1. Cell Biology
  2. Structural Biology and Molecular Biophysics
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EGFR transactivates RON to drive oncogenic crosstalk

  1. Carolina Franco Nitta
  2. Ellen W Green
  3. Elton D Jhamba
  4. Justine M Keth
  5. Iraís Ortiz-Caraveo
  6. Rachel M Grattan
  7. David J Schodt
  8. Aubrey C Gibson
  9. Ashwani Rajput
  10. Keith A Lidke
  11. Bridget S Wilson
  12. Mara P Steinkamp
  13. Diane S Lidke  Is a corresponding author
  1. Department of Pathology, University of New Mexico, United States
  2. Department of Physics & Astronomy, University of New Mexico, United States
  3. Department of Surgery, University of New Mexico, United States
  4. Comprehensive Cancer Center, University of New Mexico, United States
Research Article
Cite this article as: eLife 2021;10:e63678 doi: 10.7554/eLife.63678
8 figures, 1 table and 2 additional files

Figures

Figure 1 with 4 supplements
Crosstalk between EGFR and RON is EGF-driven.

(A and B) HEKRON/EGFR or A431RON cells were treated with ± 5 nM MSP or 50 nM EGF for 5 min at 37 °C. Representative immunoblots showing PY1068 and EGFR on cell lysates (A) or pan-phosphotyrosine (PY) and RON on samples immunoprecipitated (IP) with anti-HA antibody (B). (C and D) A431RON cells were stimulated with ± 20 nM EGF, 20 nM MSP or both for 5 min at 37 °C and immunoblotted as in (A and B). Triplicate biological experiments are quantified in the bar graphs to the right, shown as mean ± SD. (E) Representative immunoblots of a phosphorylation time course for A431RON cells treated with 5 nM EGF or 5 nM MSP and immunoblotted as in (A and B). Graphed values (right) are from triplicate biological experiments, normalized to maximal activation, and presented as mean ± SD. * p < 0.05; ** p < 0.01.

Figure 1—source data 1

Full raw western blots and blots with relevant bands labeled, corresponding to Figure 1A, B, C, D and E.

https://cdn.elifesciences.org/articles/63678/elife-63678-fig1-data1-v2.zip
Figure 1—source data 2

Source data for quantification of blots in Figure 1C, D, and E.

https://cdn.elifesciences.org/articles/63678/elife-63678-fig1-data2-v2.xlsx
Figure 1—figure supplement 1
A431RON cells were treated with increasing levels of EGF for 5 min.

Immunoblots demonstrating detection of PY1068 and EGFR on cell lysates or detection of PY and RON on samples IP with anti-HA (RON) (n = 1).

Figure 1—figure supplement 1—source data 1

Full raw western blots and blots with relevant bands labelled, corresponding to Figure 1—figure supplement 1.

https://cdn.elifesciences.org/articles/63678/elife-63678-fig1-figsupp1-data1-v2.zip
Figure 1—figure supplement 2
A431RON cells were treated with increasing levels of MSP for 5 min.

Immunoblots demonstrating detection of PY1068 and EGFR on cell lysates or detection of PY and RON on samples IP with anti-HA (RON) (n = 1).

Figure 1—figure supplement 2—source data 1

Full raw western blots and blots with relevant bands labeled, corresponding to Figure 1—figure supplement 2.

https://cdn.elifesciences.org/articles/63678/elife-63678-fig1-figsupp2-data1-v2.zip
Figure 1—figure supplement 3
HEKRON/EGFR cells were treated± EGF or MSP for 5 min.

Representative immunoblots demonstrating detection of total PY (PY20/PY99 cocktail), PY1068, PY845, PY1148, and EGFR on cell lysates. Triplicate biological experiments are quantified and graphed as mean ± SD.

Figure 1—figure supplement 3—source data 1

Full raw western blots and blots with relevant bands labeled, corresponding to Figure 1—figure supplement 3.

https://cdn.elifesciences.org/articles/63678/elife-63678-fig1-figsupp3-data1-v2.zip
Figure 1—figure supplement 3—source data 2

Source data for quantification of blots in Figure 1—figure supplement 3.

https://cdn.elifesciences.org/articles/63678/elife-63678-fig1-figsupp3-data2-v2.xlsx
Figure 1—figure supplement 4
HEKRON cells transiently transfected with EGFR-WT or EGFR-Δ998± EGF treatment for 5 min.

