From the Departments of Protein Chemistry and
§ Protein Engineering, Genentech, Inc.,
South San Francisco, California 94080
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ABSTRACT |
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Individual residues of the heregulin (HRG)
egf domain were mutated to alanine and displayed
monovalently on phagemid particles as gene III fusion proteins. Wild
type HRG
egf domain displayed on phage was properly
folded as evidenced by its ability to bind ErbB3 and ErbB4 receptor-IgG
fusion proteins with affinities close to those measured for bacterially
produced HRG
egf domain. Binding to ErbB3 and ErbB4
receptors was affected by mutation of residues throughout the
egf domain; including the NH2 terminus
(His2 and Leu3), the two
-turns
(Val15-Gly18 and
Gly42-Gln46), and some discontinuous residues
(including Leu3, Val4, Phe13,
Val23, and Leu33) that form a patch on the
major
-sheet and the COOH-terminal region (Tyr48 and
Met50-Phe53). Binding affinity was least
changed by mutations throughout the
-loop and the second strand of
the major
-sheet. More mutants had greater affinity loss for ErbB3
compared with ErbB4 implying that it has more stringent binding
requirements. Many residues important for HRG binding to its receptors
correspond to critical residues for epidermal growth factor (EGF) and
transforming growth factor
binding to the EGF receptor. Specificity
may be determined in part by bulky groups that prevent binding to the
unwanted receptor. All of the mutants tested were able to induce
phosphorylation and mitogen-activated protein kinase activation through
ErbB4 receptors and were able to modulate a transphosphorylation signal from ErbB3 to ErbB2 in MCF7 cells. An understanding of binding similarities and differences among the EGF family of ligands may facilitate the development of egf-like analogs with broad
or narrow specificity.
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INTRODUCTION |
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Members of the ErbB (also known as the human epidermal growth factor receptor or HER) family of receptor tyrosine kinases play a central role in embryonic development as evidenced by observations that mice lacking these receptors die in utero or soon after birth (1). Well defined experimental systems have shown that EGFR1 and ErbB4 ostensibly behave as fully functional ligand-binding and signaling receptors (2, 3). In contrast, ErbB2 is not activated directly by any known ligand whereas ErbB3 is devoid of intrinsic tyrosine kinase activity (4). Transactivation of ErbB2 is a common and perhaps obligatory step in ligand-activated processes involving EGFR, ErbB3, and ErbB4 (5). Importantly, human cancers of epithelial origin are especially prone to expressing dysregulated ErbB receptors with overexpression of EGFR or ErbB2 being the most common molecular alteration encountered (6, 7). These observations taken together with the combinatorial nature of the receptor-signaling pathways suggest that the relative levels of receptor expression and control of their activation are critical in maintaining normal homeostasis.
Neuregulins, also known as heregulins (HRGs) or neu
differentiation factors, are a family of ligands that bind with low
affinity to ErbB3 or ErbB4. In the presence of ErbB2 a high affinity
heteromeric receptor complex is formed (8, 9). However, the mechanism of affinity site conversion and stoichiometry of oligomerization are
uncertain. Many HRG isoforms have been identified and all are splice
variants encoded by a single gene (10-12). The egf domain is necessary and sufficient to bind ErbB3 and ErbB4 and for all known
biological activities of the HRGs (11). Two types of egf domains have been identified, and
, which differ by four of eight residues between the 5th and 6th cysteine and in the region carboxyl-terminal to the 6th cysteine (11, 12).
The solution structure of the HRG egf domain was recently
solved to high resolution using NMR spectroscopy (13, 14). The molecule
contains an NH2-terminal 3 stranded
-sheet and a smaller
2 stranded
-sheet near the COOH terminus. The relative orientation
of the 2 sheets is well defined and stabilized by four hydrogen bonds
(3 of which involve Arg44). The NH2- and
COOH-terminal residues (1-2 and 50-63) and the
-loop (24-30) are
disordered in the structure and have been shown to be highly flexible
from 15N-relaxation
measurements.2 Overall, the
structure of the HRG egf domain is similar to EGF, although
they share limited amino acid identity (14). Despite these strong
structural similarities, the binding specificity of EGF and HRG are
distinct and mutually exclusive. Substitution of a block of HRG
residues (1-5) into EGF created a molecule capable of binding both EGF
and ErbB2/ErbB3 in SKBR3 cells, indicating that the NH2
terminus is important for receptor specificity (15).
