title: LUMIER: A Discovery Tool for Mammalian Protein Interaction Networks.
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.
authors.affiliation: Center for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON, Canada, M5G 1X5. miriam@lunenfeld.ca.
authors.forename: Miriam
authors.initials: M
authors.lastname: Barrios-Rodiles
authors.affiliation: Donnelly Centre, University of Toronto, Toronto, ON, Canada, M5S 3E1.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Ellis
authors.affiliation: Donnelly Centre, University of Toronto, Toronto, ON, Canada, M5S 3E1.|Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Blencowe
authors.affiliation: Center for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON, Canada, M5G 1X5.|Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.|Breast Cancer Research, Mary Janigan Chair in Molecular Cancer Therapeutics, Toronto, ON, Canada.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Wrana
mesh_terms: D000818:Animals; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D047468:Immunoprecipitation; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*; D008322:Mammals; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction; D013223:Statistics as Topic; D014162:Transfection; D057188:Workflow
chemical_list: D011993:Recombinant Fusion Proteins; D008156:Luciferases
keywords: Binary complex; LUMIER; Mammalian cells; Protein–protein interaction; Signaling pathways; Ternary complex; Transmembrane proteins