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Proximity labeling reveals molecular determinants of FGFR4 endosomal transport Ellen Margrethe Haugsten, Vigdis Sørensen, Michaela Kunová Bosakova, Gustavo Antonio de Souza, Pavel Krejci, Antoni Wiedlocha, and Jørgen Wesche J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00652 • Publication Date (Web): 11 Sep 2016 Downloaded from http://pubs.acs.org on September 13, 2016

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Journal of Proteome Research

Proximity labeling reveals molecular determinants of FGFR4 endosomal transport

Ellen Margrethe Haugsten1,2, Vigdis Sørensen1,2, Michaela Kunova Bosakova3, Gustavo Antonio de Souza4,5, Pavel Krejci3,6, Antoni Wiedlocha1,2 and Jørgen Wesche1,2,*

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Department of Molecular Cell Biology, Institute for Cancer Research, The Norwegian Radium

Hospital, Oslo University Hospital, Montebello, 0379 Oslo, Norway 2

Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Montebello, 0379

Oslo, Norway 3

Department of Biology, Faculty of Medicine, Masaryk University, Kamenice 5,

625 00, Brno-Bohunice, Czech Republic 4

Dept. of Immunology, Oslo University Hospital - Rikshospitalet and University of Oslo, 0027,

Oslo, Norway 5

The Brain Institute, Universidade Federal do Rio Grande do Norte, UFRN, Natal-RN, Brazil

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International Clinical Research Center, St. Anne's University Hospital, Brno, Czech Republic

*Author to whom correspondence should be addressed: Jørgen Wesche Tel: +47 22 78 19 31 Fax: +47 22 78 18 45 Email: [email protected] Running title: Proteomics reveal FGFR4 intracellular transport pathways

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Abbreviations

FGF1 FGFR FGFR4 FRS2 ERC MS TGN RTK 3D-SIM Tf

Fibroblast growth factor 1 Fibroblast growth factor receptor Fibroblast growth factor receptor 4 FGF receptor substrate 2 Endocytic recycling compartment Mass Spectrometry Trans-Golgi network Receptor tyrosine kinase Three-dimensional structured illumination microscopy Transferrin

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ABSTRACT The fibroblast growth factor receptors (FGFRs) are important oncogenes promoting tumor progression in many types of cancer, such as breast, bladder, and lung cancer as well as multiple myeloma and rhabdomyosarcoma. However, little is known about how these receptors are internalized and down-regulated in cells. We have here applied proximity biotin labeling to identify proteins involved in FGFR4 signaling and trafficking. For this purpose we fused a mutated biotin ligase, BirA*, to the C-terminal tail of FGFR4 (FGFR4-BirA*) and the fusion protein was stably expressed in U2OS cells. Upon addition of biotin to these cells, proteins in proximity to the FGFR4-BirA* fusion protein became biotinylated and could be isolated and identified by quantitative mass spectrometry. We identified in total 291 proteins, including 80 proteins that were enriched in samples where the receptor was activated by the ligand (FGF1), among them several proteins previously found to be involved in FGFR signaling (e.g. FRS2, PLCγ, RSK2 and NCK2). Interestingly, many of the identified proteins were implicated in endosomal transport and by precise annotation we were able to trace the intracellular pathways of activated FGFR4. Validating the data by confocal and three-dimensional structured illumination microscopy analysis, we concluded that FGFR4 uses clathrin-mediated endocytosis for internalization and is further sorted from early endosomes to the recycling compartment and the trans-Golgi network. Depletion of cells for clathrin heavy chain led to accumulation of FGFR4 at the cell surface and increased levels of active FGFR4 and PLCγ, while AKT and ERK signaling was diminished, demonstrating that functional clathrin-mediated endocytosis is required for proper FGFR4 signaling. Thus, this study reveals proteins and pathways involved in FGFR4 transport and signaling that provide possible targets and opportunities for therapeutic intervention in FGFR4 aberrant cancer.

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KEYWORDS: FGFR4, FGF1, BioID, Quantitative MS, confocal microscopy, three-dimensional structured illumination microscopy, Endocytosis, Clathrin, recycling compartment, trans-Golgi network.