In this study, a comprehensive mutational analysis of the
egf domain of HRG was conducted to determine areas
critical for binding receptors and initiating signal transduction.
Individual amino acids of the HRG
egf domain were changed
to alanine to identify loss of binding. To facilitate this, mutants
were expressed monovalently on phagemids and analyzed for binding to
ErbB3 and ErbB4 receptor-IgG fusion proteins in an ELISA format.
Selected mutants were expressed in Escherichia coli as
thioredoxin (Trx) fusion proteins for further characterization. We
identified regions of the molecule critical for the preservation of
binding to the receptors. Mutation of some residues had similar effects
on binding to both receptors, while other changes had differential
effects on ErbB3 and ErbB4 binding. Comparison of the HRG binding data and mutagenesis studies of EGF indicates that there are both
similarities and differences in how these ligands interact with their
receptors. We also characterized receptor binding and phosphorylation
by many of the alanine mutants on cells expressing ErbB receptors. All
of the mutants tested were able to induce phosphorylation and MAPK
activation through ErbB4 receptors and were able to modulate a
transphosphorylation signal from ErbB3 to ErbB2 in MCF7 cells.
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EXPERIMENTAL PROCEDURES |
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Reagents--
The egf domain of HRG (177-244) was
expressed in bacteria. This form of HRG
was radioiodinated as
described previously (8). Preparation of receptor-IgGs was described by
Fitzpatrick et al.3
Phagemid Construction, Kunkel Mutagenesis, and Phage
Display--
Alanine mutants were generated by Kunkel mutagenesis (16)
using the phagemid display vector pHRG2-g3 as a template (43). pHRG2-g3
contains residues 177-244 of the egf domain of HRG
(hereafter referred to as residues 1-68) attached to the COOH terminus
of the pIII gene. Phagemids displaying HRG
mutants were produced by
the addition of M13KO7 helper phage to XL1-Blue cells (Stratagene, Inc.) containing the mutated recombinant plasmid (17). Phagemid stocks
were made by precipitating cell culture broths after 18-24 h growth
with 20% PEG (8000), 2.5 M NaCl. Phagemids were
resuspended in PBS (0.01 M sodium phosphate, 0.1 M NaCl, pH 7.5).
ELISA Measurement of Phagemid Affinities--
Microtiter plates
(Nunc, Maxi-sorb 96-well) were coated overnight with 0.5 µg of rabbit
anti-human IgG, Fc fragment-specific antibodies (Jackson
Immunoresearch) in 100 µl of 0.05 M NaCO3, pH
9.6, at 4 °C. Plates were blocked with PBS + 0.1% BSA, washed with
PBS + 0.05% Tween 20 (wash buffer), then wells were coated with 0.1 or
0.05 µg of ErbB receptor-IgG in PBS + 0.1% BSA + 0.05% Tween 20 (binding buffer) for 1 h and washed again. Serial dilutions of
receptor-IgG (competitor) and a concentration of phage, predetermined to give 60% saturation without competitor, were added to wells in 100 µl of binding buffer and incubated for 2 h to overnight at room
temperature. Following incubation, plates were washed thoroughly,
incubated with 1:900 dilution of anti-M13 horseradish peroxidase
conjugate (Pharmacia) for 20 min. The level of phagemid bound was
assayed using o-phenylenediamine dihydrochloride substrate solution (Sigma). EC50 values were calculated with a
4-parameter fit equation and based on the concentration of soluble
receptor-IgG needed to displace 50% of the phagemid from the plate.
Assays on both receptors were carried out with the same phage
preparation, on the same day. This served as a control of phage
expression because numerous mutants showed little to no affinity for
ErbB3, but good displacement curves could be generated for ErbB4
binding.
Trx-HRG Vector Preparation and Mutagenesis--
Selected HRG
mutants were expressed in a soluble form as Trx fusion proteins. The
parent vector, pET23a (Novagen), was digested with NdeI and
HindIII and Trx (bases 2722-3180, pTrxFus
vector, Invitrogen) was inserted. HRG alanine mutants were initially generated by site-directed mutagenesis in the vector pRK5.gDhrgB1 (18).
