Letter | Published:

Genetic wiring maps of single-cell protein states reveal an off-switch for GPCR signalling

Nature volume 546, pages 307311 (08 June 2017) | Download Citation


As key executers of biological functions, the activity and abundance of proteins are subjected to extensive regulation. Deciphering the genetic architecture underlying this regulation is critical for understanding cellular signalling events and responses to environmental cues. Using random mutagenesis in haploid human cells, we apply a sensitive approach to directly couple genomic mutations to protein measurements in individual cells. Here we use this to examine a suite of cellular processes, such as transcriptional induction, regulation of protein abundance and splicing, signalling cascades (mitogen-activated protein kinase (MAPK), G-protein-coupled receptor (GPCR), protein kinase B (AKT), interferon, and Wingless and Int-related protein (WNT) pathways) and epigenetic modifications (histone crotonylation and methylation). This scalable, sequencing-based procedure elucidates the genetic landscapes that control protein states, identifying genes that cause very narrow phenotypic effects and genes that lead to broad phenotypic consequences. The resulting genetic wiring map identifies the E3-ligase substrate adaptor KCTD5 (ref. 1) as a negative regulator of the AKT pathway, a key signalling cascade frequently deregulated in cancer. KCTD5-deficient cells show elevated levels of phospho-AKT at S473 that could not be attributed to effects on canonical pathway components. To reveal the genetic requirements for this phenotype, we iteratively analysed the regulatory network linked to AKT activity in the knockout background. This genetic modifier screen exposes suppressors of the KCTD5 phenotype and mechanistically demonstrates that KCTD5 acts as an off-switch for GPCR signalling by triggering proteolysis of Gβγ heterodimers dissociated from the Gα subunit. Although biological networks have previously been constructed on the basis of gene expression2,3, protein–protein associations4,5,6, or genetic interaction profiles7,8, we foresee that the approach described here will enable the generation of a comprehensive genetic wiring map for human cells on the basis of quantitative protein states.

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We thank J. Goedhart, L. Wessels, B. van Steensel, S. Nijman, and members of the Brummelkamp, Perrakis, and Sixma laboratories for discussions. We thank R. Spaapen for providing CUL3 knockout cells, P. Celie and M. Stadnik for assistance with the recombinant protein expression, as well as E. Fessler and J. P. Medema for generation of WNT3A/R-spondin-conditioned medium. This work was supported by the Dutch Cancer Society (NKI 2015-7609), the Cancer Genomics Center, an Ammodo KNAW Award 2015 for Biomedical Sciences to T.R.B., by the Netherlands Organization for Scientific Research (NWO) as part of the National Roadmap Large-scale Research Facilities of the Netherlands, Proteins@Work (project number 184.032.201) to O.B.B. and A.F.M.A., and by a Vidi grant (723.012.102) to A.F.M.A.

Author information

Author notes

    • Lucas T. Jae

    Present address: Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Straße 25, 81377 Munich, Germany.

    • Markus Brockmann
    •  & Vincent A. Blomen

    These authors contributed equally to this work.


  1. Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.

    • Markus Brockmann
    • , Vincent A. Blomen
    • , Joppe Nieuwenhuis
    • , Elmer Stickel
    • , Matthijs Raaben
    • , Onno B. Bleijerveld
    • , A. F. Maarten Altelaar
    • , Lucas T. Jae
    •  & Thijn R. Brummelkamp
  2. Biomolecular Mass Spectrometry and Proteomics, Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584CH Utrecht, The Netherlands.

    • A. F. Maarten Altelaar
  3. CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria.

    • Thijn R. Brummelkamp
  4. Cancergenomics.nl, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.

    • Thijn R. Brummelkamp


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M.B., V.A.B., J.N., M.R. L.T.J., and T.R.B. were responsible for the overall design of the study. V.A.B. and E.S. performed the bioinformatics. E.S. developed the Phenosaurus platform. O.B.B. and A.F.M.A. designed, performed, and analysed the proteomics experiments. M.B., V.A.B., L.T.J., and T.R.B. wrote the manuscript; all authors commented on it.

Competing interests

T.R.B. is co-founder and shareholder of Haplogen GmbH and Scenic Biotech BV, and M.B., V.B; J.N., M.R., L.T.J., and T.R.B. are listed as inventors on a patent application related to the technology.

Corresponding authors

Correspondence to Lucas T. Jae or Thijn R. Brummelkamp.

Reviewer Information Nature thanks J. Moffat and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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