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INTRODUCTION Similarly to many receptor tyrosine kinases (RTKs), the fibroblast growth factor receptors (FGFRs) are often found overexpressed and mutated in cancer and constitute important therapeutic targets1-2. Important examples include the 8p11-12 amplicon in breast cancer that causes robust overexpression of FGFR13 and activating mutations found in FGFR2 in endometrial cancer4-5. Activating mutations in FGFR3 have also been shown in many cases of bladder cancer6. Furthermore, FGFR4 is often found mutated or overexpressed in the childhood cancer rhabdomyosarcoma and in some types of lung cancer7-8. The importance of FGFR4 as an oncogene has been validated by inhibiting or depleting the receptor in model cell lines, which caused tumor growth inhibition and decreased metastasis in a mouse model7. Thus, FGFRs constitute a class of oncogenes that are important drivers of cancer progression in many cases1-2. In order to develop novel strategies for targeted therapeutics to these receptors, it will be important to elucidate in detail how they signal and how they are down-regulated. Despite their importance as oncogenes, little is known about how these receptors are internalized and transported. It has been shown that FGFR1 is internalized and degraded in lysosomes after activation, which efficiently terminates its signaling9-10. FGFR4, however, was shown to be efficiently recycled back to the cell-surface and was not degraded, leading to prolonged signaling9-10. It is possible that this mechanism could contribute to the oncogenicity of FGFR4, as the recycling pathway bypasses the efficient termination of signaling in lysosomes, which is the normal attenuation pathway for RTKs. We have previously found that Rab11 is involved in the recycling of FGFR4 and that the level of ubiquitination seems to play a role11. Apart from this, little is known about the molecular details of FGFR4 transport within cells.

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In order to understand better the intracellular signaling and transport of FGFR4, we applied the BioID system, a newly developed technique of biotin tagging12. The recent advances in biotin tagging of proximal proteins have opened up new possibilities to map both potential interacting partners and the surrounding molecular environment of a protein of interest. Advantages of this system include the possibility to catch transient interactions and to identify membrane proteins that are difficult to pull-down by classical techniques. Here, we applied BioID on a receptor that travels to and from the plasma membrane and through several intracellular compartments. We hypothesized that by identifying proteins proximal to FGFR4 over time, we could trace the intracellular pathways the receptor follows. We fused the mutated biotin ligase (BirA-R118G denoted BirA*) to the intracellular part of FGFR4 and used quantitative proteomics to identify proteins that were proximal to FGFR4 in the absence or presence of FGF1 that activates the receptor. We identified by this approach 291 proteins, among them were 80 proteins enriched in samples of the activated FGFR4. The identity of these proteins was subjected to detailed analysis to map the subcellular localizations of FGFR4. We found that FGFR4 uses clathrin-mediated endocytosis for internalization and confirmed the recycling of FGFR4 through the recycling compartment. Interestingly, we also found that FGFR4 may pass through the trans-Golgi network (TGN). Moreover, interfering with FGFR4 endocytosis led to altered signaling. Thus, proper endocytosis is required for accurate FGFR4 signaling.

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Experimental procedures Antibodies and Compounds The following antibodies were used: mouse anti-EEA1 from BD transduction laboratories; rabbit anti-FGFR4, mouse anti-flotillin-1, mouse anti-PLCγ and rabbit antiphospho-PLCγ from Santa Cruz Biotechnology; mouse anti-phospho-FGFR, rabbit antiphospho-AKT, rabbit anti-AKT, rabbit anti-ERK1/2, and mouse anti-phospho-ERK1/2 from Cell Signaling; rabbit anti-phospho-RSK1/2 from R&D Systems Europe; mouse anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase)-HRP from Abcam; rabbit anti-HA epitope tag from Rockland; mouse anti-HA epitope tag and rabbit anti-Giantin from Covance; sheep antiTGN46 from Abd Serotec; rabbit anti-Rab11 from Life Technologies; mouse anti-LAMP1 from Developmental Studies Hybridoma Bank; mouse anti-γ-tubulin from Sigma-Aldrich; mouse anticlathrin heavy chain (CHC) from RDI division of Fitzgerald Industries; rabbit anti-Scyl2 was a generous gift from Prof. Scottie Robinson13; HRP-Streptavidin, Alexa488-Streptavidin and secondary antibodies from Jackson Immuno-Research Laboratories. Protease inhibitor cocktail tablets (ethylenediaminetetraacetic acid (EDTA)-free, complete) were from Roche Diagnostics. DyLight 550 NHS Ester and Ez-link Sulfo-NHS-SSbiotin were from Thermo Scientific. Alexa 647-transferrin (Tf) and Hoechst 33342 were purchased from Life Technologies. Streptavidin Sepharose High Performance was from GE Healthcare Life Sciences. Dyngo-4a was from AbCam. Mowiol, biotin, heparin, phosphatase inhibitors and Ponceau S were from Sigma-Aldrich. FGF1 was prepared as previously described14. FGF1 was labeled with DyLight 550 (Life Technologies) following the manufacturer's procedures.

