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Direct identification of ligand-receptor interactions on living cells and tissues


Many cellular responses are triggered by proteins, drugs or pathogens binding to cell-surface receptors, but it can be challenging to identify which receptors are bound by a given ligand. Here we describe TRICEPS, a chemoproteomic reagent with three moieties—one that binds ligands containing an amino group, a second that binds glycosylated receptors on living cells and a biotin tag for purifying the receptor peptides for identification by quantitative mass spectrometry. We validated this ligand-based, receptor-capture (LRC) technology using insulin, transferrin, apelin, epidermal growth factor, the therapeutic antibody trastuzumab and two DARPins targeting ErbB2. In some cases, we could also determine the approximate ligand-binding sites on the receptors. Using TRICEPS to label intact mature vaccinia viruses, we identified the cell surface proteins AXL, M6PR, DAG1, CSPG4 and CDH13 as binding factors on human cells. This technology enables the identification of receptors for many types of ligands under near-physiological conditions and without the need for genetic manipulations.

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Figure 1: TRICEPS enables LRC.
Figure 2: Workflow for the LRC and identification on living cells.
Figure 3: LRC identifies receptors and receptor panels for ligands ranging from peptides to intact viruses on living cells and tissues.

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We greatly acknowledge T. Clough and O. Vitek at Purdue University for help with statistical data analysis. We are grateful to A. Hofmann, T. Bock, D. Bausch-Fluck, F. Cerciello, A. Jacobs and A. Leitner for suggestions and support at all stages of the project. We acknowledge S. Dettwiler, P. Schraml, M. Tinguely, H. Moch and the Laboratory for In situ Technologies, University Hospital Zurich, for preparation and staining of breast cancer tissues. This work was supported by funding from National Center of Competence in Research (NCCR) Neural Plasticity and Repair (to B.W.), Swiss National Science Foundation (SNSF) (to B.W.), (to B.W.), SNSF Ambizione (to J.M.), and European Research Council (ERC) (to R.A.) and and ERC (to S.K. on behalf of A. Helenius). Immortalized murine pre-adipocytes were kindly provided by M. Rosenwald and C. Wolfrum (ETH Zurich). ErbB2-negative breast carcinoma tissue cut into 50 μm slices was kindly provided by the tissue biobank of the Institute of Surgical Pathology, University Hospital Zurich.

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Authors and Affiliations



A.P.F. and B.W. designed the project and wrote the paper. A.P.F. performed experiments and analyzed all data. A.P.F., B.W., O.-Y.J. and E.M.C. designed TRICEPS and O.-Y.J. synthesized the reagents. J.M. and S.K. designed and performed vaccinia virus experiments and J.M. edited the manuscript. C.J. and A.P. designed DARPin experiments and performed ELISAs. R.A., H.M. and L.M.H. contributed ideas and performed experiments. All authors discussed the results and implications and commented on the manuscript at all stages.

Corresponding author

Correspondence to Bernd Wollscheid.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Note 1 (PDF 1786 kb)

Supplementary Table 1

LRC with human insulin on murine adipocytes (XLS 57 kb)

Supplementary Table 2

LRC competition experiment with human insulin on Jurkat T lymphocytes (XLS 204 kb)

Supplementary Table 3

LRC with transferrin and apelin on U-2 OS cells (XLS 90 kb)

Supplementary Table 4

LRC with EGF and trastuzumab on U251 cells (XLS 54 kb)

Supplementary Table 5

LRC with DARPin 9.01 and DARPin H14 on BT-474 cells (XLS 50 kb)

Supplementary Table 6

LRC with trastuzumab on primary breast cancer tissue (XLS 207 kb)

Supplementary Table 7

LRC with vaccinia virus on HeLa CCL2 cells (XLS 57 kb)

Supplementary Table 8

siRNA sequences used for target protein depletion (XLS 26 kb)

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Frei, A., Jeon, OY., Kilcher, S. et al. Direct identification of ligand-receptor interactions on living cells and tissues. Nat Biotechnol 30, 997–1001 (2012).

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