Monitoring activities of receptor tyrosine kinases using a universal adapter in genetically encoded split TEV assays

  • Jan P. Wintgens
  • Sven P. Wichert
  • Luksa Popovic
  • Moritz J. Rossner
  • Michael C. WehrEmail author
Original Article


Receptor tyrosine kinases (RTKs) play key roles in various aspects of cell biology, including cell-to-cell communication, proliferation and differentiation, survival, and tissue homeostasis, and have been implicated in various diseases including cancer and neurodevelopmental disorders. Ligand-activated RTKs recruit adapter proteins through a phosphotyrosine (p-Tyr) motif that is present on the RTK and a p-Tyr-binding domain, like the Src homology 2 (SH2) domain found in adapter proteins. Notably, numerous combinations of RTK/adapter combinations exist, making it challenging to compare receptor activities in standardised assays. In cell-based assays, a regulated adapter recruitment can be investigated using genetically encoded protein–protein interaction detection methods, such as the split TEV biosensor assay. Here, we applied the split TEV technique to robustly monitor the dynamic recruitment of both naturally occurring full-length adapters and artificial adapters, which are formed of clustered SH2 domains. The applicability of this approach was tested for RTKs from various subfamilies including the epidermal growth factor (ERBB) family, the insulin receptor (INSR) family, and the hepatocyte growth factor receptor (HGFR) family. Best signal-to-noise ratios of ligand-activated RTK receptor activation was obtained when clustered SH2 domains derived from GRB2 were used as adapters. The sensitivity and robustness of the RTK recruitment assays were validated in dose-dependent inhibition assays using the ERBB family-selective antagonists lapatinib and WZ4002. The RTK split TEV recruitment assays also qualify for high-throughput screening approaches, suggesting that the artificial adapter may be used as universal adapter in cell-based profiling assays within pharmacological intervention studies.


Cell-based assay Receptor tyrosine kinases TEV protease Split TEV recruitment assay Lapatinib 



Epidermal growth factor


EGF-like domain of NRG1


Epidermal growth factor receptor


Erb-B2 receptor tyrosine kinase 2


Erb-B2 receptor tyrosine kinase 3


Erb-B2 receptor tyrosine kinase 4


Growth factor receptor bound protein 2


High-throughput screening


Insulin growth factor 1 receptor


Mesenchymal epithelial transition proto-oncogene, receptor tyrosine kinase


Neuregulin 1


Phosphoinositide-3-kinase regulatory subunit 1


Receptor tyrosine kinase


Src homology 2 domain-containing adaptor protein 1


Src homology 2


Tobacco etch virus



We thank Barbara Meisel, Monika Rübekeil, Johanna Zach, and Nadia Gabellini for excellent technical support.

Author contributions

Designed experiments and analysed data: JPW, MCW; performed experiments: JPW, LP; supported assay development with laboratory automation technology: SPW; provided essential reagents and promoted the study: MJR; wrote the manuscript: JPW, MCW; conceived and orchestrated the study: MCW.


M.C.W. was supported by the Deutsche Forschungsgemeinschaft (WE 5683/1-1). Systasy Bioscience GmbH was a beneficiary of the PDZnet project that has received funding from the European Union’s H2020 Framework Programme under the Marie Sklodowska-Curie Grant agreement no. 675341.

Compliance with ethical standards

Conflict of interest

The authors declare competing financial interest.

Supplementary material

18_2018_3003_MOESM1_ESM.pdf (2.3 mb)
Supplementary material 1 (PDF 2340 kb)


