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.

Department of Biology Faculty of Medicine Masaryk University Kamenice 5 625 00 Brno Bohunice Czech Republic

Department of Biology Faculty of Medicine Masaryk University Kamenice 5 625 00 Brno Bohunice Czech Republic International Clinical Research Center St Anne's University Hospital 656 91 Brno Czech Republic

Department of Immunology Oslo University Hospital Rikshospitalet and University of Oslo 0027 Oslo Norway The Brain Institute Universidade Federal do Rio Grande do Norte UFRN Natal RN 59078 Brazil

Department of Molecular Cell Biology Institute for Cancer Research The Norwegian Radium Hospital Oslo University Hospital Montebello 0379 Oslo Norway Centre for Cancer Biomedicine Faculty of Medicine University of Oslo Montebello 0379 Oslo Norway

Department of Molecular Cell Biology Institute for Cancer Research The Norwegian Radium Hospital Oslo University Hospital Montebello 0379 Oslo Norway Centre for Cancer Biomedicine Faculty of Medicine University of Oslo Montebello 0379 Oslo Norway Department of Core Facilities Institute for Cancer Research The Norwegian Radium Hospital Oslo University Hospital Montebello 0379 Oslo Norway

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