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Deconvolution of complex G protein–coupled receptor signaling in live cells using dynamic mass redistribution measurements

Nature Biotechnology volume 28, pages 943–949 (2010)Cite this article

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Abstract

Label-free biosensor technology based on dynamic mass redistribution (DMR) of cellular constituents promises to translate GPCR signaling into complex optical 'fingerprints' in real time in living cells. Here we present a strategy to map cellular mechanisms that define label-free responses, and we compare DMR technology with traditional second-messenger assays that are currently the state of the art in GPCR drug discovery. The holistic nature of DMR measurements enabled us to (i) probe GPCR functionality along all four G-protein signaling pathways, something presently beyond reach of most other assay platforms; (ii) dissect complex GPCR signaling patterns even in primary human cells with unprecedented accuracy; (iii) define heterotrimeric G proteins as triggers for the complex optical fingerprints; and (iv) disclose previously undetected features of GPCR behavior. Our results suggest that DMR technology will have a substantial impact on systems biology and systems pharmacology as well as for the discovery of drugs with novel mechanisms.

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Figure 1: Dynamic mass redistribution enables analysis of differential receptor-mediated G protein activation in CHO cells.
Figure 2: Dynamic mass redistribution visualizes signaling along the G12/G13 pathway.
Figure 3: Dynamic mass redistribution enables measurement of differential receptor-mediated G protein activation in HEK293 cells.
Figure 4: Parallel visualization of all signaling pathways unveils an additional signaling route of the free fatty acid receptor FFA1.
Figure 5: Dynamic mass redistribution enables analysis of GPCR functionality in immortalized and primary human keratinocytes.
Figure 6: The muscarinic M3 receptor adapts to adenylyl cyclase activation with a changed signaling repertoire.

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Acknowledgements

We thank U. Rick, M. Vasmer-Ehses and T. Kögler for expert technical assistance and Corning Inc. for providing us with the Epic system. This work was supported by the DFG (Deutsche Forschungsgemeinschaft) grants KO 1582/3-1 to E.K., MO 821/2-1 to K.M. and WE 4428/1-1 to J.W. and by the Dr. Hilmer Foundation (PhD fellowship to S.H.). A.K. is a member of the DFG-funded Research Training School GRK 677. N.J.S is an NHMRC/NHF of Australia Overseas Research Fellow. We thank M. De Amici and U. Holzgrabe (University of Milan and University of Würzburg) for kindly providing Hybrid 1 (ref. 47), T. Ulven (University of Southern Denmark) for TUG424 (ref. 38) and Astellas Pharma Inc. (Osaka, Japan) for providing us with YM-254890 (ref. 30).

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  1. Ralf Schröder and Nicole Janssen: These authors contributed equally to this work.

Authors and Affiliations

  1. Molecular, Cellular, and Pharmacobiology Section, Institute of Pharmaceutical Biology, University of Bonn, Bonn, Germany

    Ralf Schröder, Johannes Schmidt, Nicole Merten, Stephanie Hennen, Anke Müller, Stefanie Blättermann, Jesús Gomeza, Christel Drewke & Evi Kostenis

  2. Pharmacology and Toxicology Section, Institute of Pharmacy, University of Bonn, Bonn, Germany

    Nicole Janssen, Anna Kebig, Marion Mohr-Andrä & Klaus Mohr

  3. Department of Dermatology, University of Bonn, Bonn, Germany

    Sabine Zahn & Jörg Wenzel

  4. Molecular Pharmacology Group, Neuroscience and Molecular Pharmacology, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK

    Nicola J Smith & Graeme Milligan

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Contributions

R.S., N.J., J.S., A.K., N.M., S.H., A.M., S.B., M.M.-A. and N.J.S. designed and performed the experiments; E.K. and K.M. designed the research and wrote the manuscript; S.Z., J.W. and G.M. provided important biological samples or research tools; C.D., J.G., N.J.S., G.M. and R.S. provided important ideas and edited the manuscript.

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Correspondence to Klaus Mohr or Evi Kostenis.

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Competing interests

The Epic biosensor used in the procedure described in this article was provided to E.K. by Corning Inc.

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Schröder, R., Janssen, N., Schmidt, J. et al. Deconvolution of complex G protein–coupled receptor signaling in live cells using dynamic mass redistribution measurements. Nat Biotechnol 28, 943–949 (2010). https://doi.org/10.1038/nbt.1671

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