Cell lysates were immunoprecipitated (IP) with anti-RON and probed for EGFR, RON, or RON PY1238/39. Arrows highlight the location of the 180 kDa unprocessed RON (pro-RON), and the 145 kDa RONβ band (mature RON). Truncated EGFR-Δ998 appears as a lower molecular weight band in the anti-EGFR immunoblot, as expected. However, EGFR-WT co-migrates with unprocessed RON (180 kDa). Therefore, all experiments in this study quantify the bottom RON band (RONβ). Additionally, the top band in the PY1238/39 immunoblot for EGF-treated EGFR-WT cells is absent in the EGFR-Δ998 samples, confirming this antibody cross-reacts with phosphorylated EGFR. Experiments to quantify RON phosphorylation were therefore first IP for HA (RON), and then probed with PY antibodies.

Figure 1—figure supplement 4—source data 1

Full raw western blots and blots with relevant bands labeled, corresponding to Figure 1—figure supplement 4.

https://cdn.elifesciences.org/articles/63678/elife-63678-fig1-figsupp4-data1-v2.zip
Figure 2 with 2 supplements
RON and EGFR co-cluster in plasma membrane nanodomains.

(A) HEKRON/EGFR cells were first labeled for RON using ⍺-HA-FITC Fab fragment (green), treated with 10 nM EGF-AF647 (magenta) for 5 min on ice and then fixed. Representative images from three biological replicates show colocalization of RON and EGFR at the plasma membrane. Scale bars, 10 μm (cross-section) and 2 μm (apical membrane). (B) Top row: Membrane sheets were prepared from A431RON cells ± 50 nM EGF for 2 and 5 min. Sheets were labeled on the cytoplasmic face using antibodies to RON (6 nm gold) and EGFR (12 nm gold). Circles indicate co-clusters of RON and EGFR in representative images from three biological replicates; arrowheads indicate clusters containing RON (green) or EGFR (magenta) only. Scale bar, 100 nm. Bottom row: Ripley’s K bivariant function was used to evaluate co-clustering. The experimental values for L(r)-r (corresponding to EM image directly above) are shown in magenta and the 99% confidence window for complete spatial randomness is plotted as dashed lines. In each case, experimental values are seen to fall above the confidence window, indicating co-clustering.

Figure 2—figure supplement 1
Comparison of RON and EGFR colocalization using fluorescent EGF or anti-EGFR (R1, Santa Cruz) antibody in A431RON cells.

Control sample of EGF-AF647 and anti-EGFR-AF488 shows the expected high colocalization (top left). Labeling of EGFR by either EGF-AF647 (ligand-bound, top right) or anti-EGFR-AF647 (no ligand, bottom left) both show high colocalization with HA-RON (anti-HA-AF488). Pearson’s Coefficient for the cell shown is indicated in the bottom left corner of the respective image. Five cells were analyzed per condition and averages ± SD are shown in the bar graph. Scale bars, 5 µm.

Figure 2—figure supplement 2
Additional EM Analysis.

(A–C) Examples of membrane sheets prepared from A431RON ± 50 nM EGF for 2 and 5 min. Sheets were labeled on the cytoplasmic face using antibodies to RON (6 nm gold) and EGFR (12 nm gold). Hopkin’s analysis (right) was performed to determine extent of homoclustering. In all cases, the data (blue bars) is shifted to the right of random distribution (pink line), indicating receptors are homoclustered on the membrane. Green line is the fit of the data to a Gaussian to extract peak Hopkin’s value for averaging as seen in (D). Scale bar, 100 nm. (D) Mean Hopkin’s Statistic across conditions, where values are taken from fitting the data to a Gaussian distribution as shown in (A–C) (green line). Values > 0.5 indicate non-random, homoclustered distribution. (E) Calculation of the percent of each receptor found within a cluster. RON and EGFR both have high propensity to homocluster, with >50% of each receptor found within homoclusters. (F) Calculation of the percent of receptors found within a heterocluster. ~30% of all EGFR molecules are identified as coclustered with RON, while ~70% of all RON molecules are coclustered with EGFR. Analysis for D, E performed as described in Espinoza et al., 2012.

Figure 3 with 2 supplements
Crosstalk occurs at the plasma membrane.

