A system for quantifying dynamic protein
interactions defines a role for Herceptin
in modulating ErbB2 interactions
T. S. Wehrman, W. J. Raab, C. L. Casipit, R. Doyonnas, J. H. Pomerantz, and H. M. Blau†
Baxter Laboratory in Genetic Pharmacology, Department of Microbiology and Immunology, Stanford University School of Medicine,
Stanford, CA 94305-5175
Edited by Joseph Schlessinger, Yale University School of Medicine, New Haven, CT, and approved October 5, 2006 (received for review June 21, 2006)
The orphan receptor tyrosine kinase ErbB2 is activated by each of
the EGFR family members upon ligand binding. However, difficul-
ties monitoring the dynamic interactions of the membrane recep-
tors have hindered the elucidation of the mechanism of ErbB2
activation. We have engineered a system to monitor protein–
protein interactions in intact mammalian cells such that different
sets of protein interactions can be quantitatively compared. Ap-
plication of this system to the interactions of the EGFR family
showed that ErbB2 interacts stably with the EGFR and ErbB3, but
fails to spontaneously homooligomerize. The widely used anti-
cancer antibody Herceptin was found to effectively inhibit the
interaction of the EGFR and ErbB2 but not to interfere with the
ErbB2 with Herceptin results in increased EGFR homooligomeriza-
tion in the presence of EGF and a subsequent rapid internalization
and down-regulation of the EGFR. In summary, the protein inter-
interactions within the biological context of the plasma membrane
and provides insight into the mechanism of Herceptin action on
cells overexpressing ErbB2.
anti-cancer ? EGF receptor
activated in response to ligand-induced dimerization. ErbB2
(HER2?Neu) does not itself bind any known ligand, and activation
of this receptor is believed to be mediated through heterodimer-
ization with any of the other EGF family members. Physical
characterization of this process has proven difficult using conven-
tional biochemical methods, but it is of considerable interest
because of the role of ErbB2 in breast cancer pathogenesis.
associated with a malignant phenotype and poor prognosis, espe-
cially if coexpressed with the EGF receptor (EGFR) (1–3). For a
subset of breast cancer patients whose tumors overexpress ErbB2,
the monoclonal antibody Herceptin has revolutionized treatment
by extending lifespan and decreasing recurrence rate in an unprec-
edented manner (4–6). Although there is evidence that Herceptin
targets tumor cells for destruction by the immune system (7), the
in vitro independent of an immune response (8). Herceptin is not
known to block the formation of heterodimers of ErbB2, yet its
inhibitory effects on cell proliferation suggest that it interferes with
signal transduction by the ErbB family of tyrosine kinases. One
reason that the mechanism of action of Herceptin has remained
elusive is the difficulty in monitoring the interactions of the ErbB
receptors in a quantitative manner using available biochemical
methods, including purified or coimmunoprecipitated receptors
We postulated that the ?-gal system we recently developed for
assays of protein translocation (12) could enable a comparative
analysis of the combinatorial interactions of the ErbB family
members associated with breast cancer. By using this system the
he EGF family of receptor tyrosine kinases consists of four
members, EGFR, ErbB2, ErbB3, and ErbB4, that become
interaction of two proteins is measured as a function of comple-
mentation of low-affinity mutant subunits of the ?-gal enzyme
can be assayed, the signal-to-noise ratio is high, and receptor
homodimers and heterodimers can be compared in a quantitative
manner in the plasma membranes of large polyclonal cell popula-
tions. This combination of features is not found in other protein
interaction detection systems based on energy transfer (13, 14) or
split enzymes including dihydrofolate reductase (15), ?-lactamase
Investigation of the oligomerization properties of the EGFR,
ErbB2, and ErbB3 using ?-gal complementation yielded quantita-
tive data about the interaction of each of these receptors in basal
and stimulated conditions. The interaction of ErbB2 with the
EGFR and ErbB3 is readily detected in the presence of ligand
basal interactions of each of the family members appears similar, in
contrast to the hypothesis that ErbB2 readily forms spontaneous
homodimers. In accord with previous reports, we find that Her-
ceptin is ineffective in blocking ErbB2–ErbB3 interactions. How-
ever, we show that Herceptin does efficiently inhibit the interaction
of the EGFR and ErbB2. These results reveal a mechanism for
Herceptin action and clarify the specificity of homooligomerization
and heterooligomerization of the EGFR, ErbB2, and ErbB3.
