-50 Clones Isolated for Selective
ErbB-2/ErbB-3 Heterodimer Formation--
In an inverse
strategy, phage T1E variants were isolated that selectively bound to
ErbB-2/ErbB-3 heterodimers but failed to bind to ErbB-3 homodimers.
Thereto negative selection on homodimeric ErbB-3-IgG fusion proteins
was performed before the selection on MDA-MB-453 cells. Ligands that
ultimately depended on ErbB-2 for high affinity binding were eluted
from the cell surface by treatment with ErbB-2 antibodies. Control cell
panning confirmed the specific elution of wild-type T1E phages by this
method (data not shown). After four rounds of selection, single clones
were sequenced and analyzed for binding properties in ELISAs in a
similar way as described above for the ErbB-3 selectants (Table
II). All T1E clones selected for
preferential ErbB-2/ErbB-3 binding (ErbB-23 selectants) were found to
bind strongly to both D23 cells and ErbB-23-IgG ectodomains, confirming
that selection had been achieved. When the ErbB-23 selectants were
analyzed for their ability to bind to ErbB-3 homodimers, half of the
clones lacked detectable binding for ErbB-3-IgG, similar to phage T1E
(group 1), whereas the remaining were still able to bind with gradual
affinity to ErbB-3 homodimers (group 2). Thus, only a fraction of the
ErbB-23 selectants was able to discriminate against ErbB-3 as
dimerization partner in the ErbB-3 complex.
When comparing the sequences of the two distinct groups of ErbB-23
selectants, both differences and similarities became apparent (Table
II). The consensus sequence of group 1 ErbB-23 selectants could be
assigned as Tyr-46-Leu-47-Xaa-48-apolar-49-Asp-50,
whereas the consensus sequence of group 2 strongly resembled that of
the ErbB-3 selectants in Table I. The diminished ability to bind to
ErbB-3 homodimers of the group 1 clones correlated with a shift of the
acidic Asp from position 48 (72% group 2) to position 50 (52% group
1). Predominantly large hydrophobic residues were found at positions
46, 47, and 49 in all ErbB-23 selectants, similarly as observed for the
ErbB-3 selectants, although the exact nature of the side chains
differed. Taken together, the overall consensus sequence of ErbB-23
selectants displays remarkable overlap with the consensus sequence for
ErbB-3 selectants.
Selected T1E46-50 Variants Display Gradual Differences
in ErbB-3 Binding--
To gain further insight into the relative
contribution of specific amino acids to the ability of ligands to bind
ErbB-3, we subjected a number of selected T1E46-50 phage
variants to extended analysis using dose-response experiments. Based on
the similarity and divergence from the consensus sequences, individual
T1E selectants that harbor acidic Asp residues (at position 48 or 50 or
at both positions) combined with distinct hydrophobic residues were
chosen and produced as large scale cultures. Phage T1E served as a
positive control for ErbB-2/ErbB-3 binding, and phage EGF/WV served as
a positive control for ErbB-3 homodimer binding, whereas phage EGF was
used as a negative control.
Although no absolute binding affinities could be determined by the used
multivalent phage display system, the relative affinities could readily
be compared between the distinct clones. All clones, whether they were
isolated as ErbB-3 selectants (Fig.
2A) or as ErbB-23 selectants
(Fig. 2B), invariably were found to bind with similar strong
affinity to heterodimeric ErbB-23-IgG complexes. The binding of
wild-type T1E to the ErbB-23-IgGs was relatively low in comparison to
all other T1E variants and EGF/WV. This may be attributed to the
flexible linker region that is present in the T1E46-50
phages but absent in the wild-type T1E phage. Conversely, the binding
affinity of the various T1E46-50 clones to ErbB-3-IgGs
showed strong variation between the clones, and in general the ErbB-3
selectants (Fig. 2C) were superior to the ErbB-23 selectants
(Fig. 2D). Interestingly, the ErbB-3 selectants with
C-terminal sequences IFDWA, IADIQ, and YYDID showed significantly higher affinity for ErbB-3 than the positive control EGF/WV (Fig. 2C), indicating that C-terminal sequences strongly
contribute to enhanced ErbB-3 affinity. When comparing the ErbB-23
selectants, a more or less gradual decrease in ErbB-3 binding affinity
was observed in the order YYDID > YLEID = YLQMN = EGF/WV > YLDIS > YLTLD > T1E > YLSTD, and YLALH
In addition, the abilities of the distinct T1E variants to activate
ErbB-2/ErbB-3 heterodimers were assessed by measuring the
interleukin-3-independent proliferation of D23 cells in an MTT assay.
Fig. 3 shows that all T1E variants are
equipotent to phage EGF/WV and superior to phage T1E itself in inducing
proliferative responses in D23 cells, indicating that functional
ErbB-2/ErbB-3 heterodimers are formed in response to all selectants.
