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

Nature methods (2014-03-25)
Julia Petschnigg, Bella Groisman, Max Kotlyar, Mikko Taipale, Yong Zheng, Christoph F Kurat, Azin Sayad, J Rafael Sierra, Mojca Mattiazzi Usaj, Jamie Snider, Alex Nachman, Irina Krykbaeva, Ming-Sound Tsao, Jason Moffat, Tony Pawson, Susan Lindquist, Igor Jurisica, Igor Stagljar
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.

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O-Phospho-L-tyrosine