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Plasmids and siRNAs pcDNA3.1-FGFR4-BirA* was generated by cloning a PCR fragment containing the FGFR4 open reading frame and AgeI-HF and EcoRI-HF flanking sites into pcDNA3.1 MCSBirA(R118G)-HA cut with AgeI-HF and EcoRI-HF. pcDNA3.1 MCS-BirA(R118G)-HA was a gift from Kyle Roux (Addgene plasmid # 36047)12. pcDNA3-hFGFR4 was used as template9. pEGFP-Rab11a, pEGFP-C3-RCP (Fip1), pEGFP-C1-Fip2, pEGFP-C1-Rip11, was a generous gift from Prof. Jim Norman and are as described previously15. Scyl2-GFP was a generous gift from Prof. Ernst J. Ungewickell16. pmTq2-C1-CLC (Clathrin Light Chain) was cloned by replacing mCherry of pmCherry-CLC with mTq2 using the NheI and BsrGI restriction sites. pmTurquoise2-C1 (pmTw-2-C1) was a gift from Dorus Gadella (Addgene plasmid # 60560)17. CLC-pmCherry-C1 was a gift from Christien Merrifield (Addgene plasmid # 27680)18. pcDNA3.1 hemagglutinin epitope (HA) tagged K44A mutant constructs of dynamin 1 was a generous gift from Prof. Sandra L. Schmid19. pcDNA3.1/Zeo-BirA* was made by cloning a PCR fragment contaning BirA*-HA from the plasmid pcDNA3.1MCS-BirA(R118G)-HA with NheI and PmeI flanking sites into pcDNA3.1/Zeo cut with the same enzymes. A start codon and Kozak sequence was introduced at the beginning and a stop codon was introduced at the end of BirA*-HA. siRNA oligos targeting clathrin heavy chain (CHC-1) (s475), FGFR4 (s5177), SCYL2 (s31240) and Silencer® Negative Control No. 2 siRNA (scr) were purchased from Ambion, Life Technologies. siRNA oligos targeting clathrin heavy chain (CHC-2) (sense sequence: 5′GCAAUGAGCUGUUUGAAGATT-3′) was purchased from MWG Biotech.

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Cells and Transfection Fugene liposomal transfection reagent was used according to the manufacturer’s protocol to obtain U2OS cells stably expressing FGFR4-BirA* (U2OS-R4-BirA*) and U2OS-FGFR4 stably expressing BirA* (U2OS-R4 + BirA). Clones were selected with 1 mg/ml geneticin or 0.2 mg/ml Zeocin respectively. Clones were chosen based on their receptor/BirA* expression levels analyzed by immunofluorescence and western blotting. U2OS cells stably expressing FGFR4 have been described previously10. RH30 cells were a generous gift from Prof. Ola Myklebost (Department of Tumor Biology, The Norwegian Radium Hospital). U2OS cells and RH30 cells were propagated in DMEM or RPMI (respectively), supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin in a 5% CO2 atmosphere at 37°C. siRNA transfection was performed using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Life Technologies) according to the manufacturer’s protocol. 10 nM of siRNA was used and the experiments were performed 72-96 hours after transfection. Transient expression of different plasmids was performed by transfecting cells with plasmid DNA using Fugene 6 Transfection Reagent (Roche Diagnostics) according to the manufacturer’s protocol.

Affinity Capture of Biotinylated Proteins Cells were incubated for 24 h in complete media supplemented with 50 µM biotin in the absence or presence of 100 ng/ml FGF1 and 10 U/ml heparin. After one PBS wash, cells (approximately 1 × 108 cells) were scraped in PBS supplemented with 100 mM Glycine. Cells were pelleted by centrifugation for 10 minutes at 4000 rpm and lysed in 1 ml lysis buffer (0.1 M NaCl, 10 mM Na2PO4, 1% triton X-100, and 1 mM EDTA, pH 7.4, supplemented with protease and phosphatase inhibitors). Lysates were then centrifuged at 14 000 rpm for 10 minutes at 4°C.

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Supernatants were incubated with 300 µl Streptavidin-Sepharose High Performance for 2 hours. Beads were collected and washed twice for 5 min at 4°C in 1% Triton X-100, 500 mM NaCl, 1 mM EDTA, twice for 5 min at 4°C in PBS with 0.1% Triton X-100, twice for 5 min at room temperature in 2% SDS, twice for 5 min at room temperature in 6 M Urea in PBS, and six times for 5 min at room temperature in 1 M NaCl, 25% Acetonitrile and twice for 5 min at room temperature in 20% Acteonitrile. Bound proteins were trypsin-digested on the beads.

