Signaling cascades integrate extracellular stimuli primarily through regulated protein-protein interactions (PPIs). Intracellular signal transduction strictly depends on PPIs occurring at the membrane and in the cytosol. To monitor constitutive and regulated protein interactions within living mammalian cells, we have developed a biological assay termed split TEV. We engineered inactive fragments of the NIa protease from the tobacco etch virus (TEV protease) that regain activity only when coexpressed as fusion constructs with interacting proteins. Functional reconstitution of TEV protease fragments can be monitored with 'proteolysis-only' reporters, which can be previously silent fluorescent and luminescent reporter proteins. Additionally, proteolytically cleavable inactive transcription factors can be combined with any downstream reporter gene of choice to yield 'transcription-coupled' reporter systems. Thus, split TEV combines the advantages of split enzyme– and reporter gene–mediated assays, and provides full flexibility with regard to the final readout. In a first biological application, we monitored neuregulin-induced ErbB2/ErbB4 receptor tyrosine kinase heterodimerization.
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We acknowledge the excellent technical assistance from F. Herzog with cell cultures, and H. Böhli and J. Ohsam for cloning expression constructs. The GST-Nrg-1β fusion constructs were a kind gift of C. Lai (The Scripps Institute). Her2 and mouse ErbB4 templates were kindly provided by A. Ullrich (Max Planck Institute for Biochemistry) and C. Lai.
Summary of principles and properties of all membrane reporters.
Comparsion of GV-ER and GV-2ER activation by full-length TEV.
Western blotting of model membrane Split-TEV fusion constructs.
Analysis of TM-Luc reporter activation by transmembrane and cytosolic GCN4/GBR1a/GBR2 cc domain fragment pairs.
Time-dependent analysis of the rapamycin-induced FKBP-FRB interactions monitored with LucER.
Specificity of PPIs in the cytosol measured with LucER in CHO cells.
Comparsion of TM-GV with GV-2ER and GV-ER activated by transmembrane and cytosolic GCN4cc-TEV fragment pairs.
Western blot of Split-TEV interaction pair dependent cleavage of GV-2ER.
Comparing the kinetics of the rapamycin-induced FKBP-FRB interactions monitored with the GV-2ER and LucER.
TEV protease fragments suited for transcomplementation.
A recombinase reporter system for permanent reporter activation.
Fluorescent 'Proteolysis-only' TEV-Reporters.