To facilitate cloning of these mutants into the Trx
containing vector, a KpnI site was engineered into the
pRK5.gDhrgB1 vector immediately upstream of the NdeI site at
position 5407. The modified parental HRG was cleaved from this vector
and inserted at the carboxyl terminus of Trx at the KpnI and
BamHI cloning sites. Subsequently, additional mutants were
generated in pRK5.gDhrgB1, digested with NdeI and
BamHI, and the resultant 313-base pair fragment was ligated
in-frame to the carboxyl terminus of Trx. The vector also contains an
enterokinase protease recognition site (DDDDK) between Trx and
HRG.
Expression and Purification of Trx-HRG Protein--
Trx-HRG
expression was driven by an inducible T7 promoter. Cloning, cell
growth, and expression were carried out as described in the Novagen pET
system manual. Briefly, cloning was done in XL1-Blue cells and
expression of soluble protein in BL21DE3 host cells. BL21DE3 cells
containing the appropriate mutant plasmid were grown at 37 °C in LB
medium until the OD550 reached 0.3-0.6, then protein was
induced by 0.4 nM
isopropyl-1-thio--D-galactopyranoside and growth was
allowed to continue for 2-4 h at 28 °C. Cells were collected by
centrifugation, resuspended in 0.02 M Tris-HCl, 0.025 M EDTA, pH 7.5 (1/20 cell culture volume). Cells were lysed
by freezing on dry ice, thawing at 37 °C, and vigorous sonication. The freeze, thaw, and sonication cycle was repeated 3 times. Protein was further solubilized in 6 M guanidine HCl, 0.1 M Tris-HCl, pH 8.8, then sulfitolized by the addition of
0.1 M Na2SO3 and 0.2 M
Na2S4O6, and stirred at room
temperature for 1.5 h. Protein was dialyzed into 0.05 M Tris-HCl, pH 7.5, 0.01 M methionine. After
dialysis, the insoluble material was removed by centrifugation at
35,000 × g for 15 min. The supernatants were purified by
Fast Flow Q Sepharose (Pharmacia) chromatography using a 15-ml column equilibrated with 0.01 M Tris-HCl, pH 7.5, and protein was
eluted by a NaCl gradient. The Trx-HRG mutants eluted between 0.5 and 0.6 M NaCl. Trx-HRG was refolded overnight at room
temperature after addition of 1 mM cysteine. Finally, the
protein was dialyzed into 0.05 M Tris-HCl, pH 7.5. Each
protein preparation was visualized on Coomassie-stained gels for purity
and quantified by amino acid analysis.
Enterokinase Cleavage of Trx-HRG-- Trx-HRG protein was dialyzed into EKMax reaction buffer (Invitrogen, San Diego, CA). EKMax enzyme was added to a final ratio of 0.5 units per 20 µg of protein and incubated at 37 °C for 4 h. EKMax was inactivated with soybean trypsin inhibitor resin (Sigma) for 2 h at room temperature. The cleavage products were purified by reverse phase high performance liquid chromatography, then analyzed by mass spectrometry and protein sequence determination. One peak contained a product of approximately 9 kDa, this corresponded to a truncation at residue Lys55.
Affinity Measurement of Soluble Mutants for ErbB3 and
ErbB4--
Receptor-IgGs were coated on plates (Maxisorp C break apart
strip wells, Nunc) as described for phage ELISA. Assays were carried out with a constant amount of 125I-labeled HRG1
(residues 1-68) and varied concentrations of unlabeled Trx-HRG fusion
protein. Following incubation, plates were washed and bound
radiolabeled HRG was counted on a
-counter (Isodata). ErbB4-IgG
assays were conducted in PBS, 1% BSA for blocking and fusion protein
binding and PBS, 0.05% Tween 20 for washes. For the ErbB3-IgG assays,
the wash buffer was TBST (0.025 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.02% Tween 20), the blocking buffer was TBST, 1%
BSA, and the binding buffer (RPMI binding buffer) used was RPMI 1640 cell culture medium (Life Technologies, Inc.), 2 mM
glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES buffer, pH 7.2, 0.2% BSA.
