Testini Chiara
Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.
Methods Mol Biol. 2022;2475:97-111. doi: 10.1007/978-1-0716-2217-9_6.
Vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling pathways are tightly regulated multistep chain reactions that involve a wide range of molecular interactions and enzymatic activities. The first signal induced by VEGF binding to VEGFR2, is the activation of the receptor tyrosine kinase and autophosphorylation of intracellular tyrosine residues of the receptor. In endothelial cells, five tyrosine residues in the VEGFR2 intracellular domain are essential in signal transmission and in the respective regulation of cellular processes. Because of their number and their localization on the receptor, it is challenging to locate the proteins with which these tyrosine residues interact that result in further downstream signaling cascades. In this chapter, we describe a method to precipitate phosphotyrosine binding proteins using phosphotyrosine-containing synthetic peptides immobilized to magnetic beads. The identity of the precipitated proteins is determined by mass spectrometry and the findings validated by Western blot. Using this method, we identified and verified two proteins, growth factor receptor binding-2 (GRB2) and phosphoinositide 3'-kinase (PI3Kp85), binding to the tyrosine 1214 of VEGFR2. Thereby, we can predict the signaling pathways downstream of pY1214.
血管内皮生长因子（VEGF）/血管内皮生长因子受体2（VEGFR2）信号通路是受到严格调控的多步链式反应，涉及广泛的分子相互作用和酶活性。VEGF与VEGFR2结合诱导的第一个信号是受体酪氨酸激酶的激活以及受体细胞内酪氨酸残基的自磷酸化。在内皮细胞中，VEGFR2细胞内结构域中的五个酪氨酸残基在信号传导以及细胞过程的各自调节中至关重要。由于它们的数量以及在受体上的定位，定位与这些酪氨酸残基相互作用从而导致进一步下游信号级联反应的蛋白质具有挑战性。在本章中，我们描述了一种使用固定在磁珠上的含磷酸酪氨酸的合成肽沉淀磷酸酪氨酸结合蛋白的方法。通过质谱确定沉淀蛋白的身份，并通过蛋白质印迹法验证结果。使用这种方法，我们鉴定并验证了两种与VEGFR2的酪氨酸1214结合的蛋白质，即生长因子受体结合蛋白2（GRB2）和磷酸肌醇3'-激酶（PI3Kp85）。由此，我们可以预测pY1214下游的信号通路。