(A) Single particle tracking of QD605-HA-RON was used to quantify RON mobility on A431RON cells ± ligand. Ensemble mean squared displacement (MSD) shows reduction in slope of the MSD with ligand stimulation, indicating a reduced mobility. Treatment with EGFR kinase inhibitor prevents RON slow down with EGF. The number of jumps fit for each condition range from 42,183 to 898,300. (B) Corresponding distribution of diffusion coefficients, D, for individual cells is plotted for arange of 39 to 517 cells per condition; *** p < 0.001. (C) HEKRON/EGFR cells were labeled for RON with anti-HA-FITC Fab fragment (green), treated with 10 nM EGF-AF647 (magenta) for 5 min on ice followed by 10 min at 37°C, then fixed and labeled with an antibody to EEA1 (early endosomes, blue). Representative images from three biological replicates show that EGF-positive endosomes (arrows) primarily do not contain RON. Pearson’s coefficient for the image shown and colocalization with EEA1 is shown in the bottom left corner. (D) Alternative labeling method for monitoring endosome content where HEKRON/EGFR cells were treated with 50 nM EGF for 10 min at 37°C, fixed and then antibodies were used to label RON (anti-HA, green) or EGFR (magenta). Further quantification for C, D is in Figure 3—figure supplement 2. (E) Membrane sheets prepared from A431RON cells ± 50 nM EGF for 5 min were labeled for RON (6 nm gold) or EGFR (12 nm gold). TEM images show clathrin-coated pit lattices on the cell membranes containing EGFR, but not RON. Scale bars, 50 nm.

Figure 3—source data 1

Source data for diffusion coefficient distributions in Figure 3B.

https://cdn.elifesciences.org/articles/63678/elife-63678-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
RON mobility is reduced in response to bothlow/physiological and high/saturating doses of ligand.

HA-RON was tracked asdescribed in Figure 3A, B. (A) Ensemble mean square displacement (MSD) isplotted for range of 16,966 to 153,259 jumps fit per condition, and (B) diffusioncoefficient distribution across cells is plotted for a range of 20 to 171 cells percondition. ** p<0.01.

Figure 3—figure supplement 1—source data 1

Source data for diffusion coefficient distributions in Figure 3—figure supplement 1, panel B.

https://cdn.elifesciences.org/articles/63678/elife-63678-fig3-figsupp1-data1-v2.xlsx
Figure 3—figure supplement 2
EGFR is more readily found in EEA1-positive early endosomes than RON after EGF stimulation.

(A) Colocalization analysis for cells corresponding to treatment in Figure 3C, where RON is labeled live with anti-HA-Fab-FITC before addition of EGF-AF647. After 10 min of EGF-AF647 treatment, EGF is more colocalized with endosomes (EGF/EEA1) than RON (HAFab/EEA1). (B) Colocalization analysis for cells corresponding to Figure 3D. Here, cells are treated with ±50 nM EGF, then fixed and labeled with fluorescently-labeled primary antibodies to RON (anti-HA) or EGFR. Colocalization with EEA1 is low for both receptors in the resting state (No Tx). After EGF treatment, a significant increase in Pearson’s Correlation coefficient is seen for EGFR/EEA1 colocalization, but not for RON/EEA1. (C) Calculation of the fraction of endosomes containing either EGFR or RON or both, corresponding to (B). The fraction of RON-positive endosomes does not increase with EGF stimulation. 180 (No treatment) and 174 (50 nM EGF) endosomes were analyzed across 14 cells for each condition.

Figure 4 with 10 supplements
Two-color single QD tracking captures interactions between RON and EGFR.

Two color SPT results for resting RON receptor interactions (A–C) and ligand-bound EGFR interactions with RON (D–F). (A) Schematic representation of two-color (anti-HA-QD605 and anti-HA-QD655) RON SPT. (B) Representative 3D trajectory (top) and time series (bottom) for a RON homo-interaction lasting ~5 s (blue) with accompanying Figure 4—video 1. Scale bar, 500 nm. (C) Ensemble correlated motion plot for all two-color RON tracking. The number of jumps for each data point range from 2,068–15,649. (D) Schematic representation of two-color SPT of EGF-bound EGFR (QD655-EGF) and RON (anti-HA-QD605). (E) Sample 3D trajectory (top) and time series (bottom) showing EGF-EGFR and RON interacting for ~1.5 s (blue) with accompanying Figure 4—video 2. Scale bar, 500 nm. (F) Ensemble correlated motion plot for all EGF-EGFR and RON tracking. The number of jumps for each data point range from 1,500–16,794.