Characterization of the Enzyme Complementation System. We re-
cently described a proximity-based low-affinity enzyme comple-
mentation system for monitoring protein translocation using ?-gal.
the mutants obtained, the histidine-to-arginine mutant at position
ability to spontaneously complement the M15 deletion mutant (?)
but high signal-to-noise ratio upon induction of complementation.
Because of their low affinity, the interaction of the ?* and ? ?-gal
fragments is not sufficiently strong to maintain a complemented
enzyme. As a result, the ?-gal activity obtained at any given time is
a measure of the dynamic interaction of the two fragments, a
reflection of their local concentration, which is determined by the
interaction of the proteins to which they are fused.
For the proposed studies of the interactions of the ErbB
family, the potential of the proximity-based low-affinity ?-gal
Author contributions: T.S.W. designed research; T.S.W., W.J.R., and C.L.C. performed
research; T.S.W., R.D., J.H.P., and H.M.B. analyzed data; and T.S.W., R.D., J.H.P., and H.M.B.
wrote the paper.
Conflict of interest statement: H.M.B. is a major stockholder in a company that might have
a gain or loss financially through publication of this paper. T.S.W. and H.M.B. are inventors
of the technology described in this article; a patent is pending.
This article is a PNAS direct submission.
Abbreviations: EGFR, EGF receptor; B2AR, ?2-adrenergic receptor.
†To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2006 by The National Academy of Sciences of the USA
December 12, 2006 ?
vol. 103 ?
no. 50 ?
complementation system for analyzing specific inducible pro-
tein–protein interactions (Fig. 1A) had to be validated. First,
the rapamycin-inducible interaction of FKBP12 and FRB,
cytoplasmic proteins that associate with high affinity in the
presence of rapamycin (23), was assayed by chemilumines-
cence. Treatment of C2C12 cells expressing FKBP12? and
FRB?* with rapamycin for 2 h resulted in a 10-fold increase
in ?-gal activity (Fig. 1B), demonstrating that low-affinity
?-complementation can be used to monitor protein interac-
tions with a large induction of signal. To determine whether
the ?-gal system could also be used to monitor the interaction
of lower-affinity, reversible interactions, the association of the
G protein-coupled receptor, the ?2-adrenergic receptor
(B2AR), with ?-arrestin2 was evaluated. Upon stimulation,
B2AR becomes phosphorylated and binds ?-arrestin2. Treat-
ment of cells expressing the B2AR-? and ?-arrestin2?* fusion
proteins with agonist (isoproterenol) resulted in a 4-fold
increase in enzyme activity, which was prevented by pretreat-
ment with the antagonist (propranolol) (Fig. 1 C–E). The
dose–response and EC50 obtained as a function of ?-gal
activity are in good agreement with published values (24),
indicating that low-affinity proximity-based ?-complementa-
tion can be used as a quantitative measure of protein–protein
interactions in their natural cellular context. It is important to
note that the stoichiometry of the complemented ?* and ? is
not known. Although wild-type ?-gal has been shown to form
a tetramer (25), evidence also exists for an active ?-gal dimer
(26). It is currently unclear whether a complemented monomer
retains enzyme activity. For this reason, by using the low-
affinity ?-gal complementation system it is possible to discern
the presence and absence of induced protein complexes;
however, it is not possible to distinguish between dimerization
and higher-ordered oligomerization. In light of this limitation,
data obtained by using the ?-gal complementation system
regarding receptor interactions is referred to as heterooli-
gomerization and homooligomerization as opposed to the
more specific heterodimerization and homodimerization
ErbB2 is generally regarded as the preferred heterodimerization
the characterization of ErbB2 interactions using conventional
methods has been problematic. The extracellular domain of ErbB2
has not been shown to form heterodimers in solution (11), and the
use of phosphorylation as a surrogate marker for receptor inter-
actions has led to conflicting results (27, 28). We applied the
by fusing only the extracellular and transmembrane domains of the
EGFR and ErbB2 to ? and ?*, respectively. The intracellular
domain was excluded in these constructs to prevent receptor
clustering into coated pits, down-regulation by endocytosis, and
degradation which may alter the level of complementation ob-
served (Fig. 1F). Exposure to EGF resulted in a dose-dependent
increase in enzyme activity, demonstrating that the extracellular
and transmembrane domains of these receptors are sufficient to
mediate heterooligomerization (Fig. 1G). A time course of EGF
The goal in designing the system was to generate a method that
could measure the basal and stimulated interaction states of an
entire family of proteins. It is evident from the previous experi-
ments that coexpression of the ?* and ? results in a background
expressing specified pairs of proteins fused to the ?* and ?, the
activity had to be determined. Both the ?-arrestin2 construct and
the ErbB2 construct included YFP inserted between the gene and
the ?* peptide providing a marker of protein levels.