ErbB4 Phosphorylation-- K562 cells (ATCC) were stabily transfected with ErbB4 and propagated as described previously (19). Cells were treated 4 h to overnight with 10 ng/ml phorbol 12-myristate 13-acetate (Calbiochem) prior to use. Cells (1 × 106/treatment) were stimulated with each HRG variant for 8 min. Cells were pelleted, supernatant withdrawn, and reaction stopped by addition of lysis buffer (0.025 M Tris-HCl, pH 7.5, 0.15 M NaCl, 10% glycerol, 1% Triton X-100, 1% CHAPS, 200 nM phenylmethylsulfonyl fluoride, 100 units of apoprotinin, 10 µM leupeptin, 100 µM sodium orthovanadate, 100 µM sodium pyrophosphate). ErbB4 protein was immunoprecipited from the lysate with a mixture of 5 µg each of anti-ErbB4 monoclonal antibodies, 1459 and 1461,4 and 20 µl of immobilized protein A/G (Ultralink Immobilized Protein A/G, Pierce). Following rotation at 4 °C overnight, the mixture was centrifuged, immobilized beads were washed with lysis buffer, spun again, and resuspended in reducing, SDS gel loading buffer. Material was boiled 5 min and the supernatant loaded on 4-12% Tris glycine gels (Novex). Protein was transferred from the gels to nitrocellulose and Western blotting done with chemiluminescence detection and following the manufacturer's instructions (ECD, Amersham). Blots were probed with anti-phosphotyrosine antibody conjugated to horseradish peroxidase (Transduction Laboratories) at a dilution of 1:1000.
ErbB3 and ErbB4 K562 Cell Binding--
Cells were cultured and
pretreated with phorbol 12-myristate 13-acetate as described above.
Cells were plated in 96-well plates at density of 125,000 cells/well in
final volume of 250 µl of RPMI binding buffer. Cells were incubated
with varied concentrations of unlabeled Trx-HRG and a constant amount
(200 pM) of 125I-labeled HRG. Following
overnight incubation at 4 °C, cells were collected onto 0.45-µm
polyvinylidene difluoride membranes (Multiscreen-HV Filtration Plate,
Millipore), washed 2 times with TBST, allowed to dry and the amount of
bound radioactivity was measured.
MAPK Activation Measurements-- ErbB4 transfected K562 cells were grown to stationary phase in RPMI medium containing 10% fetal bovine serum. Prior to stimulation, cells were placed in 0.1% fetal bovine serum containing medium. After 4 h, cells were washed with PBS, then stimulated with ligand for 12 min. Following stimulation, cells were lysed in reducing SDS-polyacrylamide gel electrophoresis running buffer. 2.5 × 105 cell equivalents were loaded per lane on 4-20% Tris glycine gels (Novex). Protein was transferred from the gels to nitrocellulose. Western blotting and detection were done following the manufacturer's instructions (ECD, Amersham). Activated MAPK was detected with an anti-active MAPK antibody (Promega) at a dilution of 1:20,000. The non-activated forms of ERK 1 and ERK 2 were detected with antibodies, each used at 1:2000 (Santa Cruz). Both proteins were detected with an anti-rabbit horseradish peroxidase-conjugated secondary antibody, used at 1:10,000 (Transduction Labs).
MCF7 KIRA-ELISA-- This assay was conducted as described previously (20).
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RESULTS |
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Residues Important for Binding ErbB3 and ErbB4 Are Located
Throughout the egf Domain--
Every amino acid of the HRG
(residues 1-53) egf domain, except for 6 cysteines and 2 alanines, was mutated to alanine (Fig. 1)
and displayed monovalently on filamentous phage. Each alanine mutant
displayed on phage was analyzed for binding to ErbB3 and ErbB4-IgGs in
an ELISA format. The affinity of the phage displayed HRG
was
13.6 ± 2.4 nM for ErbB3-IgG and 18.9 ± 5.4 nM for ErbB4-IgG, which was slightly weaker than HRG
(1-68) binding to the two receptors (8.2 ± 1.0 nM
for ErbB3 and 14.8 ± 2.1 nM for ErbB4) under the same
assay conditions.
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Affinity of E. coli Expressed HRG Mutants Parallel Those Displayed
on Phage--
Individual mutants that had relatively large effects on
binding to one or both receptors were selected for further analysis and
characterization. Mutants were expressed as Trx fusion proteins, containing an additional 31 amino acids of HRG preceding the egf domain. There is an enterokinase cleavage site in the
protein between Trx and HRG allowing for removal of the Trx fusion
partner. Initial experiments showed that the fusion protein was able to bind to the receptors, although the affinity was somewhat reduced compared with HRG
(1-68) on ErbB3 (Table
I).