Sample preparation for mass spectrometry Beads containing bound proteins were submitted to in solution trypsin digestion in 100 µL of 0.1% ProteaseMax (Promega), containing 3.6 µg of trypsin (Modified, Promega). Trypsin reaction was performed overnight in a wet chamber at 37oC. Reaction was quenched by adding 1% trifluoracetic acid to the mixture (final concentration). Peptides were cleaned for mass spectrometry by the STAGE-TIP method20 using a C18 resin disk (3M Empore).

Mass Spectrometry All experiments were performed on an Easy nLC1000 nano-LC system connected to a quadrupole - Orbitrap (QExactive) mass spectrometer (ThermoElectron, Bremen, Germany) equipped with a nanoelectrospray ion source (EasySpray/Thermo). For liquid chromatography separation we used an EasySpray column (C18, 2 µm beads, 100 Å, 75 µm inner diameter) (Thermo) capillary of 25 cm bed length. The flow rate used was 300 nL/min, and the solvent gradient was 2% B to 30% B in 120 minutes, then 90% B wash in 20 minutes. Solvent A was aqueous 0.1% formic acid, whereas solvent B was 100% acetonitrile in 0.1% formic acid. Column temperature was kept at 60oC.

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The mass spectrometer was operated in the data-dependent mode to automatically switch between MS and MS/MS acquisition. Survey full scan MS spectra (from m/z 400 to 1,200) were acquired in the Orbitrap with resolution R = 70,000 at m/z 200 (after accumulation to a target of 3,000,000 ions in the quadruple). The method used allowed sequential isolation of the most intense multiply-charged ions, up to ten, depending on signal intensity, for fragmentation on the HCD cell using high-energy collision dissociation at a target value of 100,000 charges or maximum acquisition time of 100 ms. MS/MS scans were collected at 17,500 resolution at the Orbitrap cell. Target ions already selected for MS/MS were dynamically excluded for 30 seconds. General mass spectrometry conditions were: electrospray voltage, 2.1 kV; no sheath and auxiliary gas flow, heated capillary temperature of 250oC, normalized HCD collision energy 25%. Ion selection threshold was set to 1e4 counts. Isolation width of 3.0 Da was used.

Protein Identification and Label-free Quantitation MS raw files were submitted to MaxQuant software version 1.4.0.821 for protein identification using its Andromeda engine. Parameters were set as follow: protein N-acetylation and methionine oxidation as variable modifications. First search error window of 20 ppm and both precursor and MS/MS main search error set to 6 ppm. Trypsin without proline restriction enzyme option was used, with two allowed miscleavages. Minimal unique peptides were set to 1, and FDR allowed was 0.01 (1%) for peptide and protein identification. Label-free quantitation was set with a retention time alignment window of 3 min. The Uniprot human database was used (download from October 2014, with 85,915 entries). Generation of reversed sequences was selected to assign FDR rates. Known contaminants as provided by MaxQuant and identified in the samples were excluded from the analysis.

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While proteins with one peptide hits are reported in the MaxQuant output provided with the article submission, such proteins were not further taken into consideration to any of the biological analysis that was performed.

Experimental Design and Statistical Rationale Six individual experiments were performed; three experiments consisting of samples C1 (U2OS-R4 cells), S1 (U2OS-R4-BirA*) and S2 (U2OS-R4-BirA* stimulated with FGF1) and three experiments consisting of samples C1 (U2OS-R4 cells), C2 (U2OS-R4 stably transfected with BirA* and C3 (U2OS-R4 cells stably transfected with BirA* and stimulated with FGF1). All three samples in each of the six individual experiments were run three times (n=3 for LC variability, n=9 total number of replicates combined, in the case of C1: n=6 for LC variability, n=18 total number of replicates combined). The mean IBAQ values were calculated for each protein in each sample (C1, C2, C3, S1, and S2). Proteins identified in C1 were considered as background and the means of S1 and S2 were compared to that of C1. Proteins were removed from the list if they were not significantly enriched at least 3 times compared to C1 in S1 and/or S2 (p3·C2, p≤0.05. Proteins in cytosol are indicated by various shapes in the schematic presentation. (B) U2OS-R4BirA* cells were allowed to bind DL550-FGF1 at 4ºC in the presence of heparin and then washed (to remove excess DL550-FGF1) and either fixed directly (0 min) or incubated for 20 min at 37ºC before fixation (20 min). Fixed cells were then stained with anti-EEA1 antibody and Hoechst and examined by confocal microscopy. Scale bar 5 µm. (C) U2OS-R4-BirA* cells were starved in serum-free media for 5 hours and then stimulated for 20 minutes with 100 ng/ml FGF1 in the presence of 20 U/ml heparin. Cells were then lysed and the cellular material was analyzed 36