Thus, the ErbB-23 selectants only ineffectively bind to ErbB-3
homodimers, similar to T1E, but are still strong inducers of
ErbB-2/ErbB-3 heterodimers. The ErbB-3 selectants effectively bind to
ErbB-3 homodimers and ErbB-2/ErbB-3 heterodimers, similar to EGF/WV, but this increased ErbB-3 affinity does not result in enhanced mitogenic potency for cells expressing ErbB-2 and ErbB-3. Therefore, it
appears that a low threshold ErbB-3 binding is already sufficient for
the efficient formation of ErbB-2/ErbB-3 heterodimers.
EGF-like ligands differ in their ability to recruit a dimerization
partner for their cognate receptor, resulting in differential potency
and mitogenic responses. This suggests that ligands contain specific residues that mediate interaction with distinct ErbB complexes, including heterodimers with the orphan ErbB-2. To
investigate the selectivity in dimer formation by EGF-related ligands,
we have applied the phage display approach to obtain ligands with modified C-terminal residues that have (i) altered selectivity and (ii)
enhanced binding affinity. Our findings indicate that EGF-like growth
factors contain multiple, independent binding domains for ErbB-3, one
of which is located in the C-terminal tail. However, no separate
binding domain for ErbB-2 could be identified in this region. Instead,
ligand-induced ErbB-2/ErbB-3 heterodimerization appears to occur as a
consequence of a low affinity interaction with ErbB-3 and subsequent
stabilization by ErbB-2.
To address the issue of dimer selectivity, distinct combinations of
positive and negative selection strategies were applied to isolate
ligands that were able to discriminate between ErbB-2 and ErbB-3 in
complex formation with ErbB-3. Two of our present observations argue
against the hypothesis that the selectivity in recruitment of the
dimerization partner is mediated by sequences in the linear C-terminal
tail of ligands. First, none of the ErbB-3 selectants had impaired
ability to form ErbB-2/ErbB-3 heterodimers despite the negative
selection for T1E variants that depended on ErbB-2 dimerization for
binding. The most likely explanation is that the linear C-terminal
region is not directly involved in the recruitment of ErbB-2 as
dimerization partner either because ligands do not harbor a separate
ErbB-2 binding site or because such a site is located in a different
region of the ligand. Hence, our data do not favor a model in which the
linear C-terminal tail harbors a secondary binding site for ErbB-2, as
previously proposed in ligand bivalence models (26, 36).
Secondly, the incomplete discrimination against ErbB-3 homodimer
formation observed for the ErbB-23 selectants might indicate that
ligands require only a low level of ErbB-3 binding affinity to allow
the efficient formation of ErbB-2/ErbB-3 heterodimers. These ligands
can thus be considered as the "second best" ErbB-3 binders.
Moreover, the observation that ligand variants positively selected for
ErbB-3 binding but negatively for ErbB-2/ErbB-3 binding are still
potent activators of heterodimers also strongly indicates that binding
to ErbB-3 is sufficient for heterodimer formation. It thus appears that
the relative binding affinity to ErbB-3 is indicative for the binding
behavior of ligands to dimeric complexes, such that low affinity
binders only interact with ErbB-2/ErbB-3 heterodimers, whereas high
affinity binders can additionally form ErbB-3 homodimers.
Our present observations argue for a separate ErbB-3 binding site
located in the linear C-terminal tail of EGF-like ligands. Previous
mutagenesis studies have assigned several non-continuous hydrophobic
and charged residues in the triple The C-terminal sequence requirements for ErbB-3 binding can be deduced
from a comparison of the consensus sequences of the selected high and
low affinity ErbB-3 binding variants. Large hydrophobic residues are
preferred at positions 46, 47, and 49 in all selected
T1E46-50 variants, whereas methionine was alternatively
allowed at position 49. In particular Ile-46 contributes to enhanced
ErbB-3 binding affinity. In addition, the presence of an acidic residue
facilitates specific binding to ErbB-3, preferentially located at
position 48 (for strong ErbB-3 binding) or, alternatively, at position 50 (for weak ErbB-3 binding). Because a combination of the polar residues Gln-48 and Asn-50 also proved sufficient (Fig. 2D),
we propose that these two side chains are involved in hydrogen bonding. Interestingly, in the structure of receptor-bound TGF- Comparing the consensus sequence of the ErbB-3 selectants with that of
the natural ErbB-3 ligands, one can conclude that the EGF linear
C-terminal region comprising Tyr-46-Asp-48-Leu-49 contributes positively to the interaction with ErbB-3. The finding that T1E effectively forms ErbB-2/ErbB-3 heterodimers but not ErbB-3 homodimers can thus not be attributed to inappropriate C-terminal sequences, as