  1. 1.
    Blume-Jensen P, Hunter T (2001) Oncogenic kinase signalling. Nature 411:355–365CrossRefGoogle Scholar
  2. 2.
    Lemmon MA, Schlessinger J (2010) Cell signaling by receptor tyrosine kinases. Cell 141:1117–1134. CrossRefGoogle Scholar
  3. 3.
    Mei L, Nave K-A (2014) Neuregulin-ERBB signaling in the nervous system and neuropsychiatric diseases. Neuron 83:27–49. CrossRefGoogle Scholar
  4. 4.
    Santos R, Ursu O, Gaulton A et al (2017) A comprehensive map of molecular drug targets. Nat Rev Drug Discov 16:19–34. CrossRefGoogle Scholar
  5. 5.
    Yaffe MB (2002) Phosphotyrosine-binding domains in signal transduction. Nat Rev Mol Cell Biol 3:177–186. CrossRefGoogle Scholar
  6. 6.
    Liu BA, Jablonowski K, Raina M et al (2006) The human and mouse complement of SH2 domain proteins—establishing the boundaries of phosphotyrosine signaling. Mol Cell 22:851–868. CrossRefGoogle Scholar
  7. 7.
    Tinti M, Kiemer L, Costa S et al (2013) The SH2 domain interaction landscape. Cell Rep 3:1293–1305. CrossRefGoogle Scholar
  8. 8.
    Klapper LN, Glathe S, Vaisman N et al (1999) The ErbB-2/HER2 oncoprotein of human carcinomas may function solely as a shared coreceptor for multiple stroma-derived growth factors. Proc Natl Acad Sci 96:4995–5000CrossRefGoogle Scholar
  9. 9.
    Kochupurakkal BS, Harari D, Di-Segni A et al (2005) Epigen, the last ligand of ErbB receptors, reveals intricate relationships between affinity and mitogenicity. J Biol Chem 280:8503–8512. CrossRefGoogle Scholar
  10. 10.
    Guy PM, Platko JV, Cantley LC et al (1994) Insect cell-expressed p180erbB3 possesses an impaired tyrosine kinase activity. Proc Natl Acad Sci 91:8132–8136CrossRefGoogle Scholar
  11. 11.
    Yarden Y, Sliwkowski MX (2001) Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2:127–137. CrossRefGoogle Scholar
  12. 12.
    Wehr MC, Rossner MJ (2016) Split protein biosensor assays in molecular pharmacological studies. Drug Discov Today 21:415–429. CrossRefGoogle Scholar
  13. 13.
    Wehr MC, Laage R, Bolz U et al (2006) Monitoring regulated protein-protein interactions using split TEV. Nat Methods 3:985–993. CrossRefGoogle Scholar
  14. 14.
    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. CrossRefGoogle Scholar
  15. 15.
    Velanac V, Unterbarnscheidt T, Hinrichs W et al (2012) Bace1 processing of NRG1 type III produces a myelin-inducing signal but is not essential for the stimulation of myelination. Glia 60:203–217. CrossRefGoogle Scholar
  16. 16.
    Wehr MC, Hinrichs W, Brzózka MM et al (2017) Spironolactone is an antagonist of NRG1-ERBB4 signaling and schizophrenia-relevant endophenotypes in mice. EMBO Mol Med. Google Scholar
  17. 17.
    Yang X, Boehm JS, Yang X et al (2011) A public genome-scale lentiviral expression library of human ORFs. Nat Methods 8:659–661. CrossRefGoogle Scholar
  18. 18.
    Ritz C, Baty F, Streibig JC, Gerhard D (2015) Dose-response analysis using R. PLoS One 10:e0146021. CrossRefGoogle Scholar
  19. 19.
    Wintgens JP, Rossner MJ, Wehr MC (2017) Characterizing dynamic protein-protein interactions using the genetically encoded split biosensor assay technique split TEV. Methods Mol Biol 1596:219–238. CrossRefGoogle Scholar
  20. 20.
    Elkins RC, Davies MR, Brough SJ et al (2013) Variability in high-throughput ion-channel screening data and consequences for cardiac safety assessment. J Pharmacol Toxicol Methods 68:112–122. CrossRefGoogle Scholar
  21. 21.
    Song RX, Barnes CJ, Zhang Z et al (2004) The role of Shc and insulin-like growth factor 1 receptor in mediating the translocation of estrogen receptor alpha to the plasma membrane. Proc Natl Acad Sci USA 101:2076–2081. CrossRefGoogle Scholar