Isoproterenol concentration (nM)
β-galactosidase activity (RLU)
Propranolol concentration (nM)
β-galactosidase activity (RLU)
0 1020 3040 50
EGF Concentration (ng/ml)
0.11 10 1001000
Fold induction (Enzyme activity)
Fold induction (Enzyme activity)
Weak activityStrong Activity
tored by low-affinity ?-complementation of
?-gal. (A) Schematic illustration of the low-
affinity ?-complementation system. Physical as-
sociation of two chimeric proteins brings mutant
?-gal fragments, M15 (?) and H31R? (?*), into
proximity, generating ?-gal activity. (B) Low-
affinity ?-complementation monitors strong
protein interactions. Cells expressing FRB?* and
FKBP12? exhibited increased ?-gal activity after
exposure to rapamycin (Rap). (C) Low-affinity
?-complementation quantitatively monitors
protein interactions such as the inducible inter-
action of the membrane-bound B2AR and cyto-
solic ?-arrestin2 in cells expressing B2AR? and
?-arrestin2?* chimeras. ?* denotes chimeric pro-
teins consisting of protein of interest [yellow
fluorescent protein (FP)] H31R? (a*) fusions. (D)
Dose–response of the interaction of B2AR? and
?-arrestin2?* chimeras 45 min after exposure to
the agonist isoproteronol assayed as ?-gal activ-
ity. (E) The B2AR? and ?-arrestin2?* interaction
was prevented in a dose-dependent manner by
the antagonist propanolol. Increasing doses of
propanolol were added to cells 10 min before
?-complementation monitors heterooligomer
formation between the EGFR and ErbB2. The
extracellular and transmembrane domains of the
EGFR and ErbB2 were used to create two chime-
ras, EGFR? and ErbB2?*. (G) Cells expressing
both EGFR? and ErbB2?* were stimulated with
increasing concentrations of EGF. The enzyme activity was measured, demonstrating a dose-responsive increase in enzyme activity in response to ligand.
(H) Cells expressing both EGFR? and ErbB2?* were stimulated with 100 ng?ml EGF, and the enzyme activity was measured, demonstrating increasing
interaction over time.
Inducible protein interactions moni-
www.pnas.org?cgi?doi?10.1073?pnas.0605218103 Wehrman et al.
expression as a marker for ?* peptide expression (Fig. 2 A and C).
The sorted populations were plated into a 96-well dish and assayed
a proportional increase in enzyme activity with increased ?*
29), the baseline ?-gal activity is proportional to the concentration
of the fusion proteins in the cell. These results validate the use of
the system to determine local protein concentrations and illustrate
the importance of controlling for protein levels when comparing
protein interactions between cell lines.
Investigation of EGFR, ErbB2, and ErbB3 Interactions. The determi-
nation of the possible interaction states of ErbB2, EGFR, and
ErbB3 was accomplished using six different cell lines; EGFR?–
EGFR?*, EGFR?–ErbB2?*, EGFR?–ErbB3?*, ErbB2?–
EGFR?*, ErbB2?–ErbB2?*, and EGFR?–ErbB3?*. If cell lines
are to be cross-compared, the ?* fusions must localize similarly. To
ensure that all of the ?* fusions were similarly expressed at the
plasma membrane the corresponding plasmids that include YFP
sandwiched between the receptor and ?* peptide were transfected
into HEK293 cells, as these cells facilitate visualization of plasma
membrane staining. All fusion proteins exhibit similar patterns of
fluorescence at the plasma membrane by confocal microscopy
indicating similar localization of the constructs (Fig. 3A).