previously thought. Rather, it reflects the presence of suboptimal residues in both its N-terminal and C-terminal region (29). The
observation that introduction of the NRG-1 Our results imply that specific residues in the C-terminal tail of
EGF-related ligands are involved in direct interaction with ErbB-3 but
not ErbB-2. This makes it unlikely that the formation of heterodimeric
ErbB complexes is driven through bivalent binding of ligands to two
different ErbB molecules. Instead, our findings support a
receptor-mediated dimerization mechanism, in which ligand binding to
ErbB-3 determines the formation of both ErbB-3 homodimers and
ErbB-2/ErbB-3 heterodimers (Fig. 4). In a
variant of this model, it has been proposed that ligands only induce
ErbB-3 homodimers, which may subsequently stabilize two ErbB-2
molecules into a tetrameric complex (37-40). Based on the analogy of
the crystal structures of ErbB-1 and ErbB-3, it is most likely that
homodimeric ErbB-3 complexes are formed through a conformation-induced
mechanism with a 2:2 stoichiometry (1, 2, 41). In this scenario the
ligand would bind domain I of ErbB-3 through the linear N-terminal and
B-loop regions, whereas its C-terminal residues would interact with
receptor domain III. In the conformation-induced model, selectivity between the formation of inactive ErbB-3 homodimers and active ErbB-2/ErbB-3 heterodimers will be achieved through the intrinsic property of a ligand to bind ErbB-3 (Fig. 4). Ligands with high affinity for domain I and/or III of ErbB-3, including NRG-1 chimera T1E as
the template molecule because it binds to ErbB-3 homodimers with low
affinity and to ErbB-2/ErbB-3 heterodimers with high affinity. Many
phage variants were selected with enhanced binding affinity for ErbB-3
homodimers, indicating that C-terminal residues contribute to the
interaction with ErbB-3. These variants were also potent ligands for
ErbB-2/ErbB-3 heterodimers despite negative selection for such
heterodimers. In contrast, phage variants positively selected for
binding to ErbB-2/ErbB-3 heterodimers but negatively selected for
binding to ErbB-3 homodimers can be considered as "second best"
ErbB-3 binders, which require ErbB-2 heterodimerization for stable
complex formation. Our findings imply that epidermal growth factor-like
ligands bind ErbB-3 through a multi-domain interaction involving at
least both linear endings of the ligand. Apparently the ErbB-3 affinity
of a ligand determines whether it can form only ErbB-2/ErbB-3 complexes
or also ErbB-3 homodimers. Because no separate binding domain for
ErbB-2 could be identified, our data support a model in which ErbB
heterodimerization occurs through a receptor-mediated mechanism and not
through bivalent ligands.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-
) has provided evidence for the
formation of homodimeric ErbB-1 complexes through a receptor-mediated dimerization mechanism (1, 2). Ligand binding to both domains I and III
of the extracellular domain of the receptor involves the transition of
ErbB-1 from a "closed" to an "open" state, which then permits
dimerization with another liganded ErbB-1 through interaction of domain
II residues within these receptors. Most likely this mechanism can
serve as a paradigm for homodimerization of other liganded ErbB
receptors, such as ErbB-3 and ErbB-4. However, it is well established
that EGF-like growth factors preferentially signal through heterodimers
of their cognate receptor with the orphan ErbB-2. The mechanism by
which EGF-like growth factors bind their receptors in heterodimeric
receptor complexes remains an open question.
primarily bind to
domain III of ErbB-1 followed by an interaction of residues in their
B-loop with domain I of the receptor (1, 2, 13-16). By contrast,
NRG-1
has been shown to bind with high affinity to a proteolytic
fragment of ErbB-3 containing only domain I (17). Moreover, NRG binding
to ErbB-1/ErbB-4 chimeras requires the presence of domain I of the
latter receptor, suggesting that ligand binding to NRG receptors
primarily involves interaction with domain I of the receptor (18).
Alanine scanning of NRG-1
revealed that hydrophobic and charged
residues in the linear N-terminal region and the B-loop, which form a
surface patch on one site of the triple
-sheet, are the major
determinants in ErbB-3 binding (19, 20). On the other hand, residues in
the C-terminal region of NRG also may play a role in receptor binding,
because the natural
and
isoforms of NRG-1 and NRG-2, which only
vary in sequences C-terminal of the fifth cysteine, strongly differ in
their ability to bind and activate distinct ErbB combinations (21-24).
Exchange studies between NRG-1
and NRG-1
show that particularly
the linear C-terminal region determines the binding properties and
mitogenic potential of these isoforms (25). Therefore, it has been
proposed that NRGs may have a bivalent character and interact with both ErbB-3 and ErbB-2 through separate binding sites (26).
