Regulation of Tie2 activation by homo- and hetero-oligomerization

摘要 受体酪氨酸激酶(Rtk)家族参与血管内稳态和血管内皮细胞生长。 血管生成。受体及其血管生成素(Ang)配体都是诱人的药理靶点。 对癌症、炎症和其他疾病状态的干预。发展治疗性技术的障碍 目前对Tie受体激活机制的认识还不完全。鉴于 众所周知，RTK如EGFR、Kit和FGFR受生长因子诱导的二聚化调控，如 到目前为止，对Tie受体的研究还没有揭示配体诱导受体激活的机制。 激活的寡聚体Ang配体都与Tie2结合，其信号结果取决于上下文和 受包括孤儿家族成员Tie1在内的辅助受体的调节。我们已经证明了细胞外的 Tie2区(ECR)形成由其膜-近端纤维连接蛋白(FN)介导的非配体依赖的二聚体 III型(FNIII)结构域，是细胞内Tie2激活所必需的。已知的低聚Ang配体 结合到Tie2的膜远端结构域。因此，Tie受体与大多数RTK的不同之处在于 寡聚体配体调节已经存在的寡聚体受体。信号是否通过变构产生 受体二聚体的变化或促进受体交联或聚集仍不清楚(但是一种 我们提案的重点)。对于膜近端带有免疫球蛋白结构域的几种RTK (KIT、PDGFR和FMS/CSF1-R)，膜近端区域之间的同型相互作用是重要的 受体二聚化和激活，以及通过抗体结合或突变破坏这些相互作用 阻断受体的激活。我们认为，涉及膜-近端FNIII结构域的相互作用 Tie受体也可以作为治疗的靶点。此外，我们认为Tie受体可能作为一种 40%具有膜近端FNIII结构域的人类RTK的原型。 为了深入了解寡聚Ang配体如何结合并激活二聚体RTK，我们将使用 结合结构、生物化学和细胞方法来解决绑定的后果 已定义的寡聚Ang配体与Tie2 ECR二聚体结合。我们还将确定配体结合和 信号转导受到Tie1和Rpppβ的影响，两者都直接与Tie2相互作用。我们的 总体目标是建立一个详细的分子理解如何由Tie2的ECR介导的相互作用 调节受体活性，并利用这一信息开发TiE受体的抗体调节剂 激活。我们的具体目标提出了三个问题： 1.血管生成素配体是如何调节Tie2的？ 2.Tie1和Rpppβ如何调节Ang配体对Tie2活性的影响？ 3.特异性抗体能选择性地阻断Tie2相互作用的亚群吗？

Abstract The Tie family of receptor tyrosine kinases (RTKs) are involved in both vascular homeostasis and in angiogenesis. Both the receptors and their angiopoietin (Ang) ligands are attractive targets for pharmacologic intervention in cancer, inflammation and other disease states. An impediment to development of therapeutic agents is the current incomplete understanding of the mechanisms of activation of Tie receptors. Whereas RTKs such as EGFR, Kit and FGFR are well known to be regulated by growth factor-induced dimerization, for the Tie receptors, studies to date have failed to reveal the mechanism of ligand-induced receptor activation. The activating, oligomeric Ang ligands all bind to Tie2, with signaling outcomes that are context dependent and are modulated by co-receptors including the orphan family member Tie1. We have shown that the extracellular region (ECR) of Tie2 forms a ligand-independent dimer that is mediated by its membrane-proximal fibronectin type III (FNIII) domains, and is essential for Tie2 activation in cells. The oligomeric Ang ligands are known to bind to the membrane distal domains of Tie2. The Tie receptors thus differ from most RTKs in that an oligomeric ligand regulates an already oligomeric receptor. Whether signaling arises through allosteric changes in a receptor dimer or the promotion of receptor crosslinking or clustering remains unclear (but is a focus of our proposal). For several RTKs with immunoglobulin domains in their membrane-proximal regions (KIT, PDGFR and Fms/CSF1-R), homotypic interactions between membrane-proximal regions are important for receptor dimerization and activation, and disruption of these interactions by antibody binding or mutation blocks receptor activation. We propose that interactions involving the membrane-proximal FNIII domains of Tie receptors can also be targeted therapeutically. Further, we suggest that Tie receptors may serve as a prototype for the 40% of human RTKs that have membrane-proximal FNIII domains. To gain insight into how the oligomeric Ang ligands bind and activated a dimeric RTK, we will use a combination of structural, biochemical and cellular approaches to address the consequences of binding of defined, oligomeric Ang ligands to the Tie2 ECR dimer. We will also determine how ligand binding and signaling are influenced by the presence of Tie1 and the RPTPβ, both of which interact directly with Tie2. Our overall goal is to build a detailed molecular understanding of how the interactions mediated by the ECR of Tie2 regulate receptor activity, and to exploit this information to develop antibody modulators of Tie receptor activation. Our specific aims ask three questions: 1. How do angiopoietin ligands regulate Tie2? 2. How do Tie1 and RPTPβ modulate the effects of Ang ligands on Tie2 activity? 3. Can specific antibodies selectively block subsets of Tie2 interactions?