title: The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells.
abstract: Cell signaling, one of key processes in both normal cellular function and disease, is coordinated by numerous interactions between membrane proteins that change in response to stimuli. We present a split ubiquitin-based method for detection of integral membrane protein-protein interactions (PPIs) in human cells, termed mammalian-membrane two-hybrid assay (MaMTH). We show that this technology detects stimulus (hormone or agonist)-dependent and phosphorylation-dependent PPIs. MaMTH can detect changes in PPIs conferred by mutations such as those in oncogenic ErbB receptor variants or by treatment with drugs such as the tyrosine kinase inhibitor erlotinib. Using MaMTH as a screening assay, we identified CRKII as an interactor of oncogenic EGFR(L858R) and showed that CRKII promotes persistent activation of aberrant signaling in non-small cell lung cancer cells. MaMTH is a powerful tool for investigating the dynamic interactomes of human integral membrane proteins.
authors.affiliation: Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Julia
authors.initials: J
authors.lastname: Petschnigg
authors.affiliation: Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Bella
authors.initials: B
authors.lastname: Groisman
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Max
authors.initials: M
authors.lastname: Kotlyar
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Mikko
authors.initials: M
authors.lastname: Taipale
authors.affiliation: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: 1] Donnelly Centre, University of Toronto, Toronto, Ontario, Canada. [2].
authors.forename: Christoph F
authors.initials: CF
authors.lastname: Kurat
authors.affiliation: Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Azin
authors.initials: A
authors.lastname: Sayad
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Ontario, Canada.
authors.forename: J Rafael
authors.initials: JR
authors.lastname: Sierra
authors.affiliation: Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Mojca
authors.initials: M
authors.lastname: Mattiazzi Usaj
authors.affiliation: Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Jamie
authors.initials: J
authors.lastname: Snider
authors.affiliation: Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Alex
authors.initials: A
authors.lastname: Nachman
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Krykbaeva
authors.affiliation: 1] Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Ontario, Canada. [2] Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada. [3] Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Ming-Sound
authors.initials: MS
authors.lastname: Tsao
authors.affiliation: 1] Donnelly Centre, University of Toronto, Toronto, Ontario, Canada. [2] Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Jason
authors.initials: J
authors.lastname: Moffat
authors.affiliation: 1] Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada. [2].
authors.forename: Tony
authors.initials: T
authors.lastname: Pawson
authors.affiliation: 1] Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Cambridge, Massachusetts, USA. [3] Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Susan
authors.initials: S
authors.lastname: Lindquist
authors.affiliation: 1] Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Ontario, Canada. [2] Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada. [3] Department of Computer Science, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Igor
authors.initials: I
authors.lastname: Jurisica
authors.affiliation: 1] Donnelly Centre, University of Toronto, Toronto, Ontario, Canada. [2] Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. [3] Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Igor
authors.initials: I
authors.lastname: Stagljar
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002470:Cell Survival; D003600:Cytosol / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D049490:Systems Biology / Q000379:methods; D014157:Transcription Factors / Q000737:chemistry; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000737:chemistry
chemical_list: D008565:Membrane Proteins; D014157:Transcription Factors; D025801:Ubiquitin; D019000:Phosphotyrosine; C512478:EGFR protein, human; D066246:ErbB Receptors