Application guide for omics approaches to cell signaling

Research in signal transduction aims to identify the functions of different signaling pathways in physiological and pathological states. Traditional techniques using biochemical, genetic or cell biological approaches have made important contributions to our understanding of cellular signaling. However, the single-gene approach does not take into account the full complexity of cell signaling. With the availability of omics techniques, great progress has been made in understanding signaling networks. Omics approaches can be classified into two categories: 'molecular profiling', including genomic, proteomic, post-translational modification and interactome profiling; and 'molecular perturbation', including genetic and functional perturbations

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

Cell signaling, one of the key processes involved in human health and disease, is coordinated by numerous membrane protein-protein interactions (PPIs) that change in response to stimuli. Currently, there is a lack of assays that can detect these changes in stimuli- and disease-related contexts. Here, we present a novel split-ubiquitin-method for the detection of integral membrane PPIs in human cells, termed Mammalian-Membrane-Two-Hybrid (MaMTH). We highlight the strength of this technology by showing that it detects stimuli (hormone/agonist)- and phosphorylationdependent PPIs. Importantly, it can detect changes in PPIs conferred by mutations such as those in oncogenic ErbB-receptor variants or by treatment with drugs like the tyrosine-kinase inhibitor erlotinib. Using MaMTH as a screening assay, we identified CRKII as an interactor of oncogenic EGFRL858R, promoting persistent activation of aberrant signaling. In conclusion, our study illustrates that MaMTH is a powerful tool for investigating dynamic interactomes of human integral membrane proteins.The work was supported by grants from the Ontario Genomics Institute (303547), Canadian Institutes of Health Research (Catalyst - NHG99091; 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, UNiversity Health Network (GL2-01-018), FWF-Erwin Schrödinger Fellowship progra