1. Complex
Formation
(Capture) (Detection)
C
ontrol
G
efitinib
Foretinib
RFU
HER2 Y1196
- EGFR
(Phospho) EGFR Y845
(Phospho) EGFR Y998
(Phospho) Met Y1234
/Y1235
(Total) EGFR
(Total) Met
120
100
80
60
40
20
0
EGFR Y998
- c-Met
C
ontrol
G
efitinib
Foretinib
Probing EGFR, HER2, and c-Met Protein-Protein Interactions Using an Antibody Array
JACOB KNOWLES, STEPHEN CAPODILUPO, RICHARD NORING, ROBERTO CAMPOS-GONZÁLEZ, JULIO HERRERA AND ZEV GECHTMAN. CELL SIGNALING TECHNOLOGY, INC. 3 TRASK LANE, DANVERS, MA 01923
The Receptor Tyrosine Kinase (RTK) family includes important dis-
ease drivers and validated drug targets. Cooperation between RTKs is
often achieved through physical interactions and is thought be a
contributing factor for some cases of tumor resistance to targeted
therapies. Epidermal Growth-Factor Receptor (EGFR), HER2/ErbB2,
and c-Met are prominent examples of disease driver RTKs that interact
to form hetero-dimers.
Current methods to detect RTK protein-protein interactions lack
proper throughput, are not quantitative, or rely on highly engineered sys-
tems and cumbersome workflows. We have developed a planar surface
antibody array-based sandwich assay that enables the detection and
relative quantification of interactions between EGFR, HER2, and c-Met.
The antibody array-based assay revealed co-existing combinatorial
receptor interactions that were unique to each cell type tested. It also
alluded to the existence of distinct topological configurations of EGFR-
c-Met and EGFR-HER2 hetero-dimers, unveiling previously undescribed
nuances of these interactions. Lastly, the array-based assay allowed
the disruption of receptor complexes using small-molecule inhibitors
to be monitored and revealed differential sensitivity of EGFR-HER2,
and EGFR-c-Met complexes to the small-molecule EGFR kinase
inhibitor gefitinib.
The antibody array-based sandwich assay described here allows a
large panel of affinity reagents to be used and enables parallel detec-
tion of RTK protein-protein interactions, allowing a more systematic
monitoring of the response to activators or inhibitors.
A planar surface antibody array was configured to
detect and quantify Receptor Tyrosine Kinase (RTK)
protein-protein interactions.
Two sets of antibody pairs were identified that
are able to distinguish between two combina-
tions of EGFR-c-Met heterodimers, which
differ in their joint conformation.
The antibody array sandwich immunoassay
allowed monitoring of the disruption of receptor
heterodimers using small-molecule inhibitors.
Zev Gechtman
Cell Signaling Technology, Inc.
3 Trask Lane, Danvers, MA 01915
Email: zgechtman@cellsignal.com
Abstract
Summary
Contact Information
Figure 1. Schematic representation of the assay set-up. Protein-protein interactions are detected by capturing a target protein,
A, with a specific antibody and detecting the interacting partner, B, with another specific antibody. In this assay configuration,
multiple capture antibodies can be printed on each pad, and a single detection antibody is applied per pad. This format is ame-
nable to multi data-point analysis. Both direct and indirect protein-protein interactions can be detected using this assay format.
Figure 2. Representative fluorescent slide images. HCC827 cells, which express mutated and constitutively active EGFR
and overexpress c-Met, were used. The specificity of the relevant capture antibodies printed on the pad is indicated (1-5).
The pad was probed with a total c-Met detection antibody so the EGFR-c-Met interaction (1and 2), c-Met phosphorylation
(3 and 4), and total c-Met levels (5) were visualized at the same time. Non-phospholysate and phospholysate indicates the
use of lysis buffer that was either devoid of or contains phosphatase inhibitors respectively. Slides were scanned using the
Odyssey®
CLx array scanner (LI-COR®
) at a wavelength of 700 nm (compatible with DyLight™
680-conjugated streptavidin).
Image studio 2.0 software was used to quantify the spot fluorescence intensity.
Figure 3. Detection of complex formation between EGFR, c-Met, and HER2 in tumor derived cell extracts using the
antibody array-based assay. The interaction between the three RTKs in HCC827 and Calu-3 cells is constitutive and the cells
were not treated prior to lysis. A431 cells were serum starved overnight then treated with 100 ng/ml EGF for 5 minutes, where in-
dicated, to induce RTK interaction. Receptor complexes were detected using the specified RTK antibodies and quantified using the
array-based assay. Slides were scanned using the Odyssey®
CLx array scanner (LI-COR®
). Image studio 2.0 software was used to
quantify the spot fluorescence intensity.
Figure 4. Western blot analysis to demonstrate activation of EGFR and c-Met by EGF and HGF in A431 cells. A431 cells
were serum starved overnight then treated with various combinations of growth-factors and/or inhibitors as indicated. Gefitinib (EGFR
inhibitor) and foretinib (c-Met inhibitor) were used at 1µM and cells were preincubated with inhibitors for 3.5 hours prior to growth-
factor treatment. Cells were treated with 100 ng/ml EGF or HGF (where indicated) for 5 minutes. Western blot analysis shows EGFR
and c-Met phosphorylation with the various growth factor/inhibitor combinations.
