• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Dec 12, 2006; 103(50): 19063–19068.
Published online Dec 5, 2006. doi:  10.1073/pnas.0605218103
PMCID: PMC1748177
Medical Sciences

A system for quantifying dynamic protein interactions defines a role for Herceptin in modulating ErbB2 interactions


The orphan receptor tyrosine kinase ErbB2 is activated by each of the EGFR family members upon ligand binding. However, difficulties monitoring the dynamic interactions of the membrane receptors 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. Application 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 interaction of ErbB2–ErbB3. Treatment of cells expressing EGFR and ErbB2 with Herceptin results in increased EGFR homooligomerization in the presence of EGF and a subsequent rapid internalization and down-regulation of the EGFR. In summary, the protein interaction system described here enabled the characterization of ErbB2 interactions within the biological context of the plasma membrane and provides insight into the mechanism of Herceptin action on cells overexpressing ErbB2.

Keywords: anti-cancer, EGF receptor

The EGF family of receptor tyrosine kinases consists of four members, EGFR, ErbB2, ErbB3, and ErbB4, that become 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 heterodimerization with any of the other EGF family members. Physical characterization of this process has proven difficult using conventional biochemical methods, but it is of considerable interest because of the role of ErbB2 in breast cancer pathogenesis.

ErbB2 is overexpressed in 30% of breast cancers and most clearly associated with a malignant phenotype and poor prognosis, especially if coexpressed with the EGF receptor (EGFR) (13). 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 unprecedented manner (46). Although there is evidence that Herceptin targets tumor cells for destruction by the immune system (7), the antibody was originally selected as an inhibitor of tumor cell growth 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 (911).

We postulated that the β-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 interaction of two proteins is measured as a function of complementation of low-affinity mutant subunits of the β-gal enzyme fused to the receptor proteins. Inducible and reversible interactions 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 populations. 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), β-lactamase (16, 17), luciferase (18), and the previously described β-gal (1921).

Investigation of the oligomerization properties of the EGFR, ErbB2, and ErbB3 using β-gal complementation yielded quantitative 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 confirming its role as the heterodimerization partner. However, the 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 Herceptin is ineffective in blocking ErbB2–ErbB3 interactions. However, 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 recently described a proximity-based low-affinity enzyme complementation system for monitoring protein translocation using β-gal. To achieve low-affinity complementation, the classic α peptide first described by Jacob and Monod (22) was truncated and mutated. Of the mutants obtained, the histidine-to-arginine mutant at position 31 of the α peptide (α*) was chosen because of its particularly weak ability to spontaneously complement the M15 deletion mutant (ω) but high signal-to-noise ratio upon induction of complementation. Because of their low affinity, the interaction of the α* and ω β-gal fragments is not sufficiently strong to maintain a complemented enzyme. As a result, the β-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 β-gal complementation system for analyzing specific inducible protein–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 chemiluminescence. Treatment of C2C12 cells expressing FKBP12ω and FRBα* with rapamycin for 2 h resulted in a 10-fold increase in β-gal activity (Fig. 1B), demonstrating that low-affinity α-complementation can be used to monitor protein interactions with a large induction of signal. To determine whether the β-gal system could also be used to monitor the interaction of lower-affinity, reversible interactions, the association of the G protein-coupled receptor, the β2-adrenergic receptor (B2AR), with β-arrestin2 was evaluated. Upon stimulation, B2AR becomes phosphorylated and binds β-arrestin2. Treatment of cells expressing the B2AR-ω and β-arrestin2α* fusion proteins with agonist (isoproterenol) resulted in a 4-fold increase in enzyme activity, which was prevented by pretreatment with the antagonist (propranolol) (Fig. 1 CE). The dose–response and EC50 obtained as a function of β-gal activity are in good agreement with published values (24), indicating that low-affinity proximity-based α-complementation 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 α* and ω is not known. Although wild-type β-gal has been shown to form a tetramer (25), evidence also exists for an active β-gal dimer (26). It is currently unclear whether a complemented monomer retains enzyme activity. For this reason, by using the low-affinity β-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 β-gal complementation system regarding receptor interactions is referred to as heterooligomerization and homooligomerization as opposed to the more specific heterodimerization and homodimerization nomenclature.

Fig. 1.
Inducible protein interactions monitored by low-affinity α-complementation of β-gal. (A) Schematic illustration of the low-affinity α-complementation system. Physical association of two chimeric proteins brings mutant β-gal ...