  22. 22.
    Liang Q, Mohan RR, Chen L, Wilson SE (1998) Signaling by HGF and KGF in corneal epithelial cells: Ras/MAP kinase and Jak-STAT pathways. Invest Ophthalmol Vis Sci 39:1329–1338Google Scholar
  23. 23.
    Vincent F, Loria P, Pregel M et al (2015) Developing predictive assays: the phenotypic screening “rule of 3”. Sci Transl Med 7:293ps15. CrossRefGoogle Scholar
  24. 24.
    Rusnak DW, Lackey K, Affleck K et al (2001) The effects of the novel, reversible epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor, GW2016, on the growth of human normal and tumor-derived cell lines in vitro and in vivo. Mol Cancer Ther 1:85–94Google Scholar
  25. 25.
    Mulvihill MJ, Cooke A, Rosenfeld-Franklin M et al (2009) Discovery of OSI-906: a selective and orally efficacious dual inhibitor of the IGF-1 receptor and insulin receptor. Future Med Chem 1:1153–1171. CrossRefGoogle Scholar
  26. 26.
    Qian F, Engst S, Yamaguchi K et al (2009) Inhibition of tumor cell growth, invasion, and metastasis by EXEL-2880 (XL880, GSK1363089), a novel inhibitor of HGF and VEGF receptor tyrosine kinases. Cancer Res 69:8009–8016. CrossRefGoogle Scholar
  27. 27.
    Davis MI, Hunt JP, Herrgard S et al (2011) Comprehensive analysis of kinase inhibitor selectivity. Nat Biotechnol 29:1046–1051. CrossRefGoogle Scholar
  28. 28.
    Fidanze SD, Erickson SA, Wang GT et al (2010) Imidazo[2,1-b]thiazoles: multitargeted inhibitors of both the insulin-like growth factor receptor and members of the epidermal growth factor family of receptor tyrosine kinases. Bioorg Med Chem Lett 20:2452–2455. CrossRefGoogle Scholar
  29. 29.
    Wang GT, Mantei RA, Hubbard RD et al (2010) Substituted 4-amino-1H-pyrazolo[3,4-d]pyrimidines as multi-targeted inhibitors of insulin-like growth factor-1 receptor (IGF1R) and members of ErbB-family receptor kinases. Bioorg Med Chem Lett 20:6067–6071. CrossRefGoogle Scholar
  30. 30.
    Ward RA, Anderton MJ, Ashton S et al (2013) Structure- and reactivity-based development of covalent inhibitors of the activating and gatekeeper mutant forms of the epidermal growth factor receptor (EGFR). J Med Chem 56:7025–7048. CrossRefGoogle Scholar
  31. 31.
    Zhou W, Ercan D, Chen L et al (2009) Novel mutant-selective EGFR kinase inhibitors against EGFR T790M. Nature 462:1070–1074. CrossRefGoogle Scholar
  32. 32.
    Birmingham A, Selfors LM, Forster T et al (2009) Statistical methods for analysis of high-throughput RNA interference screens. Nat Methods 6:569–575. CrossRefGoogle Scholar
  33. 33.
    Jones RB, Gordus A, Krall JA, MacBeath G (2006) A quantitative protein interaction network for the ErbB receptors using protein microarrays. Nature 439:168–174. CrossRefGoogle Scholar
  34. 34.
    Oh D, Ogiue-Ikeda M, Jadwin JA et al (2012) Fast rebinding increases dwell time of Src homology 2 (SH2)-containing proteins near the plasma membrane. PNAS 109:14024–14029. CrossRefGoogle Scholar
  35. 35.
    Schulze WX, Deng L, Mann M (2005) Phosphotyrosine interactome of the ErbB-receptor kinase family. Mol Syst Biol 1(2005):0008. Google Scholar
  36. 36.
    Ward CW, Gough KH, Rashke M et al (1996) Systematic mapping of potential binding sites for Shc and Grb2 SH2 domains on insulin receptor substrate-1 and the receptors for insulin, epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor. J Biol Chem 271:5603–5609. CrossRefGoogle Scholar
  37. 37.
    Pawson T (2004) Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell 116:191–203. CrossRefGoogle Scholar
  38. 38.
