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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.

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Figure 1: The MaMTH system.
Figure 2: Use of MaMTH to monitor agonist-dependent interactions of a GPCR.
Figure 3: MaMTH allows detection of PPIs in ErbB family members.
Figure 4: Monitoring phosphorylation-dependent interactions of oncogenic ErbB receptors.
Figure 5: Confirmation of MaMTH interactors and involvement of CRKII in bypassing erlotinib-mediated toxicity in NSCLC cells.

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Acknowledgements

We thank B. Andrews for providing access to equipment and C. Nislow (University of British Columbia) for providing cDNA clones, J. Moffat (University of Toronto) for providing lentiviral plasmids, M.-S. Tsao (Princess Margaret Cancer Centre) for providing erlotinib and B. Neel (Princess Margaret Cancer Centre) for providing imatinib; S. Angers, K. Blakely, J. Jin, S. Kittanakom, A. Arnoldo, M. Mendoza and Y. Fedyshyn for experimental assistance and advice; and U. Petrovic, M. Ali, M. Lam, Z. Yao, K. Sokolina for advice and/or critical review of the manuscript. This work was supported by grants from the Ontario Genomics Institute (303547), Canadian Institutes of Health Research (Catalyst-NHG94491, PPP-125785), Canadian Foundation for Innovation (IOF-LOF), Natural Sciences and Engineering Research Council of Canada (RGPIN 372393-12), Canadian Cystic Fibrosis Foundation (300348), Canadian Cancer Society (2010-700406), Novartis and University Health Network (GL2-01-018) to I.S.; J.P. was funded by a Fonds zur Förderung der wissenschaftlichen Forschung–Erwin Schrödinger postdoctoral fellowship.

Author information

Authors and Affiliations

Authors

Contributions

I.S. conceived the project, provided guidance and assisted in manuscript preparation. J.P. coordinated, managed and was actively involved in all experiments, and wrote the bulk of the manuscript. B.G. was actively involved in the design and performance of the functional and screening part of MaMTH. M.K. and I.J. provided bioinformatics predictions and analysis of EGFR interactors. M.T., I.K. and S.L. were responsible for LUMIER assays. Y.Z. and T.P. generated co-IP and MS data. C.F.K. performed knockdown confirmations and provided technical help for all experiments. A.S., J.R.S., M.-S.T. and J.M. generated and analyzed shRNA knockdown data. J.S. was actively involved in manuscript preparation and provided project guidance. M.M.U. and A.N. were involved in data analysis, bait and prey generation, and fluorescence microscopy. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Igor Stagljar.

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Competing interests

A provisional patent application was filed by the University of Toronto on 2 July 2013 to the US Patent and Trademark Office, application 61/833,304. The intellectual property has been assigned to the University of Toronto and its commercialization is being managed by the Innovations and Partnership Office.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15, Supplementary Tables 1, 4, and 6–10 (PDF 15809 kb)

Supplementary Table 2

Previously identified EGFR interactors plus PubMed-IDs and number of preys identified in various publications. (XLSX 20 kb)

Supplementary Table 3

EGFR screening luciferase data and unrelated bait interaction data. (XLSX 73 kb)

Supplementary Table 5

Previously identified EGFR interactors not detected by MaMTH. (XLSX 10 kb)

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Petschnigg, J., Groisman, B., Kotlyar, M. et al. The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells. Nat Methods 11, 585–592 (2014). https://doi.org/10.1038/nmeth.2895

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