Two parental cell lines were constructed using C2C12 cells in
which ErbB family members are expressed at very low levels (30).
These cells were engineered to express either the ErbB2? or the
were then split and transduced with each of three constructs
encoding different ?-fusion proteins, EGFR?*, ErbB2?*, and
generated simultaneously by retroviral infection of replicate cul-
tures of the same ? expressing parental cell line. As a result, the
levels and nature of the ?-fusion protein are similar for each cell
line; by contrast, the ?* fusion protein levels were not similar,
because of inherent differences in the stability and expression level
of each of the different fusion proteins. To control for such
receptor and the ?* allowing measurement of ?* chimeric protein
expression levels. Cells expressing similar YFP levels were isolated
by FACS so that the levels of ?*-fusion proteins were comparable
of the sorted cell lines shows a ?15% total variance (Fig. 3C).
to the ligand EGF that binds the EGFR or to the ligand heregulin
(HRG?1) that binds the ErbB3 receptor (31). All of the expected
interactions were observed (Fig. 4A). EGF led to homooligomer-
ization of the EGFR and heterooligomerization of EGFR with
ErbB2, whereas HRG?1 failed to induce interaction of these
receptors. When cells were compared that expressed EGFR?–
ErbB2?* or ErbB2?–EGFR?*, the responses were similar. This
finding was important, because it indicated that similar interactions
occurred irrespective of whether the receptors were fused to ?* or
?. Although the phosphorylation of ErbB3 by the EGFR has been
shown by others (28, 32), we detected no significant interaction
between these two proteins, indicating that activation of ErbB3 by
the EGFR is unlikely to be mediated by oligomerization of their
extracellular domains. The cells expressing ErbB2? and ErbB3?*
generated heterooligomers only in response to HRG?1, but not to
EGF. The cells expressing ErbB2? and ErbB2?* were not respon-
sive to either EGF or HRG?1 treatment, consistent with the
inability of ErbB2 to bind any known ligand.
The crystal structure of ErbB2 has revealed that it is in a
constitutively active conformation, suggesting that it could sponta-
supported by the observation that full activation of ErbB2 does not
occur with ErbB2 alone but requires the presence of other ErbB
receptors in the cell (35). In addition, biochemical studies have
failed to detect ErbB2 homodimers in vitro. Our studies confirm
that ErbB2 does not form spontaneous homodimers more readily
Mean Fluorescence Intensity
Mean Fluorescence Intensity
H31Rα fusion expression level (YFP)
varying levels of ?* expression, low (L) and high (H) in A and B and low,
(C) non-YFP-expressing cells (A and C). The resulting cell lines were plated at
the same density into a 96-well dish and assayed for ?-gal activity in the
absence of inducer. For each set of cell lines, the low cell line was set equal to
1 for fluorescence and enzyme activity. In all cases tested, as the expression of
the ?* is increased, the basal ?-gal activity is also increased (B and D).
Basal enzyme activity is proportional to enzyme fragment expression
Relative cell number
assessment of ErbB interactions. ErbB2?*, ErbB3?*, and EGFR?* were trans-
fected into HEK293 cells and imaged by confocal microscopy for YFP expres-
sion (A). C2C12 cells were transduced with either the EGFR? or ErbB2?
with the indicated ?* fusions. Cells were sorted twice for YFP expression to
control for the levels of ?*. (B) Histograms of the fluorescence intensity of the
resulting double stable cell lines analyzed by flow cytometry. Quantification
Normalization of protein expression levels for the quantitative
Wehrman et al. PNAS ?
December 12, 2006 ?
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than the other receptor pairs tested because the enzyme activity is
similar for all cell lines in the absence of inducer (Fig. 4B).
The EGF family of protein ligands consists of at least 11
members. To investigate the effects of different ligands on the
of EGF, betacellulin, TGF-?, and heparin-binding EGF were
applied to cells expressing EGFR? and either EGFR?* or
ErbB2?* (Fig. 5A). Cells expressing ErbB2?–ErbB3?* were
treated with Hrg?1, Hrg?1, and SMDF (Fig. 5B). Although EGF
els in both cell lines, all of the other ligands tested show an increase
in EGFR homotypic interactions and a decrease in EGFR–ErbB2
heterotypic interactions in comparison to EGF.
