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Proteomics pp 137-148 | Cite as

LUMIER: A Discovery Tool for Mammalian Protein Interaction Networks

  • Miriam Barrios-RodilesEmail author
  • Jonathan D. Ellis
  • Benjamin J. Blencowe
  • Jeffrey L. Wrana
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1550)

Abstract

Protein–protein interactions (PPIs) play an essential role in all biological processes. In vivo, PPIs occur dynamically and depend on extracellular cues. To discover novel protein–protein interactions in mammalian cells, we developed a high-throughput automated technology called LUMIER (LUminescence-based Mammalian IntERactome). In this approach, we co-express a Luciferase (LUC)-tagged fusion protein along with a Flag-tagged protein in an efficiently transfectable cell line such as HEK-293T cells. The interaction between the two proteins is determined by co-immunoprecipitation using an anti-Flag antibody, and the presence of the LUC-tagged interactor in the complex is subsequently detected via its luciferase activity. LUMIER can easily detect transmembrane protein partners, interactions that are signaling- or splice isoform-dependent, as well as those that may occur only in the presence of posttranslational modifications. Using various collections of Flag-tagged proteins, we have generated protein interaction networks for several TGF-β family receptors, Wnt pathway members, and have systematically analyzed the effect of neural-specific alternative splicing on protein interaction networks. The results have provided important insights into the physiological and functional relevance of some of the novel interactions found. LUMIER is highly scalable and can be used for both low- and high-throughput strategies. LUMIER is thus a valuable tool for the identification and characterization of dynamically regulated PPIs in mammalian systems. Here, we describe a manual version of LUMIER in a 96-well format that can be easily implemented in any laboratory.

Key words

Protein–protein interaction LUMIER Mammalian cells Signaling pathways Transmembrane proteins Binary complex Ternary complex 

Notes

Acknowledgments

This work was supported by funding from the Canadian Institutes of Health Research (CIHR) (J.L.W. and B.J.B.), the Alzheimer’s Society of Canada (B.J.B.), and the Krembil Foundation (J.L.W.). We would also like to thank Dr. Saranya Kittanakom for the plasmid schematics framework.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Miriam Barrios-Rodiles
    • 1
    Email author
  • Jonathan D. Ellis
    • 2
  • Benjamin J. Blencowe
    • 2
    • 3
  • Jeffrey L. Wrana
    • 1
    • 3
    • 4
  1. 1.Center for Systems Biology, Lunenfeld-Tanenbaum Research InstituteMount Sinai HospitalTorontoCanada
  2. 2.Donnelly CentreUniversity of TorontoTorontoCanada
  3. 3.Department of Molecular GeneticsUniversity of TorontoTorontoCanada
  4. 4.Breast Cancer ResearchMary Janigan Chair in Molecular Cancer TherapeuticsTorontoCanada

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