CHARACTERIZATION OF NEUROPILIN, A NOVEL VEGF RECEPTOR

新型 VEGF 受体 NUROPILIN 的表征

• 批准号: 7329817
• 负责人: MICHAEL KLAGSBRUN
• 金额: $ 68.3万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-01-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7329817
• 关键词: 1-Phosphatidylinositol 3-Kinase; Actins; Adult; Affect; Angiogenesis Inhibitors; Antibodies; Apoptosis; Area; Axon; Binding; Binding Sites; Blood Vessels; Boston; Cells; Cloning; Collaborations; Cultured Cells; Cytoplasmic Protein; Cytoplasmic Tail; Cytoskeleton; DTR gene; Development; EGF gene; Embryo; Ephrin-B2; Extracellular Domain; Family; Future; Gel; Gene Expression; Growth Cones; Guanosine Triphosphate Phosphohydrolases; Heparin; Heparin Binding; Human; Immigration; Incubated; Japan; LIM Domain Kinase 1; Letters; Ligand Binding; Ligands; Luciferases; Lymphatic; Malignant Neoplasms; Membrane; Microfilaments; Monitor; Mus; NRP1 gene; Neuroglia; Neurons; Neuropilins; Numbers; PGF gene; Pathway interactions; Pattern; Phorbol Esters; Phosphoinositide-3-Kinase, Catalytic, Gamma Polypeptide; Phosphoproteins; Phosphorylation Inhibition; Placental Growth Factor; Postdoctoral Fellow; Property; Protein Overexpression; Proteins; Proteomics; Proto-Oncogene Proteins c-akt; RNA Interference; Regulation; Reporter; Rodent Model; Role; Semaphorin-3A; Semaphorins; Serine; Signal Pathway; Signal Transduction; Signaling Molecule; Site-Directed Mutagenesis; Skin; Squamous cell carcinoma; Structure; Threonine; Tissues; Transgenic Mice; Tumor Angiogenesis; Tumor Cell Line; Tyrosine; Universities; VEGF165; Vascular Endothelial Growth Factor B; Vascular Endothelial Growth Factor C; Vascular Endothelial Growth Factors; Vascular Permeabilities; Western Blotting; angiogenesis; axon guidance; cell motility; cell type; cofilin; comparative; deletion analysis; diphtheria toxin receptor; heparin-binding EGF-like growth factor; in vitro Assay; inhibitor/antagonist; keratinocyte; melanoma; migration; mutant; neoplastic cell; novel; plexin; programs; promoter; receptor; research study; response; rho; transcription factor; tumor growth; tumor progression; yeast two hybrid system

1. To investigate NRP ligand binding sites. We have determined that VEGF-A, PlGF and heparin bind to the blb2 extracellular domain (ECD). VEGF-B and plexin A (which transduces the NRP signal in response to semaphorins) also bind to NRP ECD but the binding domains for these two proteins are unknown. Future studies include i) determining the VEGF-B and plexin-A binding sites on NRP using the myc-tagged domain pull-down strategy that previously determined the VEGF-A and PlGF binding sites on NRPs (Mamluk et al., JBC, 2002) and determining whether these ligand binding sites are unique or overlapping by analysis of deletion mutants and site-directed mutagenesis; ii) structural analysis of the heparin that binds NRP blb2 in collaboration with Dr John Gallagher (University of Manchester, UK); iii) crystallizing blb2, with and without VEGF165 and with heparin in collaboration with Dr. Celia Hamson (BBRI, Boston), who previously has crystallized Ephrin B2 (see letter). 2. To analyze regulation of NRP expression. Little is known about how NRP expression is regulated and whether NRPl and NRP2 are regulated differentially. We have isolated the NRPl and NRP2 promoters and have made NRP-luciferase constructs. We have found so far that phorbol ester, EGF and HB-EGF induce NRPl promoter activity in EC, tumor cells and keratinocytes. Future studies include: i) using NRP promoter-reporter constructs to screen inducers and inhibitors of NRP expression, and identifylng the transcription factors involved; ii) analysis of NRPl and NRP2 expression in the transition of vascular to lymphatic EC upon prox-Z overexpression (see letter from Dr. Michael Detmar); iii) analysis of comparative NRP and sNRP expression in cells, tissues and mouse embryos; iv) developmental and adult NRP expression patterns in NRPlLacZ and NRP2LacZ mice generated by Dr. Seiji Takashima, a former post-doc now in Osaka, Japan. 3. To analyze the non-neuronal properties of semaphorins. Semaphorins regulate axon guidance via NRPs by repelling axons and collapsing growth cones. We have found that semaphorins bind non-neuronal cells, e.g., EC, tumor cells and keratinocytes but the consequences of these interactions require further analysts. Future studies include: i) generating large amounts of Sema3A and 3F protein from cell cultures since semaphorins are not commercially available due in part to lack of stability; ii) determining whether Sema3NSema3F affect EC and tumor cell motility, proliferation, survival and apoptosis; iii) analyzing the effects of Sema3AL/3F on the actin cytoskeleton; iv) determining whether Sema 3A/ 3F are angiogenesis inhibitors. 