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by SDS-PAGE and western blotting using the indicated antibodies. A p in front of the name of the antibody indicates that it recognizes the phosphorylated form of the protein. (D) U2OS-R4BirA* and U2OS-R4 cells were treated with FGF1 as indicated and then recorded for 12 hours. The mean cell velocities were quantified as described in Experimental Procedures. The graph represents the mean ± SEM of three independent experiments. In total, approximately 150 cells were quantified per condition. n=3. **p≤ 0.01, *p≤ 0.05 (E) U2OS-R4-BirA* and U2OS-R4 cells were treated as indicated for 24 h and then the cells were lysed and the cellular material was analyzed by SDS-PAGE and western blotting. The membrane was stained with Ponceau S before Streptavidin-Hrp (Strep-HRP). (F) U2OS-R4-BirA* and U2OS-R4 cells were treated as indicated for 24 h, fixed and stained with anti FGFR4 antibody (anti-R4), Alexa 488 streptavidin (Strep) and Hoechst. Scale bar 5 µm.

Figure 2. Map of hits enriched in the FGF1-activated FGFR4-BirA* samples (S2) based on subcellular localization and biological function. Information from GeneCards, GO Consortium and literature curation (PubMed) were used to classify the hits in subcellular compartments, and in some cases cellular processes. Some hits mapped to several categories and are therefore found in several localization on the map.

Figure 3. Intracellular transport of FGF1/FGFR4. (A) U2OS-R4-BirA* cells were allowed to internalize DL550-FGF1 in the presence of heparin for 20 minutes before fixation. Fixed cells were then stained with anti-EEA1 antibody (green), anti-HA-tag to label FGFR4-BirA* (blue) and Hoechst and examined by fluorescence microscopy. A deconvolved wide-field image (a single optical section) is shown. Scale bar 4 µm. (B) U2OS-R4 cells were incubated with

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DL550-FGF1 and Alexa647-Transferrin (Tf) in the presence of heparin for indicated periods of time before fixation. Fixed cells were then stained with anti-EEA1 antibody (green) or antiLAMP1 antibody (green) and Hoechst and examined by confocal microcopy. Scale bar 5 µm. (C) U2OS-R4-BirA* cells were transfected with Rab11-GFP (for 20 hours), then treated with DL550-FGF1 and Alexa647-Transferrin (Tf) in the presence of heparin for 60 minutes. The cells were fixed, stained with Hoechst, and examined by 3D-SIM. Image shown is a single optical section from a SIM z-stack. Scalebar, 4 µm. N, nucleus. Enlarged areas are shown below.

Figure 4. Validation of identified hits in the BioID screen. U2OS-R4 cells untransfected or transfected with Scyl2-GFP, Fip1-GFP, Fip2-GFP or Fip5-GFP were allowed to internalize DL550-FGF1 in the presence of heparin for indicated periods of time before fixation. Fixed cells were stained with Hoechst (blue) and anti-flotillin antibody (green) where indicated and examined by confocal microcopy. Scale bar 5 µm.

Figure 5. Transport of FGFR4 to the TGN. (A) U2OS-R4 cells were allowed to internalize DL550-FGF1 in the presence of heparin for 90 minutes before fixation. Fixed cells were then stained with anti-TGN46 antibody (green) and Hoechst (blue) and examined by confocal microcopy. Scale bar 5 µm. (B) U2OS-R4-BirA* cells were incubated with DL550-FGF1 in the presence of heparin for 60 minutes before fixation. Fixed cells were then stained with antiTGN46 antibody (blue), anti-Giantin antibody (green) and Hoechst and examined by 3D-SIM. A single optical section from a SIM z-stack is shown. Scale bar 4 µm. N, nucleus. Enlarged areas are shown below. (C) U2OS-R4-BirA* cells were treated as in (B). Cells were stained with antiTGN46 antibody (blue), anti-Rab11 antibody (green) and Hoechst and examined by 3D-SIM. A

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single section from a z-stack is shown. Scale bar 4 µm. N, nucleus. Enlarged areas are shown below.