Figure 4—figure supplement 1
Example 3D trajectory (top) of two QD-RON receptors that engaged in repeated transient interactions (blue segments).

Still images are shown below the 3D trajectory. Scale bar, 500 nm.

Figure 4—figure supplement 2
Example 3D trajectory (top) of a long-lived (~22 s) interaction between two QD-RON receptors.

Still images are shown below the 3D trajectory. Scale bar, 500 nm.

Figure 4—figure supplement 3
Example 3D trajectory (top) of EGF-bound EGFR and RON receptors that are initially found in a dimer complex (blue) that then dissociates at 36.5 s.

Still images are shown below the 3D trajectory. Scale bar, 500 nm.

Figure 4—figure supplement 4
Example 3D trajectory (top) of a long-lived (~16 s) interaction between EGF-bound EGFR and RON receptors.

Still images are shown below the 3D trajectory. Scale bar, 500 nm.

Figure 4—video 1
Two RON receptors are engaged in an interaction from the start of the video, which lasts for ~5 s before the receptors dissociate.

This movie accompanies the interaction shown in Figure 4B. QD605 is displayed in green and QD655 in magenta. A colored tail for each QD shows a track of the previous 10 localizations. Playback speed is 20 frames/sec. Images have been brightness and contrasts enhanced for visualization.

Figure 4—video 2
A short-lived interaction between QD605-EGF-EGFR (green) and QD655-RON (magenta).

This movie accompanies the interaction shown in Figure 4E. A colored tail for each QD shows a track of the previous 10 localizations. Playback speed is 20 frames/s. Images have been brightness and contrasts enhanced for visualization.

Figure 4—video 3
Two RON receptors undergoing repeated interactions that each last 1–2 s.

This movie accompanies the interaction shown in Supplement 1. Color scheme, comet tail, and playback speed are as previously mentioned for Figure 4—video 1.

Figure 4—video 4
A long-lived RON/RON interaction.

This movie accompanies the interaction shown in Supplement 3. Color scheme, comet tail, and playback speed are as previously mentioned for Figure 4—video 1.

Figure 4—video 5
A complex of EGF-EGFR and RON is seen to break apart after a ~ 3.5 s dimer event.

This movie accompanies the interaction shown in Supplement 5. Color scheme, comet tail, and playback speed are as previously mentioned for Figure 4—video 2.

Figure 4—video 6
A long-lived interaction between EGF-EGFR and RON.

This movie accompanies the interaction shown in Supplement 7. Color scheme, comet tail, and playback speed are as previously mentioned for Figure 4—video 2.

Figure 5 with 2 supplements
Maximal EGF-induced RON phosphorylation requires kinase activity of both receptors.

(A and B) A431RON cells were pre-treated with 10 μM afatinib (Afat, pan-ErbB inhibitor) or 1 μM BMS777607 (BMS, Met family kinase inhibitor) for 20 or 15 min, respectively. Cells were then treated ± EGF or MSP for 5 min. (A) Cell lysates were used for PY1068 and EGFR immunoblots. (B) Lysates were immunoprecipitated (IP) with an anti-HA antibody and then immunoblotted for PY and RON. All samples are from the same blot, but an extraneous lane was removed for clarity. Bar graphs are corresponding mean ± SD from triplicate biological experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5—source data 1

Full raw western blots and blots with relevant bands labelled, corresponding to Figure 5A,B.

https://cdn.elifesciences.org/articles/63678/elife-63678-fig5-data1-v2.zip
Figure 5—source data 2

Source data for quantification of blots in Figure 5A,B.

https://cdn.elifesciences.org/articles/63678/elife-63678-fig5-data2-v2.xlsx
Figure 5—figure supplement 1
A431RON-K1114M (RON kinase-dead) cells were pre-treated, where indicated, with afatinib (Afat; pan-ErbB inhibitor) or BMS777607 (BMS; Met family kinase inhibitor) for 20 or 15 min, respectively.