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Soluble Mutants Bind ErbB3 and ErbB4 Expressed on K562
Cells--
We wanted to assess binding of mutants to the receptors in
the context of the natural plasma membrane. However, most cells express
multiple members of the EGFR family and many have very low levels of
ErbB4. Using K562 cells (a human erythroleukemia cell line that does
not normally express any of the EGFR family members) transfected with
either ErbB3 or ErbB4, we assessed binding to individual receptors
(19). The affinities of HRG, Trx-HRG, and selected mutants were
measured on each cell line. The relative binding affinity of selected
mutants on cells was comparable to the data generated on the
receptor-IgGs (Table III). There is some improvement of affinity on cells compared with IgGs seen not only with
the thioredoxin fusion egf domains, but also with HRG
(177-244).
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ErbB4 Autophosphorylation Does Not Always Correlate with Binding
Affinity for HRG Alanine Mutants--
To assess the receptor
activation by each mutant, we measured ErbB4 phosphorylation, the first
step in the signal transduction pathway. Stimulation of cells with
HRG and Trx-HRG(wt) resulted in a 1.6-2-fold increase in ErbB4
phosphorylation. Each Trx-HRG mutant was tested at two concentrations,
corresponding to their measured EC50 (1 ×) on ErbB4-IgG
and 10 times the EC50 (10 ×). A representative blot is
shown in Fig. 4. About half of the
mutants were able to achieve a level of phosphorylation equal to that obtained by treatment with either HRG
or Trx-HRG (Table II). There
was little or no additional stimulation at 10 × concentration. Some mutants induced ErbB4 phosphorylation, but did not reach the fold
increase seen with HRG
or Trx-HRG. Some mutants did not
phosphorylate as well as expected based exclusively on their binding
affinity and others phosphorylated despite poor binding affinity. Thus,
phosphorylation does not always correlate with the binding affinity of
the mutant. For instance, mutant R44A, which had no measurable affinity
for the IgG receptors or on K562 cells, could induce a phosphorylation
of ErbB4 in the K562 cells. It is likely that the affinity of R44A is
too low to be detected in our assay formats.
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ErbB4 Mediates MAPK Activation in K562 Cells Treated with HRG
Mutants--
ErbB4 signal transduction proceeds through association
with SHC (22) ultimately resulting in stimulation of MAPK activation. Under some conditions, other pathways of signaling are recruited (23).
We assessed the ability of the mutants to activate MAPK. Similar to the
ErbB4 tyrosine phosphorylation response, all of the mutants were able
to induce MAPK (Fig. 5, and Table II).
R44A effected the least response of any mutant, although MAPK
activation was still about half the response of HRG. All mutants
were tested at a concentration equal to their EC50 on
ErbB4-IgGs, except for Arg44 which was tested at 5 µM.
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HRG Mutants Phosphorylate ErbB2 in MCF7 Cells--
Since ErbB3 is
a weak or dead kinase (4), we could not directly monitor the
phosphorylation of ErbB3. Instead we measured phosphorylation of ErbB2
in MCF7 cells upon stimulation with the mutants in a KIRA-ELISA (20).
MCF7 cells contain normal levels of ErbB2 and ErbB3. They have very low
levels of ErbB4. All of the mutants were able to stimulate
phosphorylation of ErbB2 (Table II, Fig.
6). The EC50 values for
HRG (1-68) and for the Trx-HRG fusion protein were 0.36 (± 0.07)
and 2.25 (± 0.41) nM, respectively, thus each showed
higher affinity binding, as expected for ErbB3-ErbB2 interactions. It
appears that many of the same residues are required for binding to the
low affinity ErbB3 homomeric binding site and to the ErbB2/ErbB3
heteromeric site. In two cases, G42A and R44A, the ratio for the MCF7
KIRA was over 2-fold higher than that for the IgG binding. For other
mutants, the ratios were lower in the MCF7 KIRA, these included F13A,
N16A, V23A, R31A, and F40A.
