Impaired Glycosylation of Vascular Endothelial Growth Factor Receptor 2 in Tumor Angiogenesis

 描述(申请人提供)：血管生成(已建立的血管系统形成新的血管)对肿瘤的生长和转移是必不可少的。血管内皮生长因子受体2(VEGFR-2)是一种受体酪氨酸激酶(RTK)，在正常和病理条件下参与血管生成信号转导。肿瘤经常表达高水平的低氧诱导的血管内皮生长因子配体，以VEGFR-2为靶点，支持肿瘤的血管形成和生长。虽然包括贝伐单抗、索拉非尼和舒尼替尼在内的VEGFR-2抑制剂被用于癌症治疗，但这些药物的耐药性的发展对癌症的有效管理构成了障碍。最近，高尔基体的转运受损和配体非依赖性VEGFR-2的激活和信号转导已被证明。有趣的是，N-糖基化的中断影响VEGFR-2的运输，并可能在这一过程中发挥作用。然而，VEGFR-2糖基化对转运和受体激活的影响在很大程度上尚不清楚。我们假设，病理情况下改变的蛋白糖基化通过影响VEGFR-2的运输而促进VEGFR-2在高尔基体的持续定位，并导致配体无关的激活和高尔基体的信号传递。因此，我们建议研究VEGFR-2糖基化对VEGFR-2转运和激活的影响。我们计划通过(A)确定VEGFR-2膜和亚细胞部分的糖基化状态并检测它们对受体激活的影响，(B)筛选细胞膜和高尔基组分中依赖糖基化的VEGFR-2蛋白相互作用，以及(C)研究VEGFR-2糖基化在蛋白质循环、周转和降解中的作用，来探索这一假说。这项建议的一个主要目标是探索糖基化在VEGFR-2运输、信号转导和异常肿瘤血管生成中的作用。我将分析VEGFR-2糖肽和释放的N-糖链，并使用UV激活的交联糖通过质谱仪筛选VEGFR-2糖基化依赖的蛋白质相互作用。这项研究的结果将阐明糖基化在VEGFR-2转运和信号传递中的作用，包括：表征高尔基体和胞外部分之间糖基化的差异，评估与糖基化变化相关的受体激活和信号的变化，识别调节VEGFR-2转运的糖基化特异性VEGFR-2相互作用伙伴，以及确定受体糖基化对蛋白质循环和降解的影响。

 DESCRIPTION (provided by applicant): Angiogenesis (the development of new blood vessels from established vasculature) is essential for tumor growth and metastasis. Vascular endothelial growth factor receptor 2 (VEGFR-2) is a receptor tyrosine kinase (RTK) involved in angiogenic signaling in normal and pathological conditions. Tumors frequently express high levels of hypoxia-induced VEGF ligands that target VEGFR-2 to support tumor vascularization and growth. While VEGFR-2 inhibitors including bevacizumab, sorafenib and sunitinib are used for cancer treatment, the development of resistance to these agents poses an obstacle to effective management of cancer. Recently, impaired trafficking and ligand-independent VEGFR-2 activation and signaling in the Golgi have been demonstrated. Intriguingly, disruption of N-glycosylation affects VEGFR-2 trafficking and may play a role in this process. However, the impact of VEGFR-2 glycosylation on trafficking and receptor activation is largely unexplored. We hypothesize that altered protein glycosylation in pathological circumstances contributes to the sustained localization of VEGFR-2 to the Golgi apparatus by affecting trafficking of VEGFR-2 and leads to ligand-independent activation and signaling from the Golgi. Therefore, we propose to study the impact of VEGFR-2 glycosylation on VEGFR-2 trafficking and activation. We plan to explore this hypothesis by (A) establishing the glycosylation status of membrane and subcellular fractions of VEGFR-2 and examining their impact on receptor activation, (B) screening for glycosylation-dependent VEGFR-2 protein interactions in cell membrane and Golgi fractions, and (C) investigating the role of VEGFR-2 glycosylation in protein recycling, turnover and degradation. A major goal of this proposal is to explore the role of glycosylation in VEGFR-2 trafficking, signaling and aberrant tumor angiogenesis. I will analyze VEGFR-2 glycopeptides and released N-glycans and screen for VEGFR-2 glycosylation-dependent protein interactions using UV-activated crosslinking sugars via mass spectrometry. Results from this study will illuminate the role of glycosylation in VEGFR-2 trafficking and signaling, including: characterization of differences in glycosylation between Golgi and extracellular fractions, assessment of changes in receptor activation and signaling associated with changes in glycosylation, identification of glycosylation-specific VEGFR-2 interacting partners that modulate VEGFR-2 trafficking, and determination of the impact of receptor glycosylation on protein recycling and degradation.