Cells were subsequently treated with EGF or MSP for 5 min and lysed for immunoblot analysis. For RON and phospho-RON immunoblotting, lysates were IP with an anti-HA antibody (RON) and whole cell lysates used for EGFR and EGFR-PY1068, and their respective overlays. Triplicate experiments for EGFR (white bars) and RON (grey bars) phosphorylation are plotted as mean ± SD. *** p < 0.001.

Figure 5—figure supplement 1—source data 1

Full raw western blots and blots with relevant bands labelled, corresponding to Figure 5—figure supplement 1.

https://cdn.elifesciences.org/articles/63678/elife-63678-fig5-figsupp1-data1-v2.zip
Figure 5—figure supplement 1—source data 2

Source data for quantification of blots in Figure 5—figure supplement 1.

https://cdn.elifesciences.org/articles/63678/elife-63678-fig5-figsupp1-data2-v2.xlsx
Figure 5—figure supplement 2
A431RON or A431RON-K1114M cells were treated ± EGF for 5 min before imaging.

RON diffusion was tracked using QD605-HA-RON and measured over time, displayed as an ensemble mean squared displacement (MSD) plot. A reduction in slope of the MSD indicates a reduced mobility.

Figure 6 with 2 supplements
Crosstalk occurs through direct phosphorylation of RON by EGFR.

(A) A431RON cells were pre-treated with dasatinib (Das, Src inhibitor) for 30 min prior to stimulation with EGF for 5 min at 37 °C. Representative immunoblots of cell lysates detecting PY1068 and total EGFR (top), or PY and RON after IP with anti-HA (RON) (bottom). (B) HEKRON cells transiently transfected with EGFR-WT or EGFR-Δ998 ± EGF for 5 min. Representative immunoblots detecting PY and RON after IP with anti-RON or detection of total EGFR on cell lysates (bottom inset). (C) Kinase assay using the purified EGFR kinase domain (EGFR-KD) co-incubated with RON-K1114M IP samples ± ATP. Representative immunoblot detecting total phosphorylation (PY) of RON. All bar graphs represent mean ± SD from triplicate biological experiments. ** p < 0.01; *** p < 0.001.

Figure 6—source data 1

Full raw western blots and blots with relevant bands labelled, corresponding to Figure 6A, B, and C.

https://cdn.elifesciences.org/articles/63678/elife-63678-fig6-data1-v2.zip
Figure 6—source data 2

Source data for quantification of blots in Figure 1C, D and E.

https://cdn.elifesciences.org/articles/63678/elife-63678-fig6-data2-v2.xlsx
Figure 6—figure supplement 1
A431RON cells were pre-treated with dasatinib (Src inhibitor) at different concentrations for 30 min prior to cell lysis.

Cell lysates were probed by immunoblot for Src-PY416 and total Src, with corresponding overlays. Immunoblots show results from two biological replicates.

Figure 6—figure supplement 1—source data 1

Full raw western blots and blots with relevant bands labelled, corresponding to Figure 6—figure supplement 1.

https://cdn.elifesciences.org/articles/63678/elife-63678-fig6-figsupp1-data1-v2.zip
Figure 6—figure supplement 2
Dephosphorylation assay was conducted using HEKRON cells transiently transfected with EGFR-WT or EGFR-Δ998.

Cells were activated± EGF for 2 min, and immediately treated with 10 μM afatinib (pan-ErbB inhibitor) for 20 or 40 s. Two replicate biological experiments show detection of EGFR-PY1068 and EGFR on cell lysates or PY1238/39 and RON on samples IP with anti-RON, and their respective overlays.

Figure 6—figure supplement 2—source data 1

Full raw western blots and blots with relevant bands labelled, corresponding to Figure 6—figure supplement 2.

https://cdn.elifesciences.org/articles/63678/elife-63678-fig6-figsupp2-data1-v2.zip
Functional EGFR dimers are necessary for EGFR/RON crosstalk.

(A and B) HEKRON cells transiently expressing EGFR-WT, EGFR-I682Q (receiver-impaired), EGFR-V924R (activator-impaired) or both mutants (EGFR-I682Q + V924 R) were treated ± EGF for 5 min at 37 °C. (A) Representative immunoblot detecting PY1068 and EGFR in cell lysates. (B) Representative immunoblot showing PY and RON after IP with anti-RON. Triplicate biological experiments from (A and B) are quantified and graphed as mean ± SD. *** p < 0.001.