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DISCUSSION |
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Conserved Residues in the HRG egf Domain Are Important for Binding
to Both ErbB3 and ErbB4--
All ligands that bind members of the ErbB
family of receptor tyrosine kinases do so through egf-like
domains. This motif is defined by common six cysteine residues and a
consensus protein fold. There is limited conservation of noncysteine
residues and only three additional residues Gly18,
Gly42, and Arg44 in the HRG egf
domain are conserved in all members of the EGF family. Mutation of each
of these conserved residues had a significant effect on HRG binding to
either ErbB3 or ErbB4 (Fig. 2). In particular, the R44A mutant had the
lowest affinity of any of the alanine mutants for binding to either
ErbB3 or ErbB4 receptors. In HRG, Arg44 is situated in a
type I -turn between the 2 strands of the minor
-sheet and it
acts as a hydrogen bond donor for Thr12-Phe13
and as a hydrogen bond acceptor for Val15 (Fig. 1). These
hydrogen bonding interactions together with favorable hydrophobic
interactions between Arg44, Phe13, and
Val15 presumably stabilize the relative orientation of the
two
-sheet subdomains of HRG (14). The equivalent arginine residue
is absolutely required for EGF or TGF
binding to EGFR (25, 26).
Some Residues Appear to Direct Binding to Specific ErbB
Receptors--
The alanine scanning mutagenesis of the HRG
egf domain reported here is the first detailed functional
study of this growth factor with regard to its binding properties to
its individual receptors ErbB3 and ErbB4. No naturally occurring ligand
has been identified that binds to both EGFR and ErbB3. Such a
bifunctional ligand, biregulin, has been created synthetically by
Barbacci et al. (15). This fusion peptide was made by
substituting the first five amino acids of EGF (NSDSE) with the
corresponding residues from the HRG egf domain (SHLVK).
However, a peptide consisting of only the NH2-terminal five
amino acids of HRG had no binding affinity for the cells, suggesting
that other regions of the egf domain are also important for
binding. In agreement with these findings, we note that
His2 and Leu3 result in the greatest loss of
function when changed to alanine and the effect is more pronounced for
ErbB3 than ErbB4. Leucine at position 3 is found only in the HRGs while
histidine at position 2 is also present in hBTC, hTGF
,
neuregulin-2
and neuregulin-2
(33, 34), and neuregulin-3 (19).
The first five amino acids of the HRG egf-like domain form a
well defined
-strand (14), while this region is poorly defined or
disordered in hTGF
(35) and hEGF (32), respectively. Although the
affinity of HRG
for ErbB3 and ErbB4 is similar, there are clearly
differences in binding. Seven of the soluble mutants tested lost at
least 2-fold affinity on ErbB3 compared with ErbB4. Conversely, only 3 mutants lost more than 2-fold affinity on ErbB4 compared with ErbB3.
These data taken in conjunction with the known binding specificities of
the "natural" ligands further suggest that ErbB3 has more stringent requirements for binding than ErbB4.
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ACKNOWLEDGEMENTS |
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We thank our colleagues at Genentech, Inc.: Robert Akita for providing ErbB3 and ErbB4 transfected K562 cells and Shaily Jaini for conducting the MCF7 KIRA assays.
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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.
¶ Present address: Chiron Corp., Dept. of Biological Chemistry, 4560 Horton St., Emeryville, CA 94608.
Present address: Pharmacopeia, Inc., Dept. of Biology, 3000 East Park Blvd., Cranbury, NJ 08512.
** To whom correspondence should be addressed: Genentech, Inc. MS 63, South San Francisco, CA 94080. Tel.: 650-225-1247; Fax: 650-225-5945; E-mail: marks{at}gene.com.
1
The abbreviations used are: EGFR, epidermal
growth factor receptor; HRG, heregulin; ELISA, enzyme-linked
immunosorbent assay; Trx, thioredoxin; PBS, phosphate-buffered saline;
BSA, bovine serum albumin; TGF, transforming growth factor
;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
MAPK, mitogen-activated protein kinase.
2 Fairbrother, W. J., Liu, J., Pisacane, P. I., Sliwkowski, M. X., and Palmer, A. J., III (1998) J. Mol. Biol., in press.
4 L. Bald, personal communication.
3 V. D. Fitzpatrick, P. I. Pisacane, R. L. Vandlen, and M. X. Sliwkowski, manuscript in preparation.
5 P. Osheroff, personal communication.
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REFERENCES |
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