Abstract：
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.
摘要：
血管内皮生长因子（VEGF）/VEGF受体2（VEGFR2）信号通路是紧密调节的多步链反应，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和受体细胞内酪氨酸残基的自磷酸化。在内皮细胞中，VEGFR2胞内结构域中的五个酪氨酸残基在信号传递和细胞过程的各自调节中是必需的。由于它们的数量和在受体上的定位，定位这些酪氨酸残基与之相互作用导致进一步下游信号级联的蛋白质是具有挑战性的。在这一章中,我们描述了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。通过质谱法确定沉淀的蛋白质的身份,并通过Western印迹验证发现。使用此方法，我们鉴定并验证了两种蛋白质，生长因子受体结合-2（GRB2）和磷酸肌醇3'-激酶（PI3Kp85），结合VEGFR2的酪氨酸1214。因此，我们可以预测pY1214下游的信号通路。