(T1E)
residues gained high binding affinity to ErbB-2/ErbB-3 heterodimers,
whereas they bound only weakly to ErbB-3 alone (27, 28). Unlike
NRG-1
, both T1E and biregulin seem dependent on subsequent binding
of ErbB-2 to stabilize their low affinity interaction with ErbB-3. In a
previous study we could attribute the weak ErbB-3 interaction of the
chimera T1E in part to the presence of sub-optimal sequences in the
linear N terminus. Based on a phage display approach we enhanced the
binding affinity for ErbB-3 relative to T1E by substitution of only two
residues in EGF (D2W and S3 V/R) (29). In the present study we have
used the same approach to subject five residues in the linear
C-terminal region of T1E for randomization and selection for altered
receptor selectivity and affinity. The targeted residues in T1E
correspond to the positions in the NRG isoforms that have been
implicated in the differential activation of ErbB dimers. Here we show
that T1E can be strongly optimized for binding to ErbB-3 by the current phage display approach, indicating that residues in the linear C-terminal tail contribute to the ability of ligands to bind ErbB-3 homodimers. Moreover, despite negative selection protocols we consistently observed a direct relation between the ability of T1E
variants to bind ErbB-3 and their ability to induce ErbB-2/ErbB-3 heterodimers. Because no sequences selective for ErbB-2
heterodimerization could be identified, our findings support a
model in which ErbB heterodimerization is driven by
receptor-mediated and not by ligand-mediated interactions.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and NRG-1
by means of splice
overlap extension polymerase chain reaction. The initial PCR fragments
containing the complementary overhang sequences were made using
pEZZ/Fx/T1E, pEZZ/Fx/TGF-
, and the NRG-1
gene in pNRG8-g3 (a gift
from Genentech Inc., San Francisco, CA) as templates. The fragment
for the C-terminal region of the optimized NRG-1
(referred to as
NRG-58 (30)) was constructed using oligonucleotide primers containing
mutations encoding Asn-His and Met-Ile substitutions. Mutant gene
products were subsequently introduced in the pEZZ vector using the
BamHI/SalI sites and verified by automated cycle
sequencing. Production, purification, and characterization of
recombinant T1E mutants was essentially performed as previously described (28).
176-246 (R&D Systems, Minneapolis, MN) and
T1E were radioiodinated using the Iodogen method (Pierce) according to
the manufacturer's protocol for indirect labeling, resulting in a
specific activity of 40-80 µCi/µg of protein. Ligand displacement
analyses on 32D cells were performed as described (28).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Glu-44-Ala-50), NRG-1
(Gln-45-Ala-51) and an optimized NRG-1
mutant. This latter mutant
has been obtained from a previous phage display study on NRG-1
by
selection for high affinity binding to ErbB-3 ectodomains and contains
the mutations N47H and M50I compared with the wild-type NRG-1
EGF
domain (30). The TGF-
C terminus was included because it shows the
highest sequence similarity with optimized NRG-1
and because
previous studies indicate that introduction of TGF-
C-terminal
residues into NRG-1
strongly enhances binding affinity for
ErbB-2/ErbB-3 heterodimers on SK-BR-3 cells (35). The nomenclature used
for the T1E mutants follows previously used conventions in which T1E6N
is a chimera containing TGF-
sequences up to the first cysteine
followed by EGF sequences up to the sixth cysteine and NRG-1
residues in the C-terminal tail (Fig.
1A). All T1E mutants were
expressed as recombinant peptides in E. coli, finally purified by reverse-phase HPLC and verified for the appropriate molecular weight by matrix-assisted laser desorption ionization time-of-flight spectroscopy analysis.
View larger version (28K):
[in a new window]
Fig. 1.
Exchange of the linear C-terminal region in
the EGF/TGF- chimera T1E affects the binding
to specific ErbB combinations. A, amino acid sequences
of the T1E variants used in this study, aligned with human EGF and the
EGF domain of human NRG-1
. Numbering of the conserved cysteines
(boldface) is according to T1E. B, displacement
of 125I-labeled T1E binding on 32D cells coexpressing
ErbB-2 and ErbB-3 (D23 cells). C, displacement of
125I-labeled NRG-1
binding on 32D cells expressing
ErbB-3 (D3 cells). Cells were incubated for 2 h at 4 °C with
radiolabeled ligand (2 ng/ml) in presence of serial dilutions of
unlabeled NRG-1
(closed squares), T1E (open
circles), T1E6T (closed circles), T1E6N (open
squares), or T1E6Nopt (open diamonds).
Unbound ligand was removed by sedimentation of the cells through a
serum cushion, after which cell-bound radioactivity was determined.
Results express the mean ± S.E. of three independent experiments
performed in duplicate.
(2 ng/ml). Similar differences were
observed in the ability of these T1E mutants to stimulate proliferation of D23 cells and the neuregulin-responsive MCF-7 human breast cancer
cells, indicating that their relative binding affinity corresponded to
the ability to activate ErbB-2/ErbB-3 complexes (data not shown). Thus,
changes in the C-terminal sequences of T1E strongly affect its ability
to interact with ErbB-2/ErbB-3 heterodimers. Despite the fact that
NRG-1
is the natural activator for ErbB-2/ErbB-3 heterodimers
in vivo, its C-terminal sequences do not appear beneficial
for the interaction with ErbB-2/ErbB-3 in a T1E environment. In this
respect the EGF residues present in T1E appear more effective in
stabilizing ErbB-2/ErbB-3 complexes than the corresponding NRG-1
and
TGF-
sequences.
displacement on 32D cells solely
expressing ErbB-3 receptors (D3 cells). In agreement with cross-linking
analyses, we assume that NRG-1
is able to induce homodimeric ErbB-3
complexes in D3 cells (12, 26). Fig. 1C shows that all T1E
mutants have an affinity for ErbB-3 alone that is at least 50-fold
lower than that of NRG-1
. Compared with T1E, T1E6Nopt
shows increased binding affinity, whereas T1E6N and T1E6T have almost
similar binding affinity. Thus, replacement of the C-terminal tail of
T1E for NRG-1
sequences did not significantly improve the weak
binding of T1E to the ErbB-3 receptor. By contrast, introduction of the
C-terminal tail of the optimized NRG-1
variant increased the
relative binding affinity of T1E for both homo-and heterodimeric ErbB-3
complexes, indicating these residues improve the recruitment of ErbB-2
and ErbB-3 independent of the context of the NRG molecule. Together
these findings demonstrate that residues in the C-terminal region of
T1E indeed contribute to the ligand preferences for distinct receptor complexes.