Effects of Monoclonal Antibody Treatment on Receptor Interactions.
Three monoclonal antibodies against ErbB2 were tested for their
L87 binds the extracellular domain of ErbB2 but has no effect on
receptor activation (36). When assayed by ?-gal complementation
L87 had no effect on the interaction of ErbB2 with either EGFR
or ErbB3. Antibody 2C4 was found to prevent heterooligomeriza-
previous reports in which activity was assayed as a function of
phosphorylation (37). Notably, Herceptin markedly inhibited the
interaction of EGFR? with ErbB2?*. By contrast with 2C4,
Herceptin exhibited relatively little inhibition of the ErbB2?–
ErbB3?* interaction. These effects of Herceptin were dose-
dependent, and inhibition of the EGFR?–ErbB2?* interaction
occurred at doses on a par with 2C4 (Fig. 6 B and C).
The interaction studies indicate that Herceptin primarily inhibits
the formation of ErbB2–EGFR heterooligomers. Although it is
possible that Herceptin inhibits the formation of ErbB2–EGFR
heterooligomers more strongly than ErbB2–ErbB3 heterooli-
gomers, this seems unlikely as the extracellular domains of ErbB
receptors are highly homologous, both at the sequence and struc-
tural level. We postulate that because the EGFR readily homo-
oligomerizes, whereas ErbB3 does not (38), the propensity of the
EGFR monomers to form higher-ordered structures is in compe-
tition with the formation of ErbB2–EGFR heterooligomers. By
contrast, ErbB3 cannot homooligomerize, leaving ErbB3 mono-
mers available to interact with ErbB2, even in the presence of
sequestered in EGFR–EGFR complexes becoming increasingly
unavailable for heterooligomerization with ErbB2.
Our data, together with the known properties of the ErbB
receptors, prompted us to test whether Herceptin impacted EGFR
homooligomerization in cells expressing both the EGFR and
ErbB2. ErbB2 heterooligomers and EGFR homooligomers form
with equal efficiency, as shown in Fig. 4A. As a result, inhibition of
heterooligomer formation by Herceptin treatment should result in
a higher proportion of homooligomers (39). As a test of this
hypothesis, the wild-type ErbB2 lacking a ?-gal fragment was
overexpressed in the EGFR?*–EGFR? cell line. As expected,
exposure to EGF failed to stimulate ?-gal activity, given the strong
the cells were preincubated with the Herceptin antibody, EGF
caused an increase in ?-gal activity, because the ErbB2 bound to
Herceptin had diminished ability to interact with the EGFR. This
disruption allowed EGFR?* and EGFR? to interact. Addition of
2C4 restored the EGF-induced increase in ?-gal activity of the cell
of ErbB2 heterooligomers relative to Herceptin.
We reasoned that the increase in EGFR homooligomer forma-
tion in the presence of Herceptin would result in a decrease of
EGFR at the cell surface as the EGFR is rapidly internalized and
degraded as a homooligomer, but not as a heterooligomer with
ErbB2 (40). The wild-type EGFR and wild-type ErbB2 were
coexpressed in C2C12 cells and EGFR on the cell surface was
quantified by flow cytometry. Both Herceptin and 2C4 resulted in
different combinations of ErbB receptor chimeric proteins were plated in a
96-well dish at 2 ? 105cells per well. The cells were stimulated with the
indicated ligand for 45 min, and ?-gal activity was measured. Upon exposure
Heregulin treatment resulted only in the formation of ErbB2–ErbB3 hetero-
oligomers. (B) For each of the cell lines, the ?-gal activity was measured in the
absence of ligand as an indication of basal interaction levels. Note that ErbB2
does not exhibit an increased propensity to form homooligomers relative to
the EGFR or ErbB3.
Comparative analysis of the basal and induced interactions among
and either EGFR?* or ErbB2?* were treated with EGF, TGF-?, betacellulin
(BTC), and heparin-binding EGF (HB-EGF). (B) Cells expressing the ErbB2? and
ErbB3?* were treated with Hrg?-1, Hrg?-1, and sensory motor-neuron de-
the increase in ?-gal activity is shown.