Figure 6. Clathrin- and dynamin-mediated endocytosis of FGF1/FGFR4. (A) List of identified hits involved in clathrin-mediated endocytosis. Red color indicates that these hits were enriched in samples of activated FGFR4. (B) U2OS-R4-BirA* cells were transfected with Tq2-CLC (clathrin light chain) and kept for 60 minutes at 4°C with DL550-FGF1 and heparin to allow binding to the cell surface FGFR4. The cells were briefly transferred to 37°C to induce endocytosis and then the cells were fixed. The cells were examined by 3D-SIM and a single optical section from a z-stack is shown. Scale bar 4 µm. N, nucleus. Enlarged areas (scale bare, 1µm) are shown to the right. (C) U2OS-R4 and RH30 cells were transfected with an siRNA oligo targeting clathrin heavy chain (CHC-1), or a non-targeting siRNA control (scr) for 72-96 hours. RH30 cells were in addition transfected with an siRNA oligonucleotide targeting FGFR4. Cell surface proteins were then biotinylated and the cells were lysed. Biotinylated proteins were adsorbed to streptavidin-Sepharose before analysis by SDS-PAGE and western blotting using the indicated antibodies. One representative experiment is shown. (D) Western blots as described in B were quantified and the bands corresponding to biotinylated FGFR4 (Surface FGFR4) were normalized to loading control of the lysate. The graph represents the mean±SD of four (U2OSR4) and three (RH30) independent experiments. **p≤ 0.01, *p≤ 0.05. (E) U2OS-R4 cells transfected with an siRNA oligo targeting clathrin heavy chain (CHC-1) or a non-targeting siRNA control (scr) were allowed to bind DL550-FGF1 in the presence of heparin at 4ºC. The cells were then washed (to remove excess DL550-FGF1) and incubated for 20 min at 37ºC before fixation. Some cells (as indicated) were washed with a high salt, low pH (HSLP) buffer

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(to remove FGF1 bound to cell surface receptors). Fixed cells were then stained with anti-EEA1 antibody (green) and Hoechst and examined by confocal microscopy. Scale bar 5 µm. (F) U2OSR4 cells transfected with siRNA oligos targeting clathrin heavy chain (CHC-1 and CHC-2), Scyl2 or a non-targeting siRNA control (scr) were allowed to internalize DL550-FGF1 in the presence of heparin for 20 minutes. The cells were then washed with HSLP buffer, fixed and analyzed with a high content screening Olympus ScanR system. The graph represents the mean±SEM of four independent experiments with a total of 10887 (Scr), 6610 (CHC-1), 9759 (CHC-2) and 10448 (Scyl2) cells analyzed. n=4. ***p≤ 0.001. (G) U2OS-R4 cells transfected with HA-tagged dominant negative dynamin 1 (Dyn1 K44A) were incubated with DL550-FGF1 in the presence of heparin for 20 minutes before fixation. Fixed cells were then stained with antiHA antibody (green) and Hoechst and examined by confocal microscopy. Scale bar 5 µm. (H) DL550-FGF1 and heparin was added to U2OS-R4 cells pretreated for 30 minutes with DMSO (Ctrl) or Dyngo-4a (10 µM) and kept at 37ºC for additional 20 minutes. The cells were then washed with HSLP buffer, fixed and analyzed with a high content screening Olympus ScanR system. The graph represents the mean±SD of two independent experiments with a total of 5776 (DMSO) and 5990 (Dyngo-4a) cells analyzed. n=2. ***p≤ 0.001.

Figure 7. Consequences of Clathrin-depletion on FGFR4 signaling. U2OS-R4 cells were transfected with non-targeting (-) or clathrin heavy chain targeting (+) siRNA oligos. Seventytwo hours after transfection, the cells were kept at 4ºC with 100 ng/ml FGF1 in the presence of 20 U/ml heparin for 1 hour. Next, the cells were washed and immediately transferred to 37ºC for indicated periods of time before lysis. Cell lysate were then analyzed by SDS-PAGE and western blotting using the indicated antibodies. A p in front of the name of the antibody indicates that it

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recognizes the phosphorylated form of the protein. One representative experiment is shown. Western blots were quantified and the bands corresponding to the indicated proteins were normalized to loading control and presented as fraction of maximal response. The graph represents the mean±SD of three independent experiments. n=3. **p≤ 0.01, *p≤ 0.05.