, and therefore, the adjacent five residues
(Tyr-46-Lys-50) were targeted for mutation. Because the randomized
area is localized in close proximity to the fusion point with the pIII
minor coat protein, a flexible linker was introduced into the fUSE5
phage vector to minimize possible steric effects. The resulting phages each display 2-5 copies of the fusion proteins (32). The completeness of the T1E46-50 phage library was estimated from the
number of independent transformants yielding 1.45 ×107,
thereby covering 5 times the theoretical diversity of 3.1 ×106 possible different amino acid combinations. Sequence
analysis of an aliquot of the library revealed no deviation from the
theoretical amino acid distribution.
Sequences and binding characteristics of ErbB-3 selectants
. Substitutes found on
positions 46, 47, and 49 tended to be similar in character, being
predominantly large and hydrophobic, although at position 46 a
preference for Ile was observed (75%). At position 47 residues Tyr and
Phe were found in more than half of the ErbB-3 selectants, indicating
that aromatic side chains seem to be favored. The most frequently
selected T1E46-50 clone, designated 3.1, contained IFDWA
sequences (5/16 times). The overall consensus sequence for
T1E46-50 ErbB-3 selectants, based on the frequency of
occurrence of a certain (type of) amino acid detected at the respective
position in
50% of all individual clones analyzed, was
Ile-46-aromatic-47-Asp-48-apolar-49-Xaa-50, in
which Xaa represents any residue.
Sequences and binding characteristics of ErbB-2/ErbB-3
selectants
EGF (Fig. 2D). Thus, acidic residues seem to
contribute directly to the ErbB-3 binding affinity when located at
position 48 and to a lesser extent when present at position 50. Moreover, acidic residues present at both positions seem beneficial for
ErbB-3 binding, whereas the combination of Gln-48/Asn-50 was also shown
to be efficient. Apparently the depletion of the strongest ErbB-3
binding clones from the pool of ErbB-2/ErbB-3 selectants has resulted
in the isolation of the second best ErbB-3 binding clones.
View larger version (37K):
[in a new window]
Fig. 2.
Binding characteristics of individual phage
T1E46-50 variants selected for preferential ErbB-3
homodimer binding (ErbB-3 selectants) or preferential ErbB-2/ErbB-3
heterodimer binding (ErbB-23 selectants). A and
B, phage ELISA on ErbB-23-IgG fusion proteins. C
and D, phage ELISA on homodimeric ErbB-3-IgG fusion
proteins. Binding of ErbB-3 selectants is depicted in panels
A and C, and binding of ErbB-23 selectants is depicted
in panels B and D. Phage clones used were
T1E46-50 variants IFDWA (shaded squares), IADIQ
(shaded triangles), YYDID (shaded diamonds),
YLEID (filled circles), YLDIS (filled squares),
YLQMN (filled triangles), YLTLD (crosses), YLALH
(open diamonds), and YLSTD (open squares),
whereas phage T1E (open circles), phage EGF/W2V3 (open
triangles), and phage EGF (plus symbols) were used as
positive and negative controls, respectively. Results are presented as
mean of two experiments performed in duplicate.
View larger version (19K):
[in a new window]
Fig. 3.
Ability of individual phage clones to induce
proliferation of 32D cells expressing ErbB-2 and ErbB-3 (D23
cells). Cells were incubated for 24 h in interleukin-3 free
medium containing serial dilutions of filter-sterilized
T1E46-50 phages selected for ErbB-3 homodimer binding
(A) or ErbB-2/ErbB-3 heterodimer binding (B).
Shaded squares, IFDWA; shaded triangles, IADIQ;
shaded diamonds, YYDID; filled
circles, YLEID; filled squares, YLDIS; filled
triangles, YLQMN; crosses, YLTLD; open
diamonds, YLALH; open squares, YLSTD; open
circles, phage T1E; open triangles, phage EGF/W2V3;
plus symbol, phage EGF. Viable cells were determined using
the calorimetric MTT assay. Results are given as fold induction
over non-stimulated cells of duplicate
measurements.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet formed by the linear
N-terminal region and the B-loop region of NRG-1 as the major
determinants for ErbB-3 binding (19, 20, 27). It cannot, however, be
excluded that additional residues in the ligand may contribute to
receptor interaction. For instance, residues in the
-turn in the
A-loop of NRG-1 were also susceptible to Ala mutation, although this
might be attributed to structural disturbance as well (20). The crucial
importance of N-terminal residues was further emphasized by two phage
display studies, revealing the strong requirement for aromaticity (His
or Trp) combined with aliphatic or basic side chains for ErbB-3
interaction (29, 30). Here we show that optimized sequences in the
C-terminal linear region also directly contribute to the ability of T1E
ligands to interact with ErbB-3 and that they can compensate for the
presence of suboptimal residues in the linear N terminus of T1E.