ErbB2 is generally regarded as the preferred heterodimerization partner for each of the ligand-bound ErbB receptors (27); however, 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 interactions has led to conflicting results (27, 28). We applied the low-affinity β-gal system to the interaction of the EGFR and ErbB2 by fusing only the extracellular and transmembrane domains of the EGFR and ErbB2 to ω and α*, 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 observed (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 treatment reaches 75% maximal signal within 15 min of stimulation (Fig. 1H).

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 experiments that coexpression of the α* and ω results in a background level of β-gal activity. Thus to be able to compare between cell lines expressing specified pairs of proteins fused to the α* and ω, the relationship between protein expression level and background β-gal activity had to be determined. Both the β-arrestin2 construct and the ErbB2 construct included YFP inserted between the gene and the α* peptide providing a marker of protein levels.

The B2ARω-βarrestin2α* and EGFRω–ErbB2α* cell lines were sorted by FACS into different populations based on amount of YFP expression as a marker for α* peptide expression (Fig. 2A and C). The sorted populations were plated into a 96-well dish and assayed for β-gal activity in unstimulated conditions. In both cell lines we see a proportional increase in enzyme activity with increased α* peptide concentration (Fig. 2 B and D). Thus, like Fret and Bret (14, 29), the baseline β-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.

Fig. 2.
Basal enzyme activity is proportional to enzyme fragment expression level. The B2ARω–β-arrestin2α* and EGFRω–ErbB2α* cell lines were sorted for varying levels of α* expression, low (L) and ...

Investigation of EGFR, ErbB2, and ErbB3 Interactions.

The determination of the possible interaction states of ErbB2, EGFR, and ErbB3 was accomplished using six different cell lines; EGFRω–EGFRα*, EGFRω–ErbB2α*, EGFRω–ErbB3α*, ErbB2ω–EGFRα*, ErbB2ω–ErbB2α*, and EGFRω–ErbB3α*. If cell lines are to be cross-compared, the α* fusions must localize similarly. To ensure that all of the α* fusions were similarly expressed at the plasma membrane the corresponding plasmids that include YFP sandwiched between the receptor and α* 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).

Fig. 3.
Normalization of protein expression levels for the quantitative assessment of ErbB interactions. ErbB2α*, ErbB3α*, and EGFRα* were transfected into HEK293 cells and imaged by confocal microscopy for YFP expression (A). C2C12 cells ...

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 ErbB2ω or the EGFRω fusion proteins. Each of the ω-expressing parental cell lines were then split and transduced with each of three constructs encoding different α-fusion proteins, EGFRα*, ErbB2α*, and ErbB3α*. Each cell line expressing the α* and ω fusion proteins was generated simultaneously by retroviral infection of replicate cultures of the same ω expressing parental cell line. As a result, the levels and nature of the ω-fusion protein are similar for each cell line; by contrast, the α* 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 differences, YFP was included in each construct, between the ErbB receptor and the α* allowing measurement of α* chimeric protein expression levels. Cells expressing similar YFP levels were isolated by FACS so that the levels of α*-fusion proteins were comparable (Fig. 3B). Quantification of the mean fluorescence levels from each of the sorted cell lines shows a <15% total variance (Fig. 3C).

Ligand-stimulated enzyme activities were assessed for each of the six cell lines expressing pairs of receptors. Cells were exposed either to the ligand EGF that binds the EGFR or to the ligand heregulin (HRGβ1) that binds the ErbB3 receptor (31). All of the expected interactions were observed (Fig. 4A). EGF led to homooligomerization of the EGFR and heterooligomerization of EGFR with ErbB2, whereas HRGβ1 failed to induce interaction of these receptors. When cells were compared that expressed EGFRω–ErbB2α* or ErbB2ω–EGFRα*, the responses were similar. This finding was important, because it indicated that similar interactions occurred irrespective of whether the receptors were fused to α* or ω. 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 ErbB2ω and ErbB3α* generated heterooligomers only in response to HRGβ1, but not to EGF. The cells expressing ErbB2ω and ErbB2α* were not responsive to either EGF or HRGβ1 treatment, consistent with the inability of ErbB2 to bind any known ligand.

Fig. 4.
Comparative analysis of the basal and induced interactions among the EGFR, ErbB2, and ErbB3. (A) Aliquots of each of the six cell lines expressing different combinations of ErbB receptor chimeric proteins were plated in a 96-well dish at 2 × 10 ...

