Abstract
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.
Highlights
Receptor tyrosine kinases (RTKs) are type I transmembrane protein receptors and respond, with few exceptions, to extracellular cues
We describe the application of split TEVbased RTK recruitment assays that provide a universal adapter recruitment strategy for robust and flexible cellbased assays applicable to dose–response profiling and high-throughput screening (HTS) assays
We found in our assays using the Src homology 2 (SH2)(GRB2) adapter that lapatinib efficiently inhibits EGFR (IC50: 305 nM, p IC50: 6.52) and ERBB2/ERBB3 (IC50: 72 nM, pIC50: 7.14), and ERBB4 (IC50: 166 nM, p IC50: 6.78)
Summary
Receptor tyrosine kinases (RTKs) are type I transmembrane protein receptors and respond, with few exceptions, to extracellular cues. RTK signalling is implicated in central nervous system (CNS) and peripheral nervous system (PNS) development [3]. RTKs share a similar architecture, which consists of ligand-binding domains in the extracellular region, a single alpha-helix crossing the membrane and, in the cytoplasm, a juxtamembrane domain, a protein tyrosine kinase domain, and a carboxyl (C-) terminal regulatory region [2]. Together with the G protein-coupled receptors (GPCR), RTKs are the most important receptor class in human cells and represent the most relevant drug targets in the cell [4]. Only 3% of marketed drugs target kinases as such, suggesting that the development of cell-based assays with broader applicability and robustness may contribute to better medicines
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