Notably, the T1E46-50 variants selected for preferential
binding to ErbB-3 homodimers displayed even stronger binding to
ErbB-3-IgG than the EGF/W2V3 mutant (Fig. 2C). Thus, although residues
in the N terminus and B-loop appear to confer specificity to ErbB-3
binding, residues in the linear C-terminal tail may further enhance
receptor affinity, in line with the differential ErbB-3 binding
abilities of the distinct NRG-1 isoforms. This suggests that both
linear regions of the ligand participate independently in receptor
binding such that ligand binding to ErbB-3 occurs through a multidomain
receptor interaction.
several C-terminal residues interact with both domains I and III, among which
is Asp-47 (1). It has been suggested that these residues determine the final position of the two ligand binding domains in the
complex and thereby influence the stability of the dimer. It is
tempting to speculate that in the T1E selectants Asp-48 may serve a
similar function when binding to ErbB-3. Furthermore, all T1E variants
selected for ErbB-3 binding maintained their ability to bind to ErbB-1
expressed on 32D cells and ErbB-1-IgG ectodomains, indicating that the
requirements in the C-terminal tail for ErbB-1 and ErbB-3 interaction
may partly overlap.2 This
observation is remarkable since the majority of selected clones lacked
a Leu or Ile at position 49 in T1E (Leu-47 in EGF), which is strictly
conserved among ErbB-1 ligands and known to be highly sensitive to
site-directed mutagenesis (13, 14).
C terminus into T1E did
not increase the ErbB-3 binding affinity can be explained by the
presence of only two of four required determinants, Tyr-47 and Met-49,
with Met being a less-favored residue. In agreement, mutation of these
two residues into alanine was found to reduce the ErbB-3 binding
affinity of NRG-1
(20). Apparently, natural ligands are not
necessarily optimized for high affinity to their receptors, which is
also indicated by the increase in ErbB-3 binding affinity of optimized
NRG-1
variants (30). Their affinity enhancement was mostly
attributed to the Met to Ile substitution in the C-terminal tail, as
also shown by the T1E6Nopt mutant in our study.
Interestingly, despite the abundant occurrence of Asp residues in the
T1E variants optimized for ErbB-3 binding, acidic residues were not
observed in optimized NRG-1
variants, which may be attributed to
differences in the structural environment of T1E and NRG-1
(30). We
have recently solved the three-dimensional structure of
T1E,3 but because that of the
NRG-1
isoform is unknown, a direct comparison is impossible.
and some
of the ErbB-3 variants selected here, have a high potency to bring
ErbB-3 into the open dimerization state and will, thus, induce both
ErbB-3 homodimers and ErbB-2/ErbB-3 heterodimers. Ligands with
relatively low affinity for ErbB-3 due to suboptimal interaction with
domain I or III, such as T1E and NRG-1
, have only a low potency to
induce ErbB-3 into the open state, and only in the case the liganded
ErbB-3 is complexed by ErbB-2 is a stable complex formed. This model
presumes that the dimerization site of ErbB-2 is maintained
constitutively in the open configuration, as an explanation for the
preferential heterodimerization with ErbB-2. From an evolutionary point
of view this would be a sensible mechanism to enhance the formation of
active ErbB-2/ErbB-3 complexes and to avoid biologically inactive
ErbB-3 homodimers. In this respect NRG-1
would be the optimal
natural ligand if ErbB-2 is present in excess over ErbB-3, whereas the
low affinity NRG-1
isoform would be more effective under conditions
that ErbB-3 is present in excess over ErbB-2 (21, 24). A switch in
isoform production thus offers the cell a subtle method of control
over cellular functions.
View larger version (28K):
[in a new window]
Fig. 4.
Models for ligand-induced ErbB-2/ErbB-3
heterodimerization, including ligand-mediated (left
panel) and receptor-mediated (middle and
right panel) mechanisms. The ErbB-3 receptor is
shown in white, and ErbB-2 is shown in gray. The
orientation of the linear N- and C-terminal regions of the ligand is
indicated by N and C, respectively. The middle panel depicts
the ErbB-2/ErbB-3 heterodimeric complex induced by a ligand with low
ErbB-3 affinity and the ErbB-3 homodimeric complex induced by a ligand
with high ErbB-3 affinity. For details see
"Discussion."
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Y. Yarden and D. Harrari (Weizmann
Institute of Science, Rohovot, Israel) for the kind gift of the 32D
cells and gene constructs encoding the ErbB-IgG fusion proteins. The
NRG-1 precursor was supplied by Genentech Inc. (San Francisco, CA).