EGF-like ligands have differential effects on homooligomerization
www.pnas.org?cgi?doi?10.1073?pnas.0605218103Wehrman et al.
a rapid loss of EGFR from the cell surface upon EGF treatment by
comparison with controls (Fig. 7B). Similar experiments were
performed with the SKBR3 breast cancer cell line that is known to
overexpress both EGFR and ErbB2. The SKBR3 cells also exhib-
presence of Herceptin and 2C4 (Fig. 7C). These experiments show
that blocking the interaction of the EGFR and ErbB2 using
increased EGFR homooligomer formation followed by more effi-
cient down-regulation of activated receptors. Together, these find-
ings provide an explanation of the ability of Herceptin to directly
inhibit the growth of ErbB2-expressing cancer cells independent of
an immune response.
The development of a method for monitoring dynamic receptor
interactions in an intact membrane was pivotal to the study of the
combinatorial interactions of the ErbB family members. This assay
measures the interaction of proteins as a function of the enzyme
activity generated upon induced proximity of the ?-gal enzyme
fragments to which they are fused. By controlling the expression
levels of each fragment, the entire profile of receptor interactions
could be compared across cell lines expressing different receptor
combinations. The assay is sensitive, quantitative, inducible, and
reversible. Although applied to ErbB family interactions in this
study, the protein interaction detection system described here is
readily adaptable to other protein interactions of interest.
The ability to quantitatively study receptors in the physiological
context of the plasma membrane forms an important bridge
between structural analysis of the purified extracellular domains of
the ErbB family of proteins and the indirect measurement of their
interaction provided by phosphorylation analysis. The capacity of
structural or biochemical analysis to predict and characterize pro-
tein interactions within the two-dimensional constraints of the
plasma membrane is limited. The strong, specific, and inducible
signal obtained by using the ?-gal complementation system makes
a detailed characterization of these processes possible. Much of the
data presented in this work echo what is currently known about the
interactions of the EGFR, ErbB2, and ErbB3, providing validation
of previous work. However, the protein interaction system de-
scribed here has made it possible to extend this knowledge, gen-
erating novel information about these reactions.
Our results show that in the context of the plasma membrane
ErbB2 efficiently interacts with the EGFR and ErbB3, whereas
ErbB3 and the EGFR do not form stable oligomers. Further, our
results indicate that the basal level of ErbB2 homooligomerization
is similar to its basal heterooligomerization level. An important
caveat of extrapolation of the interaction data obtained here is our
use of truncated receptors which does not take into account the
contribution of the intracellular domain on oligomerization. We
chose to use the simplest system possible to clearly delineate the
contribution of the extracellular and transmembrane domains to
the interaction of ErbB2 with the EGFR and ErbB3.
Although Herceptin has been used clinically for more than a
decade, there has been no clear characterization of its effect on
ErbB family dimerization. We show here that Herceptin primarily
result, Herceptin exposure should inhibit signaling by (i) disruption
of ErbB2–EGFR heterodimers and (ii) reduction of total EGFR
expression on the cell surface. Herceptin directly blocks the first,
leading to an increase in EGFR homodimerization, followed by
rapid internalization and ultimately a reduction in EGFR levels.
Together, the findings in this study suggest a mechanism by which
growth: targeting the ErbB2–EGFR heterodimer.
Importantly, the in vitro findings reported here correlate well
with the recently reported ErbB2 receptor expression profiles of
tumor samples from responders and nonresponders to Herceptin.
0 102030 4050
β-galactosidase activity (RLU)
0 10 203040
tion by Herceptin and 2C4 increases EGFR homooli-
gomer formation and internalization. (A) C2C12 cells
expressing the EGFR? and EGFR?*, as well as overex-
ment), were treated with increasing concentrations of
EGF. In the absence of antibody (No Ab) heterooli-
gomer formation is favored and enzyme activity does
not increase in response to EGF. Incubation with 1
?g?ml of each antibody before EGF treatment restores
the ability of the EGFR to form homooligomers. (B and
C) Assay of EGFR internalization in response to anti-
body treatment. C2C12 cells overexpressing both the
incubated with anti-EGFR antibody (Ab-11), and analyzed by flow cytometry. For the Herceptin and 2C4 curves, 5 ?g?ml of each antibody was added 10 min
before EGF for each time point. Each antibody caused a rapid, ligand-induced decrease in EGFR on the cell surface as compared with controls (No Ab).
