Monitoring activities of receptor tyrosine kinases using a universal adapter in genetically encoded split TEV assays
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.
KeywordsCell-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
Insulin growth factor 1 receptor
Mesenchymal epithelial transition proto-oncogene, receptor tyrosine kinase
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.
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.
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