Control of Hedgehog Signal Transduction by Neuropilin

Neuropilin 对 Hedgehog 信号转导的控制

• 批准号: 8296571
• 负责人: Matthew P. Scott
• 金额: $ 30.05万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-05 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8296571
• 关键词: Affect; Alleles; Axon; Binding; Blood Island; Cell Adhesion; Cell Culture Techniques; Cell Fate Control; Cell Proliferation; Cell-Cell Adhesion; Cells; Cerebellum; Childhood; Cilia; Congenital Abnormality; Cultured Cells; Cytoplasmic Granules; Data; Defect; Development; Developmental Process; Embryo; Embryonic Development; Engineering; Erinaceidae; Event; Excision; Fibroblasts; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Epistasis; Goals; Growth; Human; Immunoprecipitation; Integral Membrane Protein; Label; Lead; Learning; Life; Ligands; Limb Bud; Malignant Neoplasms; Malignant neoplasm of brain; Mammals; Maps; Masks; Mass Spectrum Analysis; Mediating; Messenger RNA; Methods; Mitogens; Modeling; Molecular; Molecular Analysis; Monitor; Morphogenesis; Movement; Mus; Mutant Strains Mice; Mutation; Natural regeneration; Neural Tube Development; Neurons; Neuropilin-1; Neuropilins; Organ; Pathway interactions; Pattern; Phenotype; Physiologic pulse; Point Mutation; Process; Production; Property; Proteins; RNA Interference; Regulation; Role; S-nitro-N-acetylpenicillamine; Screening procedure; Semaphorin-3; Semaphorins; Signal Pathway; Signal Transduction; Structure; Subfamily lentivirinae; System; Tamoxifen; Testing; Time; Tissues; VEGF165; Vascular Endothelial Growth Factor Receptor; Vascular Endothelial Growth Factors; Virus; Work; Yolk Sac; angiogenesis; cell motility; computerized data processing; design; hedgehog signal transduction; high throughput screening; human SMO protein; in vivo; loss of function; medulloblastoma; mutant; novel; plexin; precursor cell; receptor; recombinase; research study; response; restraint; smoothened signaling pathway; tumorigenesis; vasculogenesis

DESCRIPTION (provided by applicant): Hedgehog (Hh) signaling is employed in controlling cell fates in most developing tissues and organs, as well as during many regeneration events. Defects in Hh signaling lead to birth defects and cancer. Many mechanistic mysteries remain regarding how an Hh signal is transduced. Using high-throughput RNAi screening, we identified Neuropilins (Nrp) 1 and 2 as novel, specific regulators of vertebrate Hh signaling. Nrps are single-pass transmembrane proteins implicated in the reception of a diverse set of secreted ligands, including Semaphorins and VEGF165 and in cell adhesion and cell migration. In fibroblasts the inhibition of Hh signal transduction resulting from blocking Nrps is as strong as the effect of blocking cilia formation or blocking Smoothened function. Conversely, over-production of either Nrp sensitizes cells to Hh signals. New components of the Hh pathway are uncovered infrequently; the Nrps were probably missed due to their partial redundancy. Our discovery of two proteins whose functions are required by this important morphogenic pathway has the potential to bring fundamental changes to current models of Hh signaling and to enlarge the understanding of Nrp functions in other signaling pathways. Aim 1. Determine the mechanism by which Nrps regulate Hh signal transduction. Nrps could influence Hh signal transduction by directly associating with known Hh pathway components, or mediating other signals that converge with Hh transduction, or by altering cell properties or processes that are required for Hh transduction. We will investigate each of these possibilities by determining which steps in Hh signaling are affected, whether cell adhesion or migration changes are involved in the effect of Nrps upon Hh signaling, and what proteins interact directly with Nrps. Aim 2. Determine which domains of Nrp are needed to support Hh signaling, and whether known Nrp co-receptors, ligands, or effector molecules are capable of Hh pathway cross- regulation. We will investigate which domains contribute to Hh signal transduction in two ways: engineered domain deletions, and a high-throughput screen for point mutations that interfere with Nrp support of Hh signal transduction. Nrps transduce VEGF and Semaphorin signals, acting as co-receptors for VEGF receptors and Plexins, respectively. We will test whether VEGF, VEGF receptor, Plexin receptors, or Semaphorins are involved in the effect of Nrps upon Hh signal transduction. Aim 3. Investigate how Nrps influence Hh- dependent development and tumorigenesis. Using mice that carry mutations in both Nrp genes, we will control temporal and tissue-specific removal of Nrp functions to test their involvement in several Hh-dependent developmental processes in vivo. Using newly created lentiviruses, which encode specific inhibiting RNAs that block the nrp genes, we will infect primary cultures of Hh-responsive cells and monitor effects on Hh target gene expression.

描述（由申请人提供）：Hedgehog（Hh）信号传导用于控制大多数发育中的组织和器官以及许多再生事件中的细胞命运。Hh信号缺陷导致出生缺陷和癌症。关于Hh信号是如何转导的，仍然存在许多机制上的谜团。使用高通量RNAi筛选，我们确定神经纤毛蛋白（Nrp）1和2作为新的，特定的调节脊椎动物Hh信号。Nrps是单次跨膜蛋白，涉及多种分泌配体的接收，包括脑信号蛋白和VEGF 165，以及细胞粘附和细胞迁移。在成纤维细胞中，由阻断Nrps引起的Hh信号转导的抑制与阻断纤毛形成或阻断Smoothened功能的作用一样强。相反，过量产生任一Nrp使细胞对Hh信号敏感。Hh途径的新组分很少被发现; Nrps可能由于其部分冗余而被遗漏。我们发现的两种蛋白质，其功能所需的这一重要的形态发生途径有可能带来根本性的变化，目前的模型Hh信号，并扩大了解NRP功能在其他信号通路。目标1.确定Nrps调节Hh信号转导的机制。Nrps可以通过直接与已知的Hh通路组分结合，或介导与Hh转导会聚的其他信号，或通过改变Hh转导所需的细胞特性或过程来影响Hh信号转导。我们将通过确定Hh信号传导中的哪些步骤受到影响，细胞粘附或迁移变化是否参与Nrps对Hh信号传导的影响，以及哪些蛋白质直接与Nrps相互作用来研究这些可能性。目标二。确定需要Nrp的哪些结构域来支持Hh信号传导，以及已知的Nrp共受体、配体或效应分子是否能够进行Hh途径交叉调节。我们将研究哪些结构域有助于Hh信号转导的两种方式：工程结构域缺失，和高通量筛选干扰Nrp支持Hh信号转导的点突变。Nrps抑制VEGF和脑信号蛋白信号，分别作为VEGF受体和丛状蛋白的共受体。我们将测试VEGF、VEGF受体、丛状蛋白受体或脑信号蛋白是否参与Nrps对Hh信号转导的影响。目标3。研究Nrps如何影响Hh依赖的发育和肿瘤发生。使用携带两个Nrp基因突变的小鼠，我们将控制时间和组织特异性去除Nrp功能，以测试它们在体内参与几个Hh依赖的发育过程。使用新创建的慢病毒，它编码特异性抑制RNA，阻止nrp基因，我们将感染Hh反应细胞的原代培养物，并监测对Hh靶基因表达的影响。