The crystal structure of ErbB2 has revealed that it is in a constitutively active conformation, suggesting that it could spontaneously homodimerize and signal (33, 34). However, this view is not 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 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 formation of homooligomers and heterooligomers, increasing doses of EGF, betacellulin, TGF-α, and heparin-binding EGF were applied to cells expressing EGFRω and either EGFRα* or ErbB2α* (Fig. 5A). Cells expressing ErbB2ω–ErbB3α* were treated with Hrgβ1, Hrgα1, and SMDF (Fig. 5B). Although EGF causes similar homooligomerization and heterooligomerization levels 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.

Fig. 5.
EGF-like ligands have differential effects on homooligomerization and heterooligomerization of the EGFR and ErbB2. (A) Cells expressing EGFRω and either EGFRα* or ErbB2α* were treated with EGF, TGF-α, betacellulin (BTC), ...

Effects of Monoclonal Antibody Treatment on Receptor Interactions.

Three monoclonal antibodies against ErbB2 were tested for their effects on ErbB receptor oligomerization levels (Fig. 6A). Antibody L87 binds the extracellular domain of ErbB2 but has no effect on receptor activation (36). When assayed by β-gal complementation L87 had no effect on the interaction of ErbB2 with either EGFR or ErbB3. Antibody 2C4 was found to prevent heterooligomerization of ErbB2 with either ErbB3 or EGFR, in good agreement with previous reports in which activity was assayed as a function of phosphorylation (37). Notably, Herceptin markedly inhibited the interaction of EGFRω with ErbB2α*. By contrast with 2C4, Herceptin exhibited relatively little inhibition of the ErbB2ω–ErbB3α* interaction. These effects of Herceptin were dose-dependent, and inhibition of the EGFRω–ErbB2α* interaction occurred at doses on a par with 2C4 (Fig. 6 B and C).

Fig. 6.
Monoclonal antibody treatment affects the interaction of ErbB2 and the EGFR. (A) The EGFRω–ErbB2α* and ErbB2ω–ErbB3α* cell lines were treated with 5 μg/ml of the indicated monoclonal antibodies for ...

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 heterooligomers, this seems unlikely as the extracellular domains of ErbB receptors are highly homologous, both at the sequence and structural level. We postulate that because the EGFR readily homooligomerizes, whereas ErbB3 does not (38), the propensity of the EGFR monomers to form higher-ordered structures is in competition with the formation of ErbB2–EGFR heterooligomers. By contrast, ErbB3 cannot homooligomerize, leaving ErbB3 monomers available to interact with ErbB2, even in the presence of Herceptin. As a result, unlike ErbB3, EGFR would be progressively 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 β-gal fragment was overexpressed in the EGFRα*–EGFRω cell line. As expected, exposure to EGF failed to stimulate β-gal activity, given the strong propensity for heterooligomer formation (Fig. 7A). However, when the cells were preincubated with the Herceptin antibody, EGF caused an increase in β-gal activity, because the ErbB2 bound to Herceptin had diminished ability to interact with the EGFR. This disruption allowed EGFRα* and EGFRω to interact. Addition of 2C4 restored the EGF-induced increase in β-gal activity of the cell line to a greater extent, as expected given the more potent inhibition of ErbB2 heterooligomers relative to Herceptin.

Fig. 7.
Inhibition of EGFR–ErbB2 heterooligomerization by Herceptin and 2C4 increases EGFR homooligomer formation and internalization. (A) C2C12 cells expressing the EGFRω and EGFRα*, as well as overexpressed wild-type full-length ErbB2 ...

We reasoned that the increase in EGFR homooligomer formation 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 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 exhibited a similar increase in EGF-induced EGFR internalization in the presence of Herceptin and 2C4 (Fig. 7C). These experiments show that blocking the interaction of the EGFR and ErbB2 using Herceptin would not only lead to reduced ErbB2 activation, but also increased EGFR homooligomer formation followed by more efficient down-regulation of activated receptors. Together, these findings 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 β-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 protein interactions within the two-dimensional constraints of the plasma membrane is limited. The strong, specific, and inducible signal obtained by using the β-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 described here has made it possible to extend this knowledge, generating 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 impacts ErbB2–EGFR heterodimer levels, not ErbB2–ErbB3. As a 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 Herceptin inhibits ErbB receptor signaling and therefore tumor cell 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. In patients whose tumors overexpress ErbB2, a response to Herceptin 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 β-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 sites to generate the β-gal constructs. The PCR products were fused to the N terminus of the ω fragment in a WZL retroviral construct, as described (12), and the YFPH31Rα retroviral construct. The B2AR-ω construct used has been described (21). The full coding sequence of human β-arrestin B2 was PCR-amplified from a cDNA clone and inserted into the MfeI-XhoI sites of the ω and YFPH31Rα* vectors. The complete coding sequence of FKBP12 and amino acids 2025–2114 of human FRAP were PCR-amplified and inserted into the ω and YFPH31Rα* 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 using MfeI-XhoI restriction enzymes, not fused to a β-gal fragment.