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Dutch Cancer Society and the Netherlands Organization for Advancement of Research.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. Tel.: 31-243652707;
Fax: 31-243652999; E-mail: vzoelen@sci.kun.nl.
Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M211948200
2 C. Stortelers, S. P. van der Woning, S. Jacobs-Oomen, M. Wingens, and E. J. J. van Zoelen, unpublished observations.
3 M. Wingens, G. Vuister, and E. J. van Zoelen, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: EGF, epidermal growth factor; NRG, neuregulin; TGF, transforming growth factor; ErbB-IgG, dimeric form of ErbB extracellular domains fused to the constant region of immunoglobulin G; MTT, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; tu, titrating units.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Garrett, T. P., McKern, N. M., Lou, M., Elleman, T. C., Adams, T. E., Lovrecz, G. O., Zhu, H. J., Walker, F., Frenkel, M. J., Hoyne, P. A., Jorissen, R. N., Nice, E. C., Burgess, A. W., and Ward, C. W. (2002) Cell 110, 763-773[Medline] [Order article via Infotrieve] |
2. | Ogiso, H., Ishitani, R., Nureki, O., Fukai, S., Yamanaka, M., Kim, J. H., Saito, K., Sakamoto, A., Inoue, M., Shirouzu, M., and Yokoyama, S. (2002) Cell 110, 775-787[Medline] [Order article via Infotrieve] |
3. | Yarden, Y., and Sliwkowski, M. X. (2001) Nat. Rev. Mol. Cell Biol. 2, 127-137[CrossRef][Medline] [Order article via Infotrieve] |
4. | Riese, D. J., II, and Stern, D. F. (1998) Bioessays 20, 41-48[CrossRef][Medline] [Order article via Infotrieve] |
5. | Karunagaran, D., Tzahar, E., Beerli, R. R., Chen, X., Graus-Porta, D., Ratzkin, B. J., Seger, R., Hynes, N. E., and Yarden, Y. (1996) EMBO J. 15, 254-264[Abstract] |
6. |
Graus-Porta, D.,
Beerli, R. R.,
Daly, J. M.,
and Hynes, N. E.
(1997)
EMBO J.
16,
1647-1655 |
7. | Guy, P. M., Platko, J. V., Cantley, L. C., Cerione, R. A., and Carraway, K. L., III (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8132-8136[Abstract] |
8. |
Sliwkowski, M. X.,
Schaefer, G.,
Akita, R. W.,
Lofgren, J. A.,
Fitzpatrick, V. D.,
Nuijens, A.,
Fendly, B. M.,
Cerione, R. A.,
Vandlen, R. L.,
and Carraway, K. L., III
(1994)
J. Biol. Chem.
269,
14661-14665 |
9. | Wallasch, C., Weiss, F. U., Niederfellner, G., Jallal, B., Issing, W., and Ullrich, A. (1995) EMBO J. 14, 4267-4275[Abstract] |
10. | Alimandi, M., Romano, A., Curia, M. C., Muraro, R., Fedi, P., Aaronson, S. A., Di Fiore, P. P., and Kraus, M. H. (1995) Oncogene 10, 1813-1821[Medline] [Order article via Infotrieve] |
11. | Riese, D. J., II, van Raaij, T. M., Plowman, G. D., Andrews, G. C., and Stern, D. F. (1995) Mol. Cell. Biol. 15, 5770-5776[Abstract] |
12. | Pinkas-Kramarski, R., Soussan, L., Waterman, H., Levkowitz, G., Alroy, I., Klapper, L., Lavi, S., Seger, R., Ratzkin, B. J., Sela, M., and Yarden, Y. (1996) EMBO J. 15, 2452-2467[Abstract] |
13. | Groenen, L. C., Nice, E. C., and Burgess, A. W. (1994) Growth Factors 11, 235-257[Medline] [Order article via Infotrieve] |
14. | Campion, S. R., and Niyogi, S. K. (1994) Prog. Nucleic Acid Res. Mol. Biol. 49, 353-383[Medline] [Order article via Infotrieve] |
15. | Woltjer, R. L., Lukas, T. J., and Staros, J. V. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7801-7805[Abstract] |
16. |
Summerfield, A. E.,
Hudnall, A. K.,
Lukas, T. J.,
Guyer, C. A.,
and Staros, J. V.
(1996)
J. Biol. Chem.
271,
19656-19659 |
17. |
Singer, E.,
Landgraf, R.,
Horan, T.,
Slamon, D.,
and Eisenberg, D.
(2001)
J. Biol. Chem.
276,
44266-44274 |
18. |
Kim, J. H.,
Saito, K.,
and Yokoyama, S.
(2002)
Eur. J. Biochem.
269,
2323-2329 |
19. | Jacobsen, N. E., Abadi, N., Sliwkowski, M. X., Reilly, D., Skelton, N. J., and Fairbrother, W. J. (1996) Biochemistry 35, 3402-3417[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Jones, J. T.,
Ballinger, M. D.,
Pisacane, P. I.,
Lofgren, J. A.,
Fitzpatrick, V. D.,
Fairbrother, W. J.,
Wells, J. A.,
and Sliwkowski, M. X.
