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Nat Methods. 2014 May;11(5):585-92. doi: 10.1038/nmeth.2895. Epub 2014 Mar 23.

The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells.

Author information

  • 1Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.
  • 2Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, Ontario, Canada.
  • 3Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
  • 4Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
  • 51] Donnelly Centre, University of Toronto, Toronto, Ontario, Canada. [2].
  • 61] 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.
  • 71] Donnelly Centre, University of Toronto, Toronto, Ontario, Canada. [2] Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
  • 81] Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada. [2].
  • 91] 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.
  • 101] 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.
  • 111] 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.

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.

PMID:
24658140
DOI:
10.1038/nmeth.2895
[PubMed - indexed for MEDLINE]
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