Monitoring activities of receptor tyrosine kinases using a universal adapter in genetically encoded split TEV assays
Monitoring activities of receptor tyrosine kinases using a universal adapter in genetically encoded split TEV assays
复制标题
DOI：
10.1007/s00018-018-03003-2
复制
发表时间：
2019-03-01
影响因子：
8
通讯作者：
Wehr, Michael C.
中科院分区：
文献类型：
--
作者：
Wintgens, Jan P.;Wichert, Sven P.;Wehr, Michael C.
受体酪氨酸激酶 (RTK) 在细胞生物学的各个方面发挥着关键作用，包括细胞间通讯、增殖和分化、生存和组织稳态，并与癌症和神经发育障碍等多种疾病有关。配体激活的 RTK 通过 RTK 上存在的磷酸酪氨酸 (p-Tyr) 基序和 p-Tyr 结合域（如接头蛋白中的 Src 同源 2 (SH2) 域）招募接头蛋白。值得注意的是，存在多种 RTK/适配器组合，这使得在标准化测定中比较受体活性具有挑战性。在基于细胞的测定中，可以使用基因编码的蛋白质-蛋白质相互作用检测方法（例如分离 TEV 生物传感器测定）来研究受调节的接头募集。在这里，我们应用分割 TEV 技术来稳健地监测自然发生的全长接头和人工接头的动态招募，这些接头由簇状 SH2 域形成。该方法的适用性针对来自不同亚家族的 RTK 进行了测试，包括表皮生长因子 (ERBB) 家族、胰岛素受体 (INSR) 家族和肝细胞生长因子受体 (HGFR) 家族。当使用源自 GRB2 的簇状 SH2 结构域作为接头时，获得了配体激活的 RTK 受体激活的最佳信噪比。使用 ERBB 家族选择性拮抗剂拉帕替尼和 WZ4002 在剂量依赖性抑制测定中验证了 RTK 募集测定的灵敏度和稳健性。 RTK 分裂 TEV 招募测定也符合高通量筛选方法，这表明人工适配器可用作药理学干预研究中基于细胞的分析测定的通用适配器。
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.