(1998)
J. Biol. Chem.
273,
11667-11674 |
21. |
Pinkas-Kramarski, R.,
Shelly, M.,
Glathe, S.,
Ratzkin, B. J.,
and Yarden, Y.
(1996)
J. Biol. Chem.
271,
19029-19032 |
22. |
Crovello, C. S.,
Lai, C.,
Cantley, L. C.,
and Carraway, K. L., III
(1998)
J. Biol. Chem.
273,
26954-26961 |
23. |
Wang, L. M.,
Kuo, A.,
Alimandi, M.,
Veri, M. C.,
Lee, C. C.,
Kapoor, V.,
Ellmore, N.,
Chen, X. H.,
and Pierce, J. H.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
6809-6814 |
24. | Jones, J. T., Akita, R. W., and Sliwkowski, M. X. (1999) FEBS Lett. 447, 227-231[CrossRef][Medline] [Order article via Infotrieve] |
25. | Whoriskey, J. S., Pekar, S. K., Elliott, G. S., Hara, S., Liu, N., Lenz, D. M., Zamborelli, T., Mayer, J. P., Tarpley, J. E., Lacey, D. L., Ratzkin, B., and Yoshinaga, S. K. (1998) Growth Factors 15, 307-321[Medline] [Order article via Infotrieve] |
26. |
Tzahar, E.,
Pinkas-Kramarski, R.,
Moyer, J. D.,
Klapper, L. N.,
Alroy, I.,
Levkowitz, G.,
Shelly, M.,
Henis, S.,
Eisenstein, M.,
Ratzkin, B. J.,
Sela, M.,
Andrews, G. C.,
and Yarden, Y.
(1997)
EMBO J.
16,
4938-4950 |
27. |
Barbacci, E. G.,
Guarino, B. C.,
Stroh, J. G.,
Singleton, D. H.,
Rosnack, K. J.,
Moyer, J. D.,
and Andrews, G. C.
(1995)
J. Biol. Chem.
270,
9585-9589 |
28. | Stortelers, C., Lenferink, A. E., van de Poll, M. L., Gadellaa, M., van Zoelen, C., and van Zoelen, E. J. (2002) Biochemistry 41, 4292-4301[CrossRef][Medline] [Order article via Infotrieve] |
29. | Stortelers, C., Souriau, C., van Liempt, E., van de Poll, M. L., and van Zoelen, E. J. (2002) Biochemistry 41, 8732-8741[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Ballinger, M. D.,
Jones, J. T.,
Lofgren, J. A.,
Fairbrother, W. J.,
Akita, R. W.,
Sliwkowski, M. X.,
and Wells, J. A.
(1998)
J. Biol. Chem.
273,
11675-11684 |
31. |
Souriau, C.,
Fort, P.,
Roux, P.,
Hartley, O.,
Lefranc, M. P.,
and Weill, M.
(1997)
Nucleic Acids Res.
25,
1585-1590 |
32. | Smith, G. P., and Scott, J. K. (1993) Methods Enzymol. 217, 228-257[Medline] [Order article via Infotrieve] |
33. |
Chen, X.,
Levkowitz, G.,
Tzahar, E.,
Karunagaran, D.,
Lavi, S.,
Ben-Baruch, N.,
Leitner, O.,
Ratzkin, B. J.,
Bacus, S. S.,
and Yarden, Y.
(1996)
J. Biol. Chem.
271,
7620-7629 |
34. | Klapper, L. N., Vaisman, N., Hurwitz, E., Pinkas-Kramarski, R., Yarden, Y., and Sela, M. (1997) Oncogene 14, 2099-2109[CrossRef][Medline] [Order article via Infotrieve] |
35. | Harris, A., Adler, M., Brink, J., Lin, R., Foehr, M., Ferrer, M., Langton-Webster, B. C., Harkins, R. N., and Thompson, S. A. (1998) Biochem. Biophys. Res. Commun. 251, 220-224[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Lemmon, M. A.,
Bu, Z.,
Ladbury, J. E.,
Zhou, M.,
Pinchasi, D.,
Lax, I.,
Engelman, D. M.,
and Schlessinger, J.
(1997)
EMBO J.
16,
281-294 |
37. |
Gamett, D. C.,
Pearson, G.,
Cerione, R. A.,
and Friedberg, I.
(1997)
J. Biol. Chem.
272,
12052-12056 |
38. | Huang, G. C., Ouyang, X., and Epstein, R. J. (1998) Biochem. J. 331, 113-119[Medline] [Order article via Infotrieve] |
39. |
Ferguson, K. M.,
Darling, P. J.,
Mohan, M. J.,
Macatee, T. L.,
and Lemmon, M. A.
(2000)
EMBO J.
19,
4632-4643 |
40. | Schlessinger, J. (2000) Cell 103, 211-225[Medline] [Order article via Infotrieve] |
41. |
Cho, H. S.,
and Leahy, D. J.
(2002)
Science
297,
1330-1333 |