Role of NgBR

描述(申请人提供)：内皮细胞(EC)激活和定向迁移是血管生成的第一步，血管生成涉及肿瘤生长和糖尿病视网膜病变的发病机制。我们以前发现NGBR是一种新的受体，在Nogo-B刺激的EC体外迁移中是必不可少的。最近，我们证明了NGBR在斑马鱼体内血管生成中是必不可少的。在初步结果中，我们提出了新的数据表明，NGBR与法尼化RAS(F-RAS)结合，在这样做的过程中，激活RAS-PI3K-Akt依赖的途径来诱导EC迁移。虽然RAS是一种癌基因，在癌症中已经得到了很好的研究，但它在内皮细胞生物学中的作用仍然相对不清楚。事实上，血管内皮细胞生长因子能显著增加EC中RAS的活性。结构性激活的RAS可以刺激Akt的磷酸化，Akt是EC迁移的关键角色。我们的初步数据显示，RAS基因敲除可使血管内皮生长因子诱导的内皮细胞迁移减少约50%，并完全取消Nogo-B诱导的内皮细胞迁移。提示RAS在调节EC迁移中的作用值得进一步研究。此外，考虑到NGBR基因敲除可使血管内皮生长因子诱导的EC迁移减少约60%，并完全消除Nogo-B诱导的EC迁移，NGBR介导的Ras-PI3K-Akt信号可能是比先前认为的更重要的信号通路。在本申请中，我们将确定NGBR与F-RAS相互作用以启动RAS依赖的EC迁移的机制。我们的应用假设是：NGBR通过其胞浆区域的特定位置招募F-RAS，这是Nogo-B和VEGF刺激的内皮细胞迁移的关键步骤。为了验证这一假设，我们将：1.确定NGBR细胞质结构域中哪些区域是结合F-RAS所必需的。此外，我们将利用NGBR功能突变的缺失来确定EC迁移和血管生成是否以及在多大程度上依赖于NGBR介导的RAS易位；2.确定NGBR调节Nogo-B/VEGF刺激的RAS激活的机制；3.确定NGBR在与VEGFR2协调诱导依赖RAS的EC迁移和血管生成中的作用。这一应用具有创新性，因为它首次确定了NGBR作为一种新的支架蛋白在调节RAS转位和激活RAS-PI3K-AKT途径中的作用。这项应用的成功发现将对血管生物学产生“巨大”影响，因为它将发现或揭示一条刺激EC迁移和血管生成的全新途径。虽然RAS是一个在癌症中被广泛研究的癌基因，但是RAS转移到质膜影响EC功能的确切机制尚不清楚。我们提出的研究将首次确定NGBR介导RAS激活影响EC迁移的机制。这些研究的发现将使我们能够开发新的抗血管生成疗法，以预防糖尿病视网膜病变和肿瘤血管生成。

DESCRIPTION (provided by applicant): Endothelial cell (EC) activation and directional migration is an initial step in angiogenesis that is involved in the pathogenesis of tumor growth and diabetic retinopathy. We previously identified NgBR as a new receptor that is essential for Nogo-B-stimulated EC migration in vitro. Recently, we demonstrated that NgBR is essential for in vivo angiogenesis in zebrafish. In Preliminary Results, we present new data showing that NgBR binds farnesylated Ras (F-Ras) and in so doing, activate Ras-PI3K-Akt dependent pathways to induce EC migration. Although Ras, an oncogene, is well studied in cancer, its role in endothelial cell biology remains relatively unclear. In fact, VEGF can remarkably increase Ras activation in EC. Constitutively activated Ras can stimulate the phosphorylation of Akt, a key player for EC migration. Our preliminary data show that Ras knockdown decreases VEGF-induced EC migration by about 50% and completely abolishes Nogo-B-induced EC migration. It suggests that the role of Ras in regulating EC migration deserves further investigation. Further, considering the fact that NgBR knockdown decreases VEGF-induced EC migration by about 60% and completely abolishes Nogo-B-induced EC migration, NgBR-mediated Ras-PI3K-Akt signaling likely represents a much more prominent pathway than previously considered. In this application, we will determine the mechanisms by which NgBR interacts with F-Ras to initiate Ras-dependent EC migration. The hypothesis of our application is: "NgBR recruits F-Ras via specific sites in its cytoplasmic domain, which is an essential step for Nogo-B- and VEGF-stimulated endothelial cell migration." To test this hypothesis, we will: 1. identify which regions in the cytoplasmic domain of NgBR are required for binding F-Ras. Furthermore, we will use NgBR loss of function mutants to determine if and the extent to which EC migration and angiogenesis are dependent on NgBR-mediated Ras translocation; 2. determine the mechanism by which NgBR regulates Nogo-B/VEGF- stimulated Ras activation; 3. determine the roles of NgBR in coordinating with VEGFR2 to induce Ras- dependent EC migration and angiogenesis. This application is innovative because it is the first to define the role of NgBR as a novel scaffold protein in modulating Ras translocation and in activating Ras-PI3K-akt pathway. Successful findings from this application have a "huge" impact on vascular biology because it will have uncovered or revealed an entirely new pathway for stimulating EC migration and angiogenesis. Although Ras is a well-studied oncogene in cancer, the exact mechanisms by which Ras translocates to plasma membrane to influence EC function are unknown. Our proposed studies will be the first to define the mechanisms by which NgBR mediates Ras activation to influence EC migration. Findings from these studies will allow us to develop new and novel anti-angiogenic therapies to protect against diabetic retinopathy and tumor angiogenesis.