Receptor tyrosine kinases (RTKs) form multiprotein complexes that initiate and propagate intracellular signals and determine the RTK-specific signalling patterns. Unravelling the full complexity of protein interactions within the RTK-associated complexes is essential for understanding of RTK functions, yet it remains an understudied area of cell biology. We describe a comprehensive approach to characterize RTK interactome. A single tag immunoprecipitation and phosphotyrosine protein isolation followed by mass-spectrometry was used to identify proteins interacting with fibroblast growth factor receptor 3 (FGFR3). A total of 32 experiments were carried out in two different cell types and identified 66 proteins out of which only 20 (30.3%) proteins were already known FGFR interactors. Using co-immunoprecipitations, we validated FGFR3 interaction with adapter protein STAM1, transcriptional regulator SHOX2, translation elongation factor eEF1A1, serine/threonine kinases ICK, MAK and CCRK, and inositol phosphatase SHIP2. We show that unappreciated signalling mediators exist for well-studied RTKs, such as FGFR3, and may be identified via proteomic approaches described here. These approaches are easily adaptable to other RTKs, enabling identification of novel signalling mediators for majority of the known human RTKs.

Department of Biology Faculty of Medicine Masaryk University 62500 Brno Czech Republic

Department of Human Genetics and Orthopaedic Surgery University of California Los Angeles California 90095 USA

Institute of Chemistry and Biochemistry Faculty of Science University of South Bohemia 37005 Ceske Budejovice Czech Republic

Institute of Experimental Biology Faculty of Science Masaryk University 62500 Brno Czech Republic

International Clinical Research Center St Anne's University Hospital 65691 Brno Czech Republic

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$a Balek, Lukas $u Department of Biology, Faculty of Medicine, Masaryk University, 62500 Brno, Czech Republic.

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$a Proteomic analyses of signalling complexes associated with receptor tyrosine kinase identify novel members of fibroblast growth factor receptor 3 interactome / $c L. Balek, P. Nemec, P. Konik, M. Kunova Bosakova, M. Varecha, I. Gudernova, J. Medalova, D. Krakow, P. Krejci,

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$a Receptor tyrosine kinases (RTKs) form multiprotein complexes that initiate and propagate intracellular signals and determine the RTK-specific signalling patterns. Unravelling the full complexity of protein interactions within the RTK-associated complexes is essential for understanding of RTK functions, yet it remains an understudied area of cell biology. We describe a comprehensive approach to characterize RTK interactome. A single tag immunoprecipitation and phosphotyrosine protein isolation followed by mass-spectrometry was used to identify proteins interacting with fibroblast growth factor receptor 3 (FGFR3). A total of 32 experiments were carried out in two different cell types and identified 66 proteins out of which only 20 (30.3%) proteins were already known FGFR interactors. Using co-immunoprecipitations, we validated FGFR3 interaction with adapter protein STAM1, transcriptional regulator SHOX2, translation elongation factor eEF1A1, serine/threonine kinases ICK, MAK and CCRK, and inositol phosphatase SHIP2. We show that unappreciated signalling mediators exist for well-studied RTKs, such as FGFR3, and may be identified via proteomic approaches described here. These approaches are easily adaptable to other RTKs, enabling identification of novel signalling mediators for majority of the known human RTKs.

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$a Nemec, Pavel $u Department of Biology, Faculty of Medicine, Masaryk University, 62500 Brno, Czech Republic.

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$a Konik, Peter $u Institute of Chemistry and Biochemistry, Faculty of Science, University of South Bohemia, 37005 Ceske Budejovice, Czech Republic.

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$a Kunova Bosakova, Michaela $u Department of Biology, Faculty of Medicine, Masaryk University, 62500 Brno, Czech Republic.

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$a Varecha, Miroslav $u Department of Biology, Faculty of Medicine, Masaryk University, 62500 Brno, Czech Republic; International Clinical Research Center, St. Anne's University Hospital, 65691 Brno, Czech Republic.

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$a Gudernova, Iva $u Department of Biology, Faculty of Medicine, Masaryk University, 62500 Brno, Czech Republic.

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$a Medalova, Jirina $u Institute of Experimental Biology, Faculty of Science, Masaryk University, 62500 Brno, Czech Republic.

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$a Krakow, Deborah $u Department of Human Genetics and Orthopaedic Surgery, University of California Los Angeles, California 90095, USA.

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$a Krejci, Pavel $u Department of Biology, Faculty of Medicine, Masaryk University, 62500 Brno, Czech Republic; International Clinical Research Center, St. Anne's University Hospital, 65691 Brno, Czech Republic; Department of Human Genetics and Orthopaedic Surgery, University of California Los Angeles, California 90095, USA. Electronic address: krejcip@med.muni.cz.

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