Abstract
Dynamic protein–protein interactions (PPIs) are fundamental building blocks of cellular signaling and monitoring their regulation promotes the understanding of signaling in health and disease. Genetically encoded split protein biosensor assays, such as the split TEV method, have proved to be highly valuable when studying regulated PPIs in living cells. Split TEV is based on the functional complementation of two previously inactive TEV protease fragments fused to interacting proteins and provides a robust, sensitive and flexible readout to monitor PPIs both at the membrane and in the cytosol. Thus, split TEV can be used to analyze interactomes of receptors, membrane-associated proteins, and cytosolic proteins. In particular, split TEV is useful to assay activities of relevant drug targets, such as receptor tyrosine kinases and G protein-coupled receptors, in compound screens. As split TEV uses genetically encoded readouts, including standard reporters based on fluorescence and luminescence, the technique can also be combined with scalable molecular barcode reporter systems, allowing the integration into multiplexed high-throughput assay approaches. Split TEV can be used in standard heterologous cell lines and primary cell types, including neurons, either in a transient or stably integrated format. When using cell lines, the basic protocol takes 30–96 h to complete, depending on the complexity of the experimental question addressed.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
McNeill H, Woodgett JR (2010) When pathways collide: collaboration and connivance among signalling proteins in development. Nat Rev Mol Cell Biol 11:404–13. doi:10.1038/nrm2902.
Heng BC, Aubel D, Fussenegger M (2013) An overview of the diverse roles of G-protein coupled receptors (GPCRs) in the pathophysiology of various human diseases. Biotechnol Adv 31(8):1676–94. doi:10.1016/j.biotechadv.2013.08.017.
Lemmon MA, Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell. 2010;141:1117–34. doi:10.1016/j.cell.2010.06.011.
Billingsley ML (2008) Druggable targets and targeted drugs: enhancing the development of new therapeutics. Pharmacology 82:239–244. doi:10.1159/000157624
Michnick SW, Ear PH, Manderson EN, Remy I, Stefan E (2007) Universal strategies in research and drug discovery based on protein-fragment complementation assays. Nat Rev Drug Discov 6:569–582. doi:10.1038/nrd2311
Wehr MC, Rossner MJ (2016) Split protein biosensor assays in molecular pharmacological studies. Drug Discov Today 21:415–29. doi:10.1016/j.drudis.2015.11.004.
Hubbard SR, Miller WT (2007) Receptor tyrosine kinases: mechanisms of activation and signaling. Curr Opin Cell Biol 19:117–123. doi:10.1016/j.ceb.2007.02.010
Petschnigg J, Groisman B, Kotlyar M, Taipale M, Zheng Y, Kurat CF, Sayad A, Sierra JR, Usaj MM, Snider J, Nachman A, Krykbaeva I, Tsao M-S, Moffat J, Pawson T, Lindquist S, Jurisica I, Stagljar I (2014) The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells. Nat Methods. doi:10.1038/nmeth.2895
Wehr M, Reinecke L, Botvinnik A, Rossner M (2008) Analysis of transient phosphorylation-dependent protein-protein interactions in living mammalian cells using split-TEV. BMC Biotechnol 8:55. doi:10.1186/1472-6750-8-55
Antczak C, Bermingham A, Calder P, Malkov D, Song K, Fetter J, Djaballah H (2012) Domain-based biosensor assay to screen for epidermal growth factor receptor modulators in live cells. Assay Drug Dev Technol 10:24–36. doi:10.1089/adt.2011.423
Dorsam RT, Gutkind JS (2007) G-protein-coupled receptors and cancer. Nat Rev Cancer 7:79–94. doi:10.1038/nrc2069
Lagerström MC, Schiöth HB (2008) Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov 7:339–357. doi:10.1038/nrd2518
Kohout TA, Lefkowitz RJ (2003) Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization. Mol Pharmacol 63:9–18
MacDonald ML, Lamerdin J, Owens S, Keon BH, Bilter GK, Shang Z, Huang Z, Yu H, Dias J, Minami T, Michnick SW, Westwick JK (2006) Identifying off-target effects and hidden phenotypes of drugs in human cells. Nat Chem Biol 2:329–337. doi:10.1038/nchembio790
Djannatian MS, Galinski S, Fischer TM, Rossner MJ (2011) Studying G protein-coupled receptor activation using split-tobacco etch virus assays. Anal Biochem 412:141–152. doi:16/j.ab.2011.01.042
Barnea G, Strapps W, Herrada G, Berman Y, Ong J, Kloss B, Axel R, Lee KJ (2008) The genetic design of signaling cascades to record receptor activation. Proc Natl Acad Sci 105:64–69. doi:10.1073/pnas.0710487105
Misawa N, Kafi AKM, Hattori M, Miura K, Masuda K, Ozawa T (2010) Rapid and high-sensitivity cell-based assays of protein-protein interactions using split click beetle luciferase complementation: an approach to the study of G-protein-coupled receptors. Anal Chem 82:2552–2560. doi:10.1021/ac100104q
Ghosh I, Hamilton AD, Regan L (2000) Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein. J Am Chem Soc 122:5658–5659. doi:10.1021/ja994421w
Hu C-D, Chinenov Y, Kerppola TK (2002) Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell 9:789–798
Shyu YJ, Liu H, Deng X, Hu C-D (2006) Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions. BioTechniques 40:61–66
