Site-Specific Phosphorylation of VEGFR2 Is Mediated by Receptor Trafficking: Insights from a Computational Model

Matrix-binding isoforms and non-matrix-binding isoforms of vascular endothelial growth factor (VEGF) are both capable of stimulating vascular remodeling, but the resulting blood vessel networks are structurally and functionally different. Here, we develop and validate a computational model of the binding of soluble and immobilized ligands to VEGF receptor 2 (VEGFR2), the endosomal trafficking of VEGFR2, and site-specific VEGFR2 tyrosine phosphorylation to study differences in induced signaling between these VEGF isoforms. In capturing essential features of VEGFR2 signaling and trafficking, our model suggests that VEGFR2 trafficking parameters are largely consistent across multiple endothelial cell lines. Simulations demonstrate distinct localization of VEGFR2 phosphorylated on Y1175 and Y1214. This is the first model to clearly show that differences in site-specific VEGFR2 activation when stimulated with immobilized VEGF compared to soluble VEGF can be accounted for by altered trafficking of VEGFR2 without an intrinsic difference in receptor activation. The model predicts that Neuropilin-1 can induce differences in the surface-to-internal distribution of VEGFR2. Simulations also show that ligated VEGFR2 and phosphorylated VEGFR2 levels diverge over time following stimulation. Using this model, we identify multiple key levers that alter how VEGF binding to VEGFR2 results in different coordinated patterns of multiple downstream signaling pathways. Specifically, simulations predict that VEGF immobilization, interactions with Neuropilin-1, perturbations of VEGFR2 trafficking, and changes in expression or activity of phosphatases acting on VEGFR2 all affect the magnitude, duration, and relative strength of VEGFR2 phosphorylation on tyrosines 1175 and 1214, and they do so predictably within our single consistent model framework.血管内皮生长因子（VEGF）的基质结合型异构体与非基质结合型异构体均能够刺激血管重塑，但二者所形成的血管网络在结构与功能上存在差异。本研究开发并验证了一套计算模型，用于解析可溶性配体与固定化配体结合血管内皮生长因子受体2（VEGFR2）、VEGFR2的内体运输，以及位点特异性VEGFR2酪氨酸磷酸化的过程，以探究这两类VEGF异构体所诱导的信号通路差异。该模型在捕捉VEGFR2信号转导与内体运输的核心特征后，提示不同内皮细胞系间的VEGFR2运输参数整体上保持一致。模拟实验结果显示，Y1175与Y1214位点磷酸化的VEGFR2呈现出截然不同的定位模式。本研究首次明确证实：与可溶性VEGF刺激相比，固定化VEGF刺激下的位点特异性VEGFR2激活差异，可通过VEGFR2运输模式的改变来解释，而非受体激活本身存在内在差异。模型预测神经纤毛蛋白-1（Neuropilin-1）能够改变VEGFR2在细胞表面与细胞内的分布比例。模拟实验还显示，结合配体的VEGFR2与磷酸化VEGFR2的水平在刺激后随时间推移出现分化。借助该模型，我们识别出多个关键调控节点，这些节点能够改变VEGF结合VEGFR2后所引发的多条下游信号通路的协同调控模式。具体而言，模拟实验预测：VEGF的固定化、与神经纤毛蛋白-1的相互作用、VEGFR2运输的扰动，以及作用于VEGFR2的磷酸酶的表达或活性变化，均可在本研究的统一模型框架内，可预测地影响VEGFR2在酪氨酸1175与1214位点磷酸化的强度、持续时间与相对强弱。