Effects of the Angiopoietins on Tie 2 Downregulation

描述（由申请人提供）：Tie 2是一种内皮特异性受体酪氨酸激酶（RTK），是正常胚胎血管发育和病理性血管生成所必需的。Tie 2在RTK中是独特的，因为其配体血管生成素具有明显相反的作用。血管生成素-1（Angl）促进血管成熟和稳定，部分是通过阻止内皮粘附分子表达和血管通透性的增加。相反，Ang 2似乎抑制Angl的血管稳定作用，并且在某些情况下，Ang 2是血管生成所需的。尽管已经在内皮细胞中鉴定了许多Tie 2介导的信号传导途径和细胞应答，但这些尚不能充分解释Angl和Ang 2之间的功能差异。然而，迄今为止，还没有研究调查Tie 2下调的机制。大多数RTK以泛素依赖性方式下调，并且在大多数情况下，该过程由E3泛素连接酶c-Cbl介导，c-Cbl可以直接与活化的RTK缔合，从而影响受体泛素化、内化和随后的降解或再循环。内化和降解是负调节受体功能的重要机制。此外，已经提出，受体内化是激活一些正信号通路所必需的。我们实验室的初步研究表明，Tie 2是泛素化的，并与c-Cbl在内皮细胞配体激活后。基于这些发现，我们假设Angl和Ang 2差异调节Tie 2泛素化、内化和下调，并且这些差异部分地负责血管生成素的不同功能效应。为了验证这一假设，本提案的具体目的是：1）确定Tie 2泛素化的程度及其对Tie 2半衰期和亚细胞定位的影响; 2）确定c-Cbl在Tie 2下调中的作用;和3）确定泛素化是否导致Tie 2信号传导和功能的调节。实现这些特定目标将提供对Angl和Ang 2之间的功能差异的见解。此外，了解这些蛋白质在血管生成过程中的差异作用将对我们治疗各种疾病的能力具有重要意义，包括肿瘤血管生成，糖尿病视网膜病变和缺血性心血管疾病。

DESCRIPTION (provided by applicant): Tie2 is an endothelium-specific receptor tyrosine kinase (RTK) that is required for both normal embryonic vascular development and pathological angiogenesis. Tie2 is unique among RTKs in that its ligands, the Angiopoietins, have apparently opposite actions. Angiopoietin-1 (Angl) promotes vascular maturation and stabilization, in part, by preventing increases in endothelial adhesion molecule expression and vascular permeability. In contrast, Ang2 appears to inhibit Angl's vascular stabilizing effects, and in some cases Ang2 is required for angiogenesis. Although a number of Tie2-mediated signaling pathways and cellular responses have been identified in endothelial cells, these have not sufficiently explained the functional differences between Angl and Ang2. To date, however, no studies have investigated the mechanisms by which Tie2 is downregulated. Most RTKs are downregulated in a ubiquitin-dependent fashion, and in most cases this process is mediated by the E3 ubiquitin ligase c-Cbl, c-Cbl can associate directly with activated RTKs, whereupon it effects receptor ubiquitination, internalization, and subsequent degradation or recycling. Internalization and degradation are important mechanisms by which receptor function is negatively regulated. Moreover, it has been proposed that receptor internalization is required for activation of some positive signaling pathways. Preliminary studies in our lab demonstrate that Tie2 is ubiquitinated and associates with c-Cbl in endothelial cells following ligand activation. Based on these findings, we hypothesize that Angl and Ang2 differentially regulate Tie2 ubiquitination, internalization, and downregulation, and that these differences are in part responsible for the distinct functional effects of the Angiopoietins. To test this hypothesis, the Specific Aims of this proposal are to: 1) Determine the extent of Tie2 ubiquitination and its effects on Tie2 half-life and subcellular localization; 2) Determine the role of c-Cbl in Tie2 downregulation; and 3) Determine whether ubiquitination results in modulation of Tie2 signaling and function. Accomplishing these Specific Aims will provide insights into the functional differences between Angl and Ang2. Furthermore, understanding these proteins' differential effects during angiogenesis will have important implications for our ability to treat a variety of conditions, including tumor angiogenesis, diabetic retinopathy, and ischemic cardiovascular diseases.