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Integrated analysis of receptor activation and downstream signaling with EXTassays

Nature Methods volume 7, pages 74–80 (2010)Cite this article

Abstract

The ability to measure multiple cellular signaling events is essential to better understand the underlying complex biological processes that occur in living cells. Microarray-based technologies are now commonly used to study changes in transcription. This information, however, is not sufficient to understand the regulatory mechanisms that lead to gene expression changes. Here we present an approach to monitor signaling events upstream of gene expression. We coupled different reporter gene assays to unique expressed oligonucleotide tags (EXTs) that serve as identifiers and quantitative reporters. Multiple EXT reporters can be isolated as a pool and analyzed by hybridization to microarrays. To test the feasibility of our approach, we integrated complementation assays based on a protease from tobacco etch virus (TEV protease) and transcription factor activity profiling. Thereby, we simultaneously monitored Neuregulin-dependent mouse ErbB receptor tyrosine kinase dimerization, effector recruitment and downstream signaling.

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Figure 1: Barcoding of multiple cellular assays with EXT reporter assays.
Figure 2: Structure and synthesis of EXTs.
Figure 3: EXT mismatch-discrimination profiles.
Figure 4: EXTassay performance under control conditions.
Figure 5: Integrated NRG-ErbB signaling with EXTassays.
Figure 6: Improved kinetic performance and sensitivity of EXT reporters compared to standard luciferase assays.

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Acknowledgements

We acknowledge the contributions of F. Benseler and all members of the Sequencing Core Facility (Max Planck Institute of Experimental Medicine, Götttingen) for the EXT synthesis and excellent services, and the expert support by staff of the Microarray Core Lab of the University of Göttingen, namely R. Hitt, G. Salinas-Riester and L. Opitz. We thank R. Reinhardt, S. Klages and S. Scheer for high-throughput sequencing and primary data analysis, K.-A. Nave for support, F. Melchior, E. Wimmer, as well as all lab members for stimulating discussions, and M. Wehr for critically reading the manuscript and providing valuable feedback. This study was supported by grants of the Bundesministerium für Bildung und Forschung (FKZ0315180A) and partially by the European Union (LSHM-CT-2005-018637) to M.J.R.

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Authors and Affiliations

  1. Max Planck Institute of Experimental Medicine, Göttingen, Germany

    Anna Botvinnik, Sven P Wichert, Tobias M Fischer & Moritz J Rossner

Authors
  1. Anna Botvinnik
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  2. Sven P Wichert
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  3. Tobias M Fischer
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  4. Moritz J Rossner
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Contributions

A.B. cloned EXT constructs, performed the assays, analyzed the data, contributed conceptually and wrote the manuscript. S.P.W. performed bioinformatic calculations and analyzed the data. T.M.F. cloned expression constructs. M.J.R. conceived the study, analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Moritz J Rossner.

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Competing interests

M.J.R. filed a patent application covering the principles of EXTassays.

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Supplementary Figures 1–15, Supplementary Tables 1–5, Supplementary Note 1 and Supplementary Discussion (PDF 3237 kb)

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Botvinnik, A., Wichert, S., Fischer, T. et al. Integrated analysis of receptor activation and downstream signaling with EXTassays. Nat Methods 7, 74–80 (2010). https://doi.org/10.1038/nmeth.1407

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