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

Abstract

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.

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 fragments, M15 (ω) and H31Rα (α*), into proximity, generating β-gal activity. (B) Low-affinity α-complementation monitors strong protein interactions. Cells expressing FRBα* and FKBP12ω exhibited increased β-gal activity after exposure to rapamycin (Rap). (C) Low-affinity α-complementation quantitatively monitors protein interactions such as the inducible interaction of the membrane-bound B2AR and cytosolic β-arrestin2 in cells expressing B2ARω and β-arrestin2α* chimeras. α* denotes chimeric proteins consisting of protein of interest [yellow fluorescent protein (FP)] H31Rα (a*) fusions. (D) Dose–response of the interaction of B2ARω and β-arrestin2α* chimeras 45 min after exposure to the agonist isoproteronol assayed as β-gal activity. (E) The B2ARω and β-arrestin2α* interaction was prevented in a dose-dependent manner by the antagonist propanolol. Increasing doses of propanolol were added to cells 10 min before addition of 1 μM isoproterenol, and β-gal activity was measured 45 min later. (F and G) Low-affinity α-complementation monitors heterooligomer formation between the EGFR and ErbB2. The extracellular and transmembrane domains of the EGFR and ErbB2 were used to create two chimeras, EGFRω and ErbB2α*. (G) Cells expressing both EGFRω and ErbB2α* 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 EGFRω and ErbB2α* were stimulated with 100 ng/ml EGF, and the enzyme activity was measured, demonstrating increasing interaction over time.

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 high (H) in A and B and low, medium (M), and high in C and D. The cells are shown in comparison to control (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 β-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 α* is increased, the basal β-gal activity is also increased (B and D).

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 were transduced with either the EGFRω or ErbB2ω constructs to generate parental cell lines. Each parental line was then infected with the indicated α* fusions. Cells were sorted twice for YFP expression to control for the levels of α*. (B) Histograms of the fluorescence intensity of the resulting double stable cell lines analyzed by flow cytometry. Quantification of the mean fluorescence intensity for each cell line shows similar levels of YFP expression (C).

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 × 10⁵ cells per well. The cells were stimulated with the indicated ligand for 45 min, and β-gal activity was measured. Upon exposure to EGF, EGFR homooligomers and EGFR–ErbB2 heterooligomers were formed. Heregulin treatment resulted only in the formation of ErbB2–ErbB3 heterooligomers. (B) For each of the cell lines, the β-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.

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), and heparin-binding EGF (HB-EGF). (B) Cells expressing the ErbB2ω and ErbB3α* were treated with Hrgβ-1, Hrgα-1, and sensory motor-neuron derived factor (SMDF). Cells were incubated in the presence of ligand for 1 h, and the increase in β-gal activity is shown.

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 30 min and tested for their ability to respond to 100 ng/ml EGF or Hrgβ1, respectively. The cells treated with control antibody (IgG) and ligand were scaled to 100%, and the values in the absence of ligand were set to 0%. (B and C) Cells were exposed to different doses of Herceptin (B) or 2C4 (C) before ligand addition. β-gal activity was measured as an indication of interaction.

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 (with no β-gal fragment), were treated with increasing concentrations of EGF. In the absence of antibody (No Ab) heterooligomer formation is favored and enzyme activity does not increase in response to EGF. Incubation with 1 μg/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 antibody treatment. C2C12 cells overexpressing both the wild-type EGFR and ErbB2 (B) or the breast cancer cell line SKBR3, which expresses both of these receptors (C), were stimulated with EGF at different time points, incubated with anti-EGFR antibody (Ab-11), and analyzed by flow cytometry. For the Herceptin and 2C4 curves, 5 μg/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).