Figure 7—source data 1

Full raw western blots and blots with relevant bands labelled, corresponding to Figure 7A, B.

https://cdn.elifesciences.org/articles/63678/elife-63678-fig7-data1-v2.zip
Figure 7—source data 2

Source data for quantification of blots in Figure 7.

https://cdn.elifesciences.org/articles/63678/elife-63678-fig7-data2-v2.xlsx
Author response image 1
Detection of RON and EGFR in human cancer cell lines with low, endogenous expression of both receptors.

HCT116 colorectal carcinoma cells, SKBR3 breast cancer cells and A549 lung carcinoma cells were cultured overnight and then stimulated with 50 nM EGF or 5 nM MSP for 5 min. (A) RON receptor was IP’d with an anti-RON antibody and phosphorylated proteins in the IP were detected using a pY99 antibody. Here, detection was with HRP-antibodies. (B) Total cell lysates were run on a western blot and phosphoEGFR (pY1068) and total EGFR were detected. (C) Two-color detection using fluorescently-labeled antibodies for western using HCT116 cells. The phosphorylated protein does not overlap with full-length RON in the IP samples. The phosphorylated protein could be a truncated RON isoform or a different protein that co-Ips with RON in these cells. (D) The phospho-EGFR band does overlap with the full-length EGFR band.