Figure 5. Downstream signaling nodes were activated by EGF and HGF, and this activation was inhibited by gefitinib and
foretinib. The various cell treatments are indicated. The relative phosphorylation levels of some major signaling molecules as well as
cleavage of pro-apoptotic proteins were assessed using the PathScan®
Intracellular Signaling Antibody Array Kit (Fluorescent Readout,
#7744). Raw fluorescence values, after subtraction of background fluorescence, were used for analysis and the heatmap was gener-
ated using Multi Experiment Viewer software.
Figure 6. EGF and HGF induced complexes were detected in A431 cells: different conformations of EGFR-Met heterodi-
mers were recognized using the antibody array-based assay. A431 cells were serum starved overnight then subjected to the
various treatments indicated. Cells were treated with 100 ng/ml EGF or HGF for 5 minutes. Where indicated, cells were pretreated
with 1 µM gefitinib or foretinib for 3.5 hours. Complexes between EGFR and c-Met were detected using two sets of antibody pairs.
EGFR Y998
and total c-Met (upper), and Met Y1349
and total EGFR detection antibody (lower).
Figure 7. Various constitutive EGFR-HER2 and EGFR-c-Met complexes were detected in HCC827 cells.
The specificity of the various capture antibodies is indicated below the graph. Antibodies to total EGFR and c-Met were used for
detection. RTK complexes were detected using the array-based assay. Fluorescence intensity after background subtraction was
used for the relative quantification.
Figure 8. EGFR-c-Met dimerization was inhibited by gefitinib and foretinib. HCC827 cells were either untreated or treated
with 1 µM gefitinib or foretinib for 16 hours in complete medium. The heatmap shows tyrosine phosphorylation of RTKs on indicated
sites as well as levels of the total EGFR and c-Met protein. The bar graph shows the relative levels of EGFR-c-Met and EGFR-HER2
complexes under these conditions.
Figure 9. EGFR-HER2 and EGFR-c-Met complexes show differential sensitivity to gefitinib. HCC827 cells were treated
for 2.5 hours with various concentrations of gefitinib in serum-free medium. Capture-detection antibody pairs used to detect the
complexes are indicated in the graph. Normalized fluorescence intensity was used to create the plot.
Multiple capture antibodies are printed on each pad
Single detection antibody is applied to each pad
A
B
1 5
non-phospholysate
phospholysate
2
3
4
1 5
2
3
4
1 EGFR Y998
2 EGFR Y845
3 Met Y1234/1235
Total Met
4 Met Y1349
5 Total Met
Capture Detection
12000
8000
10000
6000
RFU
Calu-3
HCC827
A431
RFURFU
4000
2000
0
70000
50000
60000
40000
30000
20000
10000
0
70000
50000
60000
40000
30000
20000
10000
0
RFU
60000
40000
50000
30000
20000
10000
0
RFU
60000
70000
40000
50000
30000
20000
10000
0
RFU
35000
40000
25000
30000
20000
15000
10000
5000
0
EGFR-HER2 complex EGFR-c-Met complex
-EGF
+EGF
-EGF
+EGF
Non-phospholysate
Phospholysate
Non-phospholysate
Phospholysate
Non-phospholysate
Phospholysate
Non-phospholysate
Phospholysate
Capture:
Detection:
EGFR Y1845
HER2
EGFR Y998
HER2
HER2 Y1196
EGFR
Capture:
Detection:
EGFR Y845
Met
EGFR Y998
Met
Met Y1349
EGFR
p-EGFR Y1068
Total EGFR
p-Met Y1349
Total Met
C
ontrol
EG
F
H
G
F
G
efitinib
G
efitinib
+
EG
F
G
efitinib
+
H
G
F
Foretinib
Foretinib
+
EG
F
Foretinib
+
H
G
F
EGF HGF EGF HGF EGF HGF EGF HGF
G
efitinib
G
efitinib
+
Foretinib
Foretinib
PRAS40
ERK
Bad
Stat1
Akt (Ser473)
GSK-3β
Stat3
HSP27
Cleaved PARP
p53
p70 S6 Kinase
Cleaved Caspase-3
18000
14000
16000
12000
10000
8000
6000
4000
2000
0
Capture: EGFR Y988
Detection : Total Met
RelativeFluorescence
EGF
c-Met
EGFR
c-Met
EGFR
HGF
Untreated
HGF
Gefitinib
Gefitinib+EGF
Gefitinib+HGF
Foretinib
Foretinib+EGF
Foretinib+HGF
EGF
45000
35000
40000
30000
25000
20000
15000
10000
5000
0
Capture: Met Y1349
Detection : Total EGFR
Untreated
HGF
Gefitinib
Gefitinib+EGF
Gefitinib+HGF
Foretinib
Foretinib+EGF
Foretinib+HGF
EGF
RelativeFluorescence
HER2-EGFR
HER2-Met
Met-EGFR
EGFR-Met
60000
70000
40000
50000
30000
20000
10000
0
Non-phospholysate
Phospholysate
Capture:
Detection:
HER2 Y1196
Total EGFR
HER2 Y1196
Total Met
EGFR Y845
Total Met
EGFR Y998
Total Met
Met Y1349
Total EGFR
Gefitinib, (µM)
Capture: EGFR Y998
Detection: Total Met
Capture: HER2 Y1196
Detection: EGFR
NormalizedFluorescence
www.cellsignal.com Cell Signaling Technology®
and PathScan®
are trademarks of Cell Signaling Technology, Inc. / LI-COR®
and Odyssey®
are registered trademarks of LI-COR, Inc. / Dylight™
is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.