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Table 1. Enrichment analysis using Gene Ontology and Panthera GO cellular component

GARP complex clathrin adaptor complex clathrin coat AP-type membrane coat adaptor complex

recycling endosome membrane

P-valuef

Total number of proteinsb

Expected Proteinsc

Identified proteinsd

Fold Enrichmente

4 27 46 41 44

0.03 0.11 0.10 0.21 0.28

3 5 8 7 4

>100 48.2 45.3 44.4 23.7

4.16E-03 1.50E-04 4.88E-03 9.44E-08 5.92E-10

25 69 231 256 207

0.10 0.27 0.89 0.98 0.80

4 6 15 8 9

41.6 33.9 13.5 12.2 10.1

2.46E-02 7.29E-08 8.40E-07 2.67E-06 1.18E-02

119 296 273 255 649

0.46 1.14 1.05 0.98 2.49

7 11 10 9 13

15.3 9.7 9.5 9.2 5.2

1.13E-03 5.25E-05 2.76E-04 1.69E-03 3.01E-03

GO biological process

endocytic recycling lysosomal transport endosomal transport vacuolar transport cytosolic transport GO molecular function

Rab GTPase binding GTPase binding small GTPase binding Ras GTPase binding protein kinase activity a

PANTHER Overrepresentation Test (release 20160321), b total number of human proteins for this category in GO Ontology database, c number of expected proteins for this category based on the reference list, d number of positive hits from activated FGFR4 BioID screen (Suppl. Table 1) , e Identified proteins divided by expected proteins number, f Bonferroni corrected.


Page 43 of 50

U2OS-R4

C2.

C3.

S1.

S2.

U2OS-R4 + BirA*

U2OS-R4 + BirA* + FGF1

U2OS-R4-BirA*

U2OS-R4-BiRA* + FGF1

FGF1

Biotin

Plasmamembrane FGFR4

R4-BirA*

BirA* Signaling

B

E

Merge

EEA1

0 min

FGF1

Biotin: FGF1:

U2OSR4-BirA* + + - +

U2OS-R4 -

+ -

+ +

KDa

Merge

EEA1

FGF1

250 150

20 min

100 75 50 37

C

FGF1: KDa 150

-

+

25 20

D

U2OS- U2OSR4-BirA* R4

p-FGFR

**

100 150 100 150

FGFR4 p-PLCg

100 75

p-RSK1/2

Strep-HRP Lane:

Velocity (µm/min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1 A C1.

*

F

4

5

6

150 100 75 0.1 0.10

50 37 25 20

p-ERK1/2 GAPDH

3

250

0.15 0.15

15 10

37

2

KDa

0.05 0.05

37

1

00

FGF1:

U2OS-R4-BirA*

-

(CM)

FGF1 (CM)

+

-

(CM)

FGF1 (CM)

+

U2OS-R4

Biotin: + + + FGF1: + + anti-R4 Strep anti-R4 Strep anti-R4 Strep anti-R4 Strep


Ponceau S

Figure 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Clathrin coated pits AGFG1 EPN1 PICALM