4. To analyze NRP signaling pathways activated in response to VEGF165 and semaphorins. VEGF165 and semaphorins bind to NRPs expressed by a number of non-neuronal cell types. However, very little is known about how NRP signals in these cell types in response to these ligands. As a guide, a number of molecules have been implicated in semaphorin-induced growth cone collapse, including Plexin A1 and A2, the Rho family of GTPases, CRMP and GSK3. Actin filament reorganization is regulated by LIM kinase and its substrate, cofilin. Future studies include incubating EC, tumor cells and keratinocytes expressing NRPl/NRP2 with VEGF165, VEGFIZI (which doesn?t bind NRP as a control), VEGF-B and PlGF (which bind NRPl), VEGF-C (which binds NRP2), Sema3A (which binds NRP1) and Sema3F (which binds NRP2), and monitoring downstream effects by i) direct examination of signaling molecules implicated in growth cone collapse as described above; ii) western. blot with antiphosphotyrosine, serine and threonine antibodies; iii) 2D gel/proteomics to detect changes in general protein and phosphoproteins profiles; iv) transcriptional profiling of these treatments by microarray. It will be determined whether VEGF165 and semaphorins use similar or different pathways. Effects on cytoskeleton will be analyzed in collaboration with Dr. Don Ingber in our department, a well known expert in this area (see letter). Another approach is to inhibit NRP expression with antisense (RNAi, morpholinos) and analyze downstream effects. 5. To analyze the function of naturally occurring soluble NRP (sNRP). sNRP binds VEGF165 and is a VEGF165 antagonist. Future studies include: i) analyzing the effects of sNRPl on VEGF165-induced migration, proliferation and survival and determining the mechanisms involved e.g., inhibition of phosphorylation of specific tyrosine residues and of factors involved in migration such as PI3 kinase and AKT; ii) analyzing effects of sNW1 on angiogenesis and vascular permeability (VPF) activity in transgenic mice overexpressing sNRP in the skin using a K14 promoter, generated in collaboration with Dr. Michael Detmar, MGH (see letter); iii) analyzing mechanisms by which sNRP inhibits tumor progression, for example by inducing tumor cell apoptosis. 6. To investigate the role of NRPs in tumor angiogenesis and cancer. Overexpression of NWli enhances tumor angiogenesis and tumor growth in rodent models. Future experiments include i) overexpression of-NRP2 in human tumor cell lines (e.g., melanoma, squamous carcinoma); ii) overexpression of NRP antagonists, e.g., sNRP and semaphorins in human tumor cell lines. 7. To develop a proteomics program including optimizing phosphoprotein analysis to study signding. The Specific Aims of the original proposal were: 1. To Investigate VEGF165 and Semaphorid Collapsin Interactions with NRPl including: a,) mechanisms of VEGFI~S/~RPI/KDinRte ractions in EC; b) semaphorins as antagonists of EC and tumor cell motility via NRPI; and c) whether VEGF165 and semaphorins are competitive inhibitors of each other for interactions with EC and tumor cells. 2. To Investigate NRPl Expression and Function in Tumor Cells including: a) whether VEGFIbj is a direct stimulator of tumor cell motility acting via NRPl and what pathways may be involved; b) NRPl expression in tumor cells and the contribution of NRPl expression to tumor cell motility and metastatic potential. 3. To Characterize the Structure, Function and Distribution of a Naturally-Occurring Soluble NRPl (sNRP1) Receptor including: a) cloning and purification of a naturally occurring soluble NRPI; b) sNRPl as an antagonist of VEGFlbj-induced cell motility, proliferation and angiogenesis; c) the distribution of sNRP1 in comparison to intact membrane-bound NRPl; d) soluble receptor assays in vitro for analyzing modulators of VEGFlsNRPl interactions. 4. To Investigate the Regulation of NRPl Gene Expression and Activity including: a) cloning the NRPI and NRP2 promoters and identifying regulators of NRP gene expression; b) identifying cytoplasmic proteins that interact with the NRPI cytoplasmic domain using the yeast two hybrid system; c) characterization of a PDZ domain-containing protein.

