Control of Hedgehog Signal Transduction by Neuropilin

Neuropilin 对 Hedgehog 信号转导的控制

基本信息

批准号：
8620669
负责人：
TOBIAS MEYER
金额：
$ 30.21万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
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/8620669
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 Malignant Brain Tumor 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 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 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 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 screening signal processing smoothened signaling pathway tumorigenesis vasculogenesis

项目摘要

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）：刺猬（Hh）信号传导用于控制大多数发育组织和器官以及许多再生事件中的细胞命运。 Hh 信号传导缺陷会导致出生缺陷和癌症。关于 Hh 信号如何转导，仍有许多机制上的谜团。通过高通量 RNAi 筛选，我们确定 Neuropilins (Nrp) 1 和 2 是脊椎动物 Hh 信号传导的新型特异性调节剂。 Nrps 是单次跨膜蛋白，参与多种分泌配体（包括信号蛋白和 VEGF165）的接收以及细胞粘附和细胞迁移。在成纤维细胞中，阻断 Nrps 导致的 Hh 信号转导抑制与阻断纤毛形成或阻断 Smoothened 功能的效果一样强。相反，任一 Nrp 的过量产生都会使细胞对 Hh 信号敏感。 Hh 通路的新成分很少被发现； Nrp 可能由于部分冗余而被遗漏。我们发现了两种蛋白质，其功能是这一重要的形态发生途径所必需的，有可能为当前的 Hh 信号传导模型带来根本性的变化，并扩大对其他信号传导途径中 Nrp 功能的理解。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 通路成分关联，或介导与 Hh 转导汇聚的其他信号，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涉及 Nrps 对 Hh 信号传导的影响以及哪些蛋白质直接与 Nrps 相互作用来研究每种可能性。目标 2. 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。我们将通过两种方式研究哪些结构域对 Hh 信号转导有贡献：工程结构域删除，以及干扰 Hh 信号转导的 Nrp 支持的点突变的高通量筛选。 Nrps 转导 VEGF 和 Semaphorin 信号，分别充当 VEGF 受体和 Plexins 的共受体。我们将测试 VEGF、VEGF 受体、Plexin 受体或 Semaphorins 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 依赖性发育和肿瘤发生。使用携带两个 Nrp 基因突变的小鼠，我们将控制 Nrp 功能的时间和组织特异性去除，以测试它们在体内几个 Hh 依赖性发育过程中的参与。使用新创建的慢病毒（编码可阻断 nrp 基因的特异性抑制 RNA），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。

项目成果

期刊论文数量（2）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Neuropilin-2 contributes to tumorigenicity in a mouse model of Hedgehog pathway medulloblastoma.

Neuropilin-2 有助于 Hedgehog 通路髓母细胞瘤小鼠模型的致瘤性。

DOI：
发表时间：
2013-11
期刊：
影响因子：
3.9
作者：
Hayden Gephart, Melanie G;Su, YouRong Sophie;Bandara, Samuel;Tsai, Feng;Hong, Jennifer;Conley, Nicholas;Rayburn, Helen;Milenkovic, Ljiljana;Meyer, Tobias;Scott, Matthew P
通讯作者：
Scott, Matthew P

A rapid and simple method for DNA engineering using cycled ligation assembly.

一种使用循环连接组装进行 DNA 工程的快速而简单的方法。