Plasma membrane A2M APBB2 ATP13A3 BAI1

PIK3C2A SCYL2

CRACR2A EGFR FGFR4 GPR176

Page 44 of 50

IGF2R ITGB5 JPH1 KCNN4

MET PVRL2 SEMA4C SERPINE1

SLITRK4 TFRC UNC5B VANGL1

Plasma membrane

Sorting endosome APPL2 FLOT1

LMTK2 ZFYVE20

Recycling endosome FLOT1 Rab11-Fip1 Rab11-Fip2

Rab11-Fip5 SCAMP1 SCYL2

Sorting endosomes Endosome ↔ Golgi AP1B1 AP1G1 CLINT1 GGA2 IGF2R SCAMP1

MVB/late endosomes

SCYL2 STX6 VPS45 VPS51 VPS53 VPS54

ERC

Late endosome/ Lysosomes FAM160A2 TPP1 VPS33A VPS41

Golgi

SCAMP1 SMPD4 VPS13B

Golgi

Autophagy C9ORF72 ULK1

ER

Lysosomes ER

CALR HSPA5 P4HA2 RPN1 SMPD4

Nucleus Signalling AURKA CCNB2 DCBLD2

ECT2 LMTK2 NCK2

PI4KA PIK3C2A PLCG1

PTPN9 RPS6KA1 RPS6KA2

RPS6KA3 RPS6KA6 SH3PXD2A

TIAM1 ULK1

Unknown/Others APBB1 BNIP2 CCSER2


CDCA3 CDCA8 HSPE1

KIAA1468 LNPEP NCKAP5L

PNPLA8 RRP12 TFB2M

Page 45 of 50


Figure 3

EEA1

FGF1

FGFR4

Merged

EEA1

FGF1

Tf

Merged

LAMP1

FGF1

Tf

Merged

Rab11-GFP

FGF1

Tf

Merged

20 min

A

90 min

15 min

B

C 60 min

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

N



Figure 4 FGF1

Merged

Flotillin-1

FGF1

Merged

Fip1-GFP

FGF1

Merged

Fip2-GFP

FGF1

Merged

Fip5-GFP

FGF1

Merged

90 min

90 min

90 min

15 min

Scyl2-GFP

90 min

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 46 of 50

Page 47 of 50


Figure 5 TGN46

FGF1

Merged

TGN46

FGF1

Giantin

90 min

A

Merged

60 min

B

C

N

TGN46

FGF1

Rab11

60 min

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Merged N


Figure 6 A

FGF1

CLTC ESYT2 ITSN2 OCRL SH3BP4 SNX9 SYNJ1 WASL

1

N

4

4

U2OS-R4 **

RH30 * 2 .5 2.5

3.03

kDa

100

100 100

100 250

250

50

50

2.02

2 .5 2.5

2.02

1 .5 1.5

1 .5 1.5

1.01

1.0 1

0 .5 0.5

0 .5 0.5

00

00 CHC

FGF1

EEA1 FGF1

FGF1

EEA1

scr

FGF1

FGF1

EEA1 FGF1

FGF1

H

G FGF1

FGF1

Dyn1 K44A

FGF1 uptake (DL550-FGF1 Intensity, rel. to ctrl)

FGF1

EEA1

sc r H C -1

*** *** 1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2

0

0.0

*** 1.01 0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2


00

Ctrl

OH-dyn

Ctrl Dyngo-4a

1

2

3

4

sc r H C C -1 H C Sc 2 yl 2

F

HSLP wash

FGF1 uptake (DL550-FGF1 Intensity, rel. to scr)

E

CHC

C

sc r H C -1

sc r

C

g -tubulin

sc r

C

kDa

Surface FGFR4 (a.u.)

C

H

C H C

sc r

sc r C H

D

C

-1

-1

RH30 C

Lysate

CHC

2 3

Clathrin

U2OS-R4

FGFR4

3

2

C

siRNA: IP: biotin WB: FGFR4

CLC 1

-1 FG + F FR GF R 4 4

AGFG1 EPN1 PICALM PIK3C2A SCYL2

Page 48 of 50

B

Clathrin-mediated endocytosis

CHC-1 siRNA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 49 of750 Figure

0'

5'

30'

60'

120'

scr CHC-1 siRNA

240' **

+

-

+

-

+

-

+

-

+

kDa 150

100

1.01

150

0.4 0.4 0.2 0.2 00

0

150

50

50

1.0

**

**

0.4 0.4 0.2 0.2 00 -60

0

50

60 120 180 240 60 120 180 240

*

1.01

**

*

0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 -60

0

37 1.2 1.2

37

*

0.8 0.8 0.6 0.6

00

37

60 120 180 240 60 120 180 240

** *

1

1.2 1.2

50

**

0.6 0.6

1.2 1.2

100

**

0.8 0.8

-60

p-PLCg (a.u.)

-

p-AKT (a.u.)

+

p-FGFR4 (a.u.)

1.2 1.2

p-ERK (a.u.)

1 2 3 4siRNA 5CHC-1: 6 7 CHC 8 9 10 FGFR4 11 12 13 14 15 p-FGFR 16 17 18 19 p-PLCg 20 21 22 23 PLCg 24 25 26 27p-AKT 28 29 30 31 AKT 32 33 p-ERK 34 35 36 37 38 ERK 39 40 41 g -tubulin 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


50

60 120 180 240 60 120 180 240

**

1.01 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2

00 -60

0

50

60 120 180 240 60 120 180 240

Time (Minutes)



Page 50 of 50

for TOC only 1 2 3 4 5 BioID of FGFR4 6 7 - FGF1 + FGF1 8 FGF1 9 Biotin 10 11 Plasma12 membrane 13 14 15 16 FGFR4 17 fused to 18 BirA* 19 Signaling 20 Endocytosis 21 22 23 24 Isolation (pulldown) and identification 25 26 (quantitative LC MS/MS) of biotinylated proteins 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

FGF1 Clathrin

FGF1 EEA1 Tf

FGF1

The identified proteins map FGFR4 endocytic transport


TGN46 EEA1

Proximity Labeling Reveals Molecular ... - ACS Publications

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