Inhibition of EGFR–ErbB2 heterooligomeriza-
Induced β-gal activity (%)
induced β-gal activity (%)
10-1 10010-3 10-210110-4
10-1100 10-310-2 10110-4
EGFR. (A) The EGFR?–ErbB2?* and ErbB2?–ErbB3?* cell lines were treated with
5 ?g?ml of the indicated monoclonal antibodies for 30 min and tested for their
control antibody (IgG) and ligand were scaled to 100%, and the values in the
an indication of interaction.
Wehrman et al. PNAS ?
December 12, 2006 ?
vol. 103 ?
no. 50 ?
In patients whose tumors overexpress ErbB2, a response to Her- Download full-text
ceptin treatment is correlated with coexpression of the EGFR and
its ligand, as opposed to ErbB3 (41, 42). Thus, the data in this study
suggest a basis for predicting a response and selecting patients who
are likely to benefit from Herceptin therapy.
Materials and Methods
Generation of ?-Gal Fusion Proteins. The extracellular domains of
EGFR (amino acids 1–679), ErbB2 (1–686), and ErbB3 (1–693)
were PCR-amplified from cDNA clones with 5? MfeI and 3? XhoI
to the N terminus of the ? fragment in a WZL retroviral construct,
as described (12), and the YFPH31R? retroviral construct. The
B2AR-? construct used has been described (21). The full coding
clone and inserted into the MfeI-XhoI sites of the ? and
YFPH31R?* vectors. The complete coding sequence of FKBP12
and amino acids 2025–2114 of human FRAP were PCR-amplified
and inserted into the ? and YFPH31R?* vectors as MfeI-XhoI
fragments. The full-length ErbB2 clone was also PCR-amplified
from a cDNA clone and inserted into an MFG retroviral vector
Virus Production and Cell Culture. Retroviral vectors were trans-
fected into the ?nx-packaging cell line (P. L. Achacoso and G. P.
Nolan, personal communication) by using Lipofectamine 2000
(Invitrogen) in six-well dishes according to the manufacturer’s
instructions. Twenty-four hours after transfection the viral super-
natant was filtered through a 0.45-?m syringe filter onto C2C12
cells. Polybrene was added at a final concentration of 4 ?g?ml, and
Cells were returned to a 37°C 5% CO2humidified incubator for
12 h, and then the medium was exchanged with fresh medium.
streptomycin. When appropriate, cells were selected with 1 ?g?ml
hygromycin (Invitrogen) or sorted for YFP expression.
Cell Treatments and Assays. Herceptin and 2C4 were generous gifts
from Genentech. All other antibodies were supplied by NeoMar-
and heparin-binding EGF were obtained from PeproTech, and
SMDF and HRG-?1 were from R & D Systems. Isoproterenol and
rapamycin were from Sigma. Isoproterenol was resuspended in an
ascorbate solution (0.3 mM) before each experiment. For mea-
surements of ?-gal activity cells were seeded at 20,000 cells per well
of a 96-well dish overnight. After the appropriate treatment,
medium was removed from the cells, and 50 ?l of buffer B mixed
with a 1:20 dilution of Galacton-Star (Gal-screen; Applied Biosys-
tems) was added. Cells were incubated at room temperature for 45
FACS and Microscopy. Cells were trypsinized and washed in a 5%
FBS?PBS solution. Cells were sorted on a Becton Dickinson
FACStar by using the argon laser (488-nm excitation). Images of
the YFP fusion proteins were obtained on a Zeiss LSM 510
confocal microscope. For the internalization assays Ab-11 (Neo-
Markers) was conjugated to Alexa Fluor 647 according to standard
procedures and used to detect the remaining EGFR at the cell
We thank Mark Sliwkowski and Matthew Franklin at Genentech for
monoclonal antibody reagents and advice regarding experiments pre-
sented in this work. T.S.W. was supported by National Institutes of
Health (NIH) Biotechnology Training Grant T32 GM08412, NIH Aging
Training Grant T32 AG0259, and a Genentech fellowship; J.H.P. was
supported by NIH National Research Service Award AF051678; and
H.M.B. was supported by NIH Grants HD018179, AG009521,
AG024987, AG020961, and DAMD17-00-1-0442 and the Baxter
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