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
cell line (Homo sapiens)A431 (female)ATCCATCC Cat# CRL-1555, RRID:CVCL_0037
cell line (Homo sapiens)HEK-293ATCCATCC Cat# CRL-1573, RRID:CVCL_0045
cell line (Homo sapiens)A431RONThis paperA431 cells stably transfected with pcDNA3.1 HA-RON plasmid
cell line (Homo sapiens)A431RON-K1114MThis paperA431 cells stably transfected with pcDNA3.1 HA-RON-K1114M plasmid
cell line (Homo sapiens)HEKRONThis paperHEK-293 cells stably transfected with pcDNA3.1 HA-RON plasmid
cell line (Homo sapiens)HEKRON/EGFRThis paperHEKRON cells stably transfected with ACP-EGFR plasmid
transfected construct (human)pcDNA3.1 HA-RONThis paperGenerated using pcDNA3.1
transfected construct (human)ACP-EGFRZiomkiewicz et al., Cytometry A, 2013
transfected construct (human)pcDNA3.1 HA-RON-K1114MThis paper
antibodyAnti-EEA1 (Rabbit monoclonal)Cell Signaling TechnologyCat# 3288 RRID:AB_2096811Clone C5B10 IF (1:200)
antibodyAnti-EGFR (Rabbit monoclonal)Cell Signaling TechnologyCat# 4267 RRID:AB_2246311Clone D38B1 WB (1:2000)
antibodyAnti-EGFR (Goat polyclonal)R&D SystemsCat# AF231 RRID:AB_355220WB (1:1000) Used when blotting for EGFR-Δ998
antibodyAnti-EGFR (Rabbit monoclonal)Cell Signaling TechnologyCat# 4405 RRID:AB_331380Clone 15 F8 WB (1:2000)
antibodyAnti-EGFR PY1068 (Mouse monoclonal)Cell Signaling TechnologyCat# 2236 RRID:AB_331792Clone 1 H12 WB (1:2000)
antibodyAnti-EGFR PY845 (Rabbit polyclonal)Santa Cruz BiotechnologyCat# sc-23420 RRID:AB_653168WB (1:500)
antibodyAnti-EGFR PY1148 (Rabbit polyclonal)Cell Signaling TechnologyCat# 4404 RRID:AB_331127WB (1:2000)
antibodyAnti-EGFR AF647 (Mouse monoclonal)Santa Cruz BiotechnologyCat# sc-101 AF647Clone R-1 FACS (5–40 ug/mL)
antibodyAnti-EGFR (Goat polyclonal)Santa Cruz BiotechnologyCat# sc-31156 RRID:AB_2096710Clone D-20 EM (1:20)
antibodyAnti-HA AF488 (Mouse Monoclonal)Cell Signaling TechnologyCat# 2350 RRID:AB_491023Clone 6E2 FACS (5–40 ug/mL)
antibodyAnti-HA magnetic bead (Rabbit monoclonal)Cell Signaling TechnologyCat# 11846 RRID:AB_2665471Clone C29F4 IP (1:100)
antibodyAnti-HA sepharose bead (Rabbit monoclonal)Cell Signaling TechnologyCat# 3956 RRID:AB_10695091Clone C29F4 IP (1:100)
antibodyAnti-HA FITC (Fab; Rat monoclonal)RocheCat# 11988506001 RRID:AB_390916Clone 3 F10 IF (1:20)
antibodyAnti-HA Biotin (Fab; Rat monoclonal)RocheCat# 12158167001 RRID:AB_390915Clone 3 F10 SPT (200 pM)
antibodyAnti-PY20 (Mouse monoclonal)Santa Cruz BiotechnologyCat# sc-508 RRID:AB_628122WB (1:500)
antibodyAnti-PY20 HRP (Mouse monoclonal)Santa Cruz BiotechnologyCat# sc-508 HRPKinase assay (1:500)
antibodyAnti-PY99 (Mouse monoclonal)Santa Cruz BiotechnologyCat# sc-7020 RRID:AB_628123WB (1:500)
antibodyAnti-PY99 HRP (Mouse monoclonal)Santa Cruz BiotechnologyCat# sc-7020 HRPKinase assay (1:500)
antibodyAnti-RON (Goat polyclonal)R&D SystemsCat# AF691 RRID:AB_355527IP (1:100)
antibodyAnti-RONβ (Rabbit polyclonal)Santa Cruz BiotechnologyCat# sc-322 RRID:AB_677390Clone C-20 WB (1:500) EM (1:20) Discontinued antibody; remainder of experiments done with Cell Signaling Technology Cat# 2,654
antibodyAnti- RONβ (Rabbit monoclonal)Cell Signaling TechnologyCat# 2654 RRID:AB_2298153Clone C81H9 WB (1:2000)
antibodyAnti- RONβ (Mouse monoclonal)Santa Cruz BiotechnologyCat# sc-74588 RRID:AB_2235711Clone E-3 WB (1:500) Used with the PY1238/39 RON antibody (R&D Systems Cat# AF1947)
antibodyAnti-RON PY1238/39 (Rabbit polyclonal)R&D SystemsCat# AF1947 RRID:AB_1152159WB (1:2000)
antibodyAnti-Src (Mouse monoclonal)Cell Signaling TechnologyCat# 2110 RRID:AB_10691385Clone L4A1 WB (1:2000)
antibodyAnti-Src PY416 (Rabbit polyclonal)Cell Signaling TechnologyCat# 2101 RRID:AB_331697WB (1:2000)
antibodyAnti-Goat IgG IRDye 800CW (Donkey polyclonal)Li-CorCat# 926–32214 RRID:AB_621846WB (1:20000)
antibodyAnti-Goat IgG 12 nm colloidal gold (Donkey polyclonal)Jackson Immuno ResearchCat# 705-205-147 RRID:AB_2340418EM (1:20)
antibodyAnti-Mouse IgG IRDye 680RD (Goat polyclonal)Li-CorCat# 926–68070 RRID:AB_10956588WB (1:20000)