    Hanke S, Mann M (2009) The phosphotyrosine interactome of the insulin receptor family and its substrates IRS-1 and IRS-2. Mol Cell Proteomics 8:519–534. CrossRefGoogle Scholar
  39. 39.
    MacDonald JIS, Gryz EA, Kubu CJ et al (2000) Direct binding of the signaling adapter protein Grb2 to the activation loop tyrosines on the nerve growth factor receptor tyrosine kinase, TrkA. J Biol Chem 275:18225–18233. CrossRefGoogle Scholar
  40. 40.
    Galinski S, Wichert SP, Rossner MJ, Wehr MC (2018) Multiplexed profiling of GPCR activities by combining split TEV assays and EXT-based barcoded readouts. Sci Rep 8:8137. CrossRefGoogle Scholar
  41. 41.
    Roepstorff K, Grandal MV, Henriksen L et al (2009) Differential effects of EGFR ligands on endocytic sorting of the receptor. Traffic 10:1115–1127. CrossRefGoogle Scholar
  42. 42.
    Vasudevan HN, Soriano P (2016) A thousand and one receptor tyrosine kinases: wherein the specificity? Curr Top Dev Biol 117:393–404. CrossRefGoogle Scholar
  43. 43.
    Freed DM, Bessman NJ, Kiyatkin A et al (2017) EGFR ligands differentially stabilize receptor dimers to specify signaling kinetics. Cell 171:683–695.e18. CrossRefGoogle Scholar
  44. 44.
    Ronan T, Macdonald-Obermann JL, Huelsmann L et al (2016) Different epidermal growth factor receptor (EGFR) agonists produce unique signatures for the recruitment of downstream signaling proteins. J Biol Chem 291:5528–5540. CrossRefGoogle Scholar
  45. 45.
    Zinkle A, Mohammadi M (2018) A threshold model for receptor tyrosine kinase signaling specificity and cell fate determination. F1000Res. Google Scholar
  46. 46.
    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. CrossRefGoogle Scholar
  47. 47.
    Zhou J, Lin J, Zhou C et al (2011) An improved bimolecular fluorescence complementation tool based on superfolder green fluorescent protein. Acta Biochim Biophys Sin (Shanghai) 43:239–244. CrossRefGoogle Scholar
  48. 48.
    Paulmurugan R, Umezawa Y, Gambhir SS (2002) Noninvasive imaging of protein–protein interactions in living subjects by using reporter protein complementation and reconstitution strategies. PNAS 99:15608–15613. CrossRefGoogle Scholar
  49. 49.
    Petschnigg J, Groisman B, Kotlyar M et al (2014) The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells. Nat Methods 11:585–592. CrossRefGoogle Scholar
  50. 50.
    Barnea G, Strapps W, Herrada G et al (2008) The genetic design of signaling cascades to record receptor activation. Proc Natl Acad Sci USA 105:64–69. CrossRefGoogle Scholar
  51. 51.
    Jares-Erijman EA, Jovin TM (2003) FRET imaging. Nat Biotechnol 21:1387–1395. CrossRefGoogle Scholar
  52. 52.
    Pfleger KDG, Eidne KA (2006) Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat Methods 3:165–174. CrossRefGoogle Scholar
  53. 53.
    Madden R, Kosari S, Peterson GM et al (2018) Lapatinib plus capecitabine in patients with HER2-positive metastatic breast cancer: a systematic review. Int J Clin Pharmacol Ther 56:72–80. CrossRefGoogle Scholar
  54. 54.
    Petrelli F, Ghidini M, Lonati V et al (2017) The efficacy of lapatinib and capecitabine in HER-2 positive breast cancer with brain metastases: a systematic review and pooled analysis. Eur J Cancer 84:141–148. CrossRefGoogle Scholar
  55. 55.
    Basu D, Richters A, Rauh D (2015) Structure-based design and synthesis of covalent-reversible inhibitors to overcome drug resistance in EGFR. Bioorg Med Chem 23:2767–2780. CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Department of Psychiatry and PsychotherapyUniversity Hospital, LMU MunichMunichGermany
  2. 2.Systasy Bioscience GmbHMunichGermany

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