Luker KE, Smith MCP, Luker GD, Gammon ST, Piwnica-Worms H, Piwnica-Worms D (2004) Kinetics of regulated protein–protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. PNAS 101:12288–12293. doi:10.1073/pnas.0404041101
Paulmurugan R, Umezawa Y, Gambhir SS (2002) Noninvasive imaging of protein-protein interactions in living subjects by using reporter protein complementation and reconstitution strategies. Proc Natl Acad Sci U S A 99:15608–15613. doi:10.1073/pnas.242594299
Kaihara A, Kawai Y, Sato M, Ozawa T, Umezawa Y (2003) Locating a protein-protein interaction in living cells via split Renilla luciferase complementation. Anal Chem 75:4176–4181
Paulmurugan R, Gambhir SS (2003) Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation. Anal Chem 75:1584–1589
Remy I, Michnick SW (2006) A highly sensitive protein-protein interaction assay based on Gaussia luciferase. Nat Methods 3:977–979. doi:10.1038/nmeth979
Kim SB, Otani Y, Umezawa Y, Tao H (2007) Bioluminescent indicator for determining protein-protein interactions using intramolecular complementation of split click beetle luciferase. Anal Chem 79:4820–4826. doi:10.1021/ac0621571
Hida N, Awais M, Takeuchi M, Ueno N, Tashiro M, Takagi C, Singh T, Hayashi M, Ohmiya Y, Ozawa T (2009) High-sensitivity real-time imaging of dual protein-protein interactions in living subjects using multicolor luciferases. PLoS ONE 4:e5868. doi:10.1371/journal.pone.0005868
Galarneau A, Primeau M, Trudeau L-E, Michnick SW (2002) Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions. Nat Biotechnol 20:619–622. doi:10.1038/nbt0602-619
Blakely BT, Rossi FM, Tillotson B, Palmer M, Estelles A, Blau HM (2000) Epidermal growth factor receptor dimerization monitored in live cells. Nat Biotechnol 18:218–222. doi:10.1038/72686
Rossi F, Charlton CA, Blau HM (1997) Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation. Proc Natl Acad Sci U S A 94:8405–8410
Johnsson N, Varshavsky A (1994) Split ubiquitin as a sensor of protein interactions in vivo. Proc Natl Acad Sci U S A 91:10340–10344
Stagljar I, Korostensky C, Johnsson N, Heesen S et al (1998) A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. PNAS 95:5187–5192
Wehr MC, Laage R, Bolz U, Fischer TM, Grünewald S, Scheek S, Bach A, Nave K-A, Rossner MJ (2006) Monitoring regulated protein-protein interactions using split TEV. Nat Methods 3:985–993. doi:10.1038/nmeth967
Hamdan FF, Audet M, Garneau P, Pelletier J, Bouvier M (2005) High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer 1-based beta-arrestin2 recruitment assay. J Biomol Screen 10:463–475. doi:10.1177/1087057105275344
Vrecl M, Jorgensen R, Pogacnik A, Heding A (2004) Development of a BRET2 screening assay using beta-arrestin 2 mutants. J Biomol Screen 9:322–333. doi:10.1177/1087057104263212
Kapust RB, Tözsér J, Fox JD, Anderson DE, Cherry S, Copeland TD, Waugh DS (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng 14:993–1000
Nunn CM, Jeeves M, Cliff MJ, Urquhart GT, George RR, Chao LH, Tscuchia Y, Djordjevic S (2005) Crystal structure of tobacco etch virus protease shows the protein C terminus bound within the active site. J Mol Biol 350:145–155. doi:10.1016/j.jmb.2005.04.013
Wehr MC, Galinski S, Rossner MJ (2015) Monitoring G Protein-Coupled Receptor Activation Using the Protein Fragment Complementation Technique Split TEV. Methods Mol Biol 1272:107–118. doi:10.1007/978-1-4939-2336-6_8
Praskova M, Khoklatchev A, Ortiz-Vega S, Avruch J (2004) Regulation of the MST1 kinase by autophosphorylation, by the growth inhibitory proteins, RASSF1 and NORE1, and by Ras. The Biochemical journal 381:453–462
Genevet A, Wehr MC, Brain R, Thompson BJ, Tapon N (2010) Kibra is a regulator of the Salvador/Warts/Hippo signaling network. Dev Cell 18:300–308. doi:10.1016/j.devcel.2009.12.011
Wehr MC, Holder MV, Gailite I, Saunders RE, Maile TM, Ciirdaeva E, Instrell R, Jiang M, Howell M, Rossner MJ, Tapon N (2013) Salt-inducible kinases regulate growth through the Hippo signalling pathway in Drosophila. Nat Cell Biol 15:61–71. doi:10.1038/ncb2658
Botvinnik A, Wichert SP, Fischer TM, Rossner MJ (2010) Integrated analysis of receptor activation and downstream signaling with EXTassays. Nat Meth 7:74–80. doi:10.1038/nmeth.1407
Ura S, Masuyama N, Graves JD, Gotoh Y (2001) Caspase cleavage of MST1 promotes nuclear translocation and chromatin condensation. Proc Natl Acad Sci USA 98:10148–10153. doi:10.1073/pnas.181161698
Stark C, Breitkreutz B-J, Reguly T, Boucher L, Breitkreutz A, Tyers M (2006) BioGRID: a general repository for interaction datasets. Nucleic Acids Res 34:D535–539. doi:10.1093/nar/gkj109
Acknowledgment
This work was supported by the Ludwig Maximilian University of Munich and the DFG grant WE 5683/1-1 to M.C.W.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Wintgens, J.P., Rossner, M.J., Wehr, M.C. (2017). Characterizing Dynamic Protein–Protein Interactions Using the Genetically Encoded Split Biosensor Assay Technique Split TEV. In: Stein, V. (eds) Synthetic Protein Switches. Methods in Molecular Biology, vol 1596. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6940-1_14
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6940-1_14
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6938-8
Online ISBN: 978-1-4939-6940-1
eBook Packages: Springer Protocols