antibodyAnti-Mouse IgG IRDye 680RD (Donkey polyclonal)Li-CorCat# 926–68072 RRID:AB_10953628WB (1:20000)
antibodyAnti-Rabbit IgG IRDye 800CW (Goat polyclonal)Li-CorCat# 926–32211 RRID:AB_621843WB (1:20000)
antibodyAnti-Rabbit IgG IRDye 680RD (Donkey polyclonal)Li-CorCat# 926–68073 RRID:AB_10954442WB (1:20000)
antibodyAnti-Rabbit IgG AF555 (Fab; Goat polyclonal)Thermo Fisher ScientificCat# A-21430 RRID:AB_2535851IF (1:500)
antibodyAnti-Rabbit IgG 6 nm colloidal gold (Donkey polyclonal)Jackson Immuno ResearchCat# 711-195-152 RRID:AB_2340609EM (1:20)
recombinant DNA reagentpDONR223-MST1RAddgeneRRID:Addgene_23942 Johannessen et al., 2010RON sequence used to make the HA-tagged RON plasmid
recombinant DNA reagentpcDNA3.1/V5-His-TOPOInvitrogenUsed as a backbone for HA-tagged RON plasmid (HA-RON and HA-RON-K1114M)
recombinant DNA reagentpcTAPAgilent
recombinant DNA reagentpcDNA3.1 HA-EGFR WTValley et al., 2015Plasmid used for generating EGFR-I682Q, EGFR-V924R, and EGFR-Δ998 with primers below
sequence-based reagentUltramer to generate HA-tagged RON plasmidThis paperCACCATGGAGCTCCTC CCGCCTCAGTCCTTCC TGTTGCTGCTGCTGTT GCCTGACAAGCCCGCG GCGGGCTATCCTTACG ACGTGCCTGACTACGCC GCAGCAGCAGAGGACT GGCAGTGCCCGCACA Has CACC ligation sequence, start codon, RON signal peptide, HA-tag, and alanine linker 5’ of the mature RON coding region
sequence-based reagentPrimers to generate RON-K1114M mutagenesisThis paperDanilkovitch-Miagkova et al., 2000Forward: GTGATGCGAC TTAGTGACATGATGGC ACATTGGATTC Reverse: GAATCCAATG TGCCATCATGTCACTAA GTCGCATCAC
sequence-based reagentPrimers to generate EGFR-KDThis paperForward: CGCCGGATCC CCAACGAATGGGCCTA AG Reverse: CGAGGTCGAC GGTATCGATAAGCTTTG CTCCAATAAATTCACTGC
sequence-based reagentPrimers to generate EGFR-I682Q mutagenesisThis paperForward: CAACCAAGCT CTCTTGAGGCAGTTG AAGGAAACTGAATTC Reverse: GAATTCAGT TTCCTTCAACTGCCTC AAGAGAGCTTGGTTGG
sequence-based reagentPrimers to generate EGFR-V924R mutagenesisThis paperForward: GATGTCTACA TGATCATGCGCAAGT GCTGGATGATA Reverse: TATCATCCAG CACTTGCGCATGATC ATGTAGACATC
sequence-based reagentPrimers to generate truncated EGFR-Δ998This paperForward: GTTAAGCTTG GTACCGAGCTCGGAT CCAGTACCCTTCACC ATGCGACCCTCCGGG AC Reverse: CCCTCTAGA CTCGAGCGGCCGCCT AGAAGTTGGAGTCTGTAGGACTTGGC
peptide, recombinant proteinHuman recombinant EGFInvitrogenCat# PHG0311
peptide, recombinant proteinHuman recombinant EGFPeproTechCat# AF-100–15
peptide, recombinant proteinHuman recombinant EGF-biotinThermo Fisher ScientificCat# E3477
peptide, recombinant proteinHuman recombinant EGF-AF647Thermo Fisher ScientificCat# E35351
peptide, recombinant proteinHuman recombinant MSPR&D SystemsCat# 4306 MS-010
commercial assay or kitBCA protein assay kitPierceCat# 23,225
commercial assay or kitECL blotting substratePierceCat# 32,106
commercial assay or kitExpi293 Expression System KitThermo Fisher ScientificCat# A14635Used for producing EGFR-KD and RON-K1114M proteins for use in kinase assay
chemical compound, drugAfatinibSelleck ChemicalsCat# S1011
chemical compound, drugBMS777607Selleck ChemicalsCat# S1561
chemical compound, drugDasatinibSanta Cruz BiotechnologyCat# sc-358114
chemical compound, drugPD153035EMD MilliporeCat# 234,491
software, algorithmMATLABMathworksRRID:SCR_001622
software, algorithmDIPImageDelf University of Technology
otherQD605 streptavidinThermo Fisher ScientificCat# Q10101MPFor use in SPT
otherQD655 streptavidinThermo Fisher ScientificCat# Q10121MPFor use in SPT
otherprotein A/G magnetic beadsPierceCat# 88,802For use in IP
otherQuantum AlexaFluor 647Bangs LaboratoriesCat# 647 AFor use in receptor quantification on FACS
otherQuantum AlexaFluor 488Bangs LaboratoriesCat# 488 AFor use in receptor quantification on FACS

Additional files

Transparent reporting form
https://cdn.elifesciences.org/articles/63678/elife-63678-transrepform1-v2.docx
Supplementary file 1

List of top proteins co-IP in A431RON cells with anti-HA antibody for RON pulldown, as analyzed by Mass Spectrometry.

https://cdn.elifesciences.org/articles/63678/elife-63678-supp1-v2.docx

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