Regulation of Tie2 activation by homo- and hetero-oligomerization

摘要 受体酪氨酸激酶（RTK）的Tie家族参与血管稳态和血管内分泌。 血管生成受体及其血管生成素（Ang）配体都是药理学研究的有吸引力的靶点。 干预癌症、炎症和其他疾病状态。阻碍治疗发展的因素 目前对Tie受体激活机制的理解不完全。而 众所周知，RTK如EGFR、Kit和FGFR受生长因子诱导的二聚化调节， Tie受体，迄今为止的研究未能揭示配体诱导的受体活化的机制。 活化的寡聚Ang配体都与Tie 2结合，信号传导结果是环境依赖性的， 由包括孤儿家族成员Tie 1的共受体调节。我们已经证明，细胞外 Tie 2的ECR区形成配体非依赖性二聚体，其由其近膜纤连蛋白介导 III型（FNIII）结构域，并且是细胞中Tie 2活化所必需的。已知寡聚Ang配体 与Tie 2的膜远端结构域结合。因此，Tie受体与大多数RTK的不同之处在于， 寡聚配体调节已经寡聚的受体。信号是否通过变构产生 受体二聚体的变化或受体交联或簇集的促进作用仍不清楚（但这是一种新的方法）。 我们的建议）。对于在其膜近端区域具有免疫球蛋白结构域的几种RTK， (KIT、PDGFR和Fms/CSF 1-R），膜近端区域之间的同型相互作用是重要的 用于受体二聚化和活化，以及通过抗体结合或突变破坏这些相互作用 阻断受体激活。我们提出，涉及FNIII近膜结构域的相互作用， Tie受体也可以在治疗上被靶向。此外，我们认为Tie受体可能作为一种 40%的人RTK具有膜近端FNIII结构域的原型。 为了深入了解寡聚体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结合的后果， Tie 2 ECR二聚体的定义的低聚Ang配体。我们还将确定如何配体结合， Tie 1和RPTPβ的存在影响信号传导，两者都直接与Tie 2相互作用。我们 总体目标是建立一个详细的分子理解如何相互作用介导的ECR的Tie 2 调节受体活性，并利用这些信息开发Tie受体的抗体调节剂 activation.我们的具体目标提出了三个问题： 1.血管生成素配体如何调节Tie 2？ 2. Tie 1和RPTPβ如何调节Ang配体对Tie 2活性的影响？ 3.特异性抗体能选择性阻断Tie 2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?