Virus Production and Cell Culture.

Retroviral vectors were transfected into the Φnx-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 supernatant was filtered through a 0.45-μm syringe filter onto C2C12 cells. Polybrene was added at a final concentration of 4 μg/ml, and the plates were spun at 1,900 rpm in a tabletop centrifuge for 30 min. Cells were returned to a 37°C 5% CO2 humidified incubator for 12 h, and then the medium was exchanged with fresh medium. C2C12 cells were grown in 20% FBS DMEM containing penicillin/streptomycin. When appropriate, cells were selected with 1 μg/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 NeoMarkers. Recombinant human EGF ligands, EGF, TGF-α, betacellulin, and heparin-binding EGF were obtained from PeproTech, and SMDF and HRG-α1 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 measurements of β-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 μl of buffer B mixed with a 1:20 dilution of Galacton-Star (Gal-screen; Applied Biosystems) was added. Cells were incubated at room temperature for 45 min. Luminescence was measured in a Tropix TR717 luminometer.

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 (NeoMarkers) was conjugated to Alexa Fluor 647 according to standard procedures and used to detect the remaining EGFR at the cell surface.


We thank Mark Sliwkowski and Matthew Franklin at Genentech for monoclonal antibody reagents and advice regarding experiments presented 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 Foundation.


EGF receptor
β2-adrenergic receptor.


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.


1. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Science. 1987;235:177–182. [PubMed]
2. Gschwantler-Kaulich D, Hudelist G, Koestler WJ, Czerwenka K, Mueller R, Helmy S, Ruecklinger E, Kubista E, Singer CF. Oncol Rep. 2005;14:305–311. [PubMed]
3. DiGiovanna MP, Stern DF, Edgerton SM, Whalen SG, Moore D, II, Thor AD. J Clin Oncol. 2005;23:1152–1160. [PubMed]
4. Romond EH, Perez EA, Bryant J, Suman VJ, Geyer CE, Jr, Davidson NE, Tan-Chiu E, Martino S, Paik S, Kaufman PA, et al. N Engl J Med. 2005;353:1673–1684. [PubMed]
5. Piccart-Gebhart MJ, Procter M, Leyland-Jones B, Goldhirsch A, Untch M, Smith I, Gianni L, Baselga J, Bell R, Jackisch C, et al. N Engl J Med. 2005;353:1659–1672. [PubMed]
6. Cobleigh MA, Vogel CL, Tripathy D, Robert NJ, Scholl S, Fehrenbacher L, Wolter JM, Paton V, Shak S, Lieberman G, Slamon DJ. J Clin Oncol. 1999;17:2639–2648. [PubMed]
7. Clynes RA, Towers TL, Presta LG, Ravetch JV. Nat Med. 2000;6:443–446. [PubMed]
8. Hudziak RM, Lewis GD, Winget M, Fendly BM, Shepard HM, Ullrich A. Mol Cell Biol. 1989;9:1165–1172. [PMC free article] [PubMed]
9. Karunagaran D, Tzahar E, Beerli RR, Chen X, Graus-Porta D, Ratzkin BJ, Seger R, Hynes NE, Yarden Y. EMBO J. 1996;15:254–264. [PMC free article] [PubMed]
10. Horan T, Wen J, Arakawa T, Liu N, Brankow D, Hu S, Ratzkin B, Philo JS. J Biol Chem. 1995;270:24604–24608. [PubMed]
11. Ferguson KM, Darling PJ, Mohan MJ, Macatee TL, Lemmon MA. EMBO J. 2000;19:4632–4643. [PMC free article] [PubMed]
12. Wehrman TS, Casipit CL, Gewertz NM, Blau HM. Nat Methods. 2005;2:521–527. [PubMed]
13. Xu Y, Piston DW, Johnson CH. Proc Natl Acad Sci USA. 1999;96:151–156. [PMC free article] [PubMed]
14. Pollok BA, Heim R. Trends Cell Biol. 1999;9:57–60. [PubMed]
15. Pelletier JN, Campbell-Valois FX, Michnick SW. Proc Natl Acad Sci USA. 1998;95:12141–12146. [PMC free article] [PubMed]
16. Galarneau A, Primeau M, Trudeau LE, Michnick SW. Nat Biotechnol. 2002;20:619–622. [PubMed]
17. Wehrman T, Kleaveland B, Her JH, Balint RF, Blau HM. Proc Natl Acad Sci USA. 2002;99:3469–3474. [PMC free article] [PubMed]
18. Paulmurugan R, Gambhir SS. Anal Chem. 2003;75:1584–1589. [PMC free article] [PubMed]
19. Rossi F, Charlton CA, Blau HM. Proc Natl Acad Sci USA. 1997;94:8405–8410. [PMC free article] [PubMed]
20. Blakely BT, Rossi FM, Tillotson B, Palmer M, Estelles A, Blau HM. Nat Biotechnol. 2000;18:218–222. [PubMed]
21. Yan YX, Boldt-Houle DM, Tillotson BP, Gee MA, D'Eon BJ, Chang XJ, Olesen CE, Palmer MA. J Biomol Screen. 2002;7:451–459. [PubMed]
22. Jacob F, Monod J. J Mol Biol. 1961;3:318–356. [PubMed]
23. Chen J, Zheng XF, Brown EJ, Schreiber SL. Proc Natl Acad Sci USA. 1995;92:4947–4951. [PMC free article] [PubMed]
24. Oakley RH, Hudson CC, Cruickshank RD, Meyers DM, Payne RE, Jr, Rhem SM, Loomis CR. Assay Drug Dev Technol. 2002;1:21–30. [PubMed]
25. Matthews BW. C R Biol. 2005;328:549–556. [PubMed]
26. Kaneshiro CM, Enns CA, Hahn MG, Peterson JS, Reithel FJ. Biochem J. 1975;151:433–434. [PMC free article] [PubMed]
27. Graus-Porta D, Beerli RR, Daly JM, Hynes NE. EMBO J. 1997;16:1647–1655. [PMC free article] [PubMed]
28. Riese DJ, II, van Raaij TM, Plowman GD, Andrews GC, Stern DF. Mol Cell Biol. 1995;15:5770–5776. [PMC free article] [PubMed]
29. Mercier JF, Salahpour A, Angers S, Breit A, Bouvier M. J Biol Chem. 2002;277:44925–44931. [PubMed]
30. Corti S, Salani S, Del Bo R, Sironi M, Strazzer S, D'Angelo MG, Comi GP, Bresolin N, Scarlato G. Exp Cell Res. 2001;268:36–44. [PubMed]
31. Hynes NE, Lane HA. Nat Rev Cancer. 2005;5:341–354. [PubMed]
32. Zhang K, Sun J, Liu N, Wen D, Chang D, Thomason A, Yoshinaga SK. J Biol Chem. 1996;271:3884–3890. [PubMed]
33. Cho HS, Mason K, Ramyar KX, Stanley AM, Gabelli SB, Denney DW, Jr, Leahy DJ. Nature. 2003;421:756–760. [PubMed]
34. Garrett TP, McKern NM, Lou M, Elleman TC, Adams TE, Lovrecz GO, Kofler M, Jorissen RN, Nice EC, Burgess AW, Ward CW. Mol Cell. 2003;11:495–505. [PubMed]
35. Yarden Y. Oncology. 2001;61(Suppl 2):1–13. [PubMed]
36. Klapper LN, Vaisman N, Hurwitz E, Pinkas-Kramarski R, Yarden Y, Sela M. Oncogene. 1997;14:2099–2109. [PubMed]
37. Agus DB, Akita RW, Fox WD, Lewis GD, Higgins B, Pisacane PI, Lofgren JA, Tindell C, Evans DP, Maiese K, et al. Cancer Cell. 2002;2:127–137. [PubMed]
38. Berger MB, Mendrola JM, Lemmon MA. FEBS Lett. 2004;569:332–336. [PubMed]
39. Hendriks BS, Wiley HS, Lauffenburger D. Biophys J. 2003;85:2732–2745. [PMC free article] [PubMed]
40. Wang Z, Zhang L, Yeung TK, Chen X. Mol Biol Cell. 1999;10:1621–1636. [PMC free article] [PubMed]
41. Hudelist G, Kostler WJ, Czerwenka K, Kubista E, Attems J, Muller R, Gschwantler-Kaulich D, Manavi M, Huber I, Hoschutzky H, et al. Int J Cancer. 2005;118:1126–1134. [PubMed]
42. Smith BL, Chin D, Maltzman W, Crosby K, Hortobagyi GN, Bacus SS. Br J Cancer. 2004;91:1190–1194. [PMC free article] [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences
PubReader format: click here to try


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...