Receptor tyrosine kinases (RTKs) form multiprotein complexes that initiate and propagate intracellular signals and determine the RTK-specific signalling patterns. Unravelling the full complexity of protein interactions within the RTK-associated complexes is essential for understanding of RTK functions, yet it remains an understudied area of cell biology. We describe a comprehensive approach to characterize RTK interactome. A single tag immunoprecipitation and phosphotyrosine protein isolation followed by mass-spectrometry was used to identify proteins interacting with fibroblast growth factor receptor 3 (FGFR3). A total of 32 experiments were carried out in two different cell types and identified 66 proteins out of which only 20 (30.3%) proteins were already known FGFR interactors. Using co-immunoprecipitations, we validated FGFR3 interaction with adapter protein STAM1, transcriptional regulator SHOX2, translation elongation factor eEF1A1, serine/threonine kinases ICK, MAK and CCRK, and inositol phosphatase SHIP2. We show that unappreciated signalling mediators exist for well-studied RTKs, such as FGFR3, and may be identified via proteomic approaches described here. These approaches are easily adaptable to other RTKs, enabling identification of novel signalling mediators for majority of the known human RTKs.

受体酪氨酸激酶 (RTK) 形成多蛋白复合物，可启动和传播细胞内信号并确定 RTK 特异性信号模式。解开 RTK 相关复合物中蛋白质相互作用的全部复杂性对于理解 RTK 功能至关重要，但它仍然是细胞生物学的一个研究不足的领域。我们描述了一种表征 RTK 交互组的综合方法。使用单标签免疫沉淀和磷酸酪氨酸蛋白分离，然后进行质谱法来鉴定与成纤维细胞生长因子受体 3 (FGFR3) 相互作用的蛋白质。在两种不同的细胞类型中进行了总共 32 项实验，并鉴定了 66 种蛋白质，其中只有 20 (30.3%) 种蛋白质是已知的 FGFR 相互作用物。使用免疫共沉淀，我们验证了 FGFR3 与衔接蛋白 STAM1、转录调节因子 SHOX2、翻译延伸因子 eEF1A1、丝氨酸/苏氨酸激酶 ICK、MAK 和 CCRK 以及肌醇磷酸酶 SHIP2 的相互作用。我们表明，对于经过充分研究的 RTK，如 FGFR3，存在未被重视的信号介质，并且可以通过此处描述的蛋白质组学方法进行识别。这些方法很容易适用于其他 RTK，从而能够识别大多数已知人类 RTK 的新型信号介质。