1. 研究NRP配体结合位点。我们已经确定 VEGF-A、PlGF 和肝素与 blb2 胞外结构域 (ECD) 结合。 VEGF-B 和丛蛋白 A（响应信号蛋白转导 NRP 信号）也与 NRP ECD 结合，但这两种蛋白的结合域尚不清楚。未来的研究包括 i) 使用 myc 标记域下拉策略确定 NRP 上的 VEGF-B 和丛蛋白-A 结合位点，该策略先前确定了 NRP 上的 VEGF-A 和 PlGF 结合位点（Mamluk 等人，JBC，2002），并通过分析缺失突变体和定点诱变确定这些配体结合位点是独特的还是重叠的； ii) 与 John Gallagher 博士（英国曼彻斯特大学）合作，对结合 NRP blb2 的肝素进行结构分析； iii) 与 Celia Hamson 博士（BBRI，波士顿）合作，在有或没有 VEGF165 和肝素的情况下结晶 blb2，他之前已经结晶了 Ephrin B2（见信件）。 2.分析NRP表达的调控。关于NRP表达如何受到调节以及NRP1和NRP2是否受到差异性调节知之甚少。我们已经分离了NRP1和NRP2启动子并制备了NRP-荧光素酶构建体。迄今为止我们已经发现佛波酯、EGF和HB-EGF诱导EC、肿瘤细胞和角质形成细胞中的NRP1启动子活性。未来的研究包括：i)使用NRP启动子-报告基因构建体来筛选NRP表达的诱导剂和抑制剂，并鉴定所涉及的转录因子； ii)在 prox-Z 过度表达后，分析血管 EC 向淋巴 EC 转变中的 NRP1 和 NRP2 表达（参见 Michael Detmar 博士的信）； iii) 细胞、组织和小鼠胚胎中 NRP 和 sNRP 表达的比较分析； iv) 由现居日本大阪的前博士后 Seiji Takashima 博士生成的 NRP1LacZ 和 NRP2LacZ 小鼠中的发育和成年 NRP 表达模式。 3. 分析信号蛋白的非神经元特性。信号蛋白通过 NRP 排斥轴突和塌陷生长锥来调节轴突引导。我们发现信号蛋白结合非神经元细胞，例如 EC、肿瘤细胞和角质形成细胞，但这些相互作用的后果需要进一步分析。未来的研究包括： i) 从细胞培养物中产生大量的 Sema3A 和 3F 蛋白，因为信号蛋白由于缺乏稳定性而无法在商业上获得； ii)确定Sema3NSema3F是否影响EC和肿瘤细胞运动、增殖、存活和凋亡； iii) 分析Sema3AL/3F对肌动蛋白细胞骨架的影响； iv)确定Sema 3A/3F是否是血管生成抑制剂。 4. 分析响应 VEGF165 和信号蛋白而激活的 NRP 信号通路。 VEGF165 和信号蛋白与许多非神经元细胞类型表达的 NRP 结合。然而，人们对 NRP 如何在这些细胞类型中响应这些配体发出信号知之甚少。作为指导，许多分子与信号蛋白诱导的生长锥塌陷有关，包括 Plexin A1 和 A2、GTPase 的 Rho 家族、CRMP 和 GSK3。肌动蛋白丝重组受 LIM 激酶及其底物丝切蛋白 (cofilin) 调节。未来的研究包括将表达 NRPl/NRP2 的 EC、肿瘤细胞和角质形成细胞与 VEGF165、VEGFIZI (作为对照，不结合 NRP)、VEGF-B 和 PlGF (结合 NRPl)、VEGF-C (结合 NRP2)、Sema3A (结合 NRP1) 和 Sema3F (结合 NRP2) 一起孵育，以及 通过 i) 如上所述直接检查与生长锥塌陷有关的信号分子来监测下游效应； ii) 西方。用抗磷酸酪氨酸、丝氨酸和苏氨酸抗体进行印迹； iii) 2D 凝胶/蛋白质组学，用于检测一般蛋白质和磷蛋白谱的变化； iv) 通过微阵列对这些处理进行转录分析。将确定 VEGF165 和信号蛋白是否使用相似或不同的途径。将与我们部门的 Don Ingber 博士（该领域的知名专家）合作分析对细胞骨架的影响（见信件）。另一种方法是用反义（RNAi、吗啉）抑制 NRP 表达并分析下游效应。 5. 分析天然存在的可溶性NRP（sNRP）的功能。 sNRP 结合 VEGF165，是 VEGF165 拮抗剂。未来的研究包括：i)分析sNRPl对VEGF165诱导的迁移、增殖和存活的影响并确定所涉及的机制，例如抑制特定酪氨酸残基的磷酸化和参与迁移的因子（例如PI3激酶和AKT）； ii) 使用与 MGH 的 Michael Detmar 博士合作生成的 K14 启动子，分析 sNW1 对皮肤中过表达 sNRP 的转基因小鼠的血管生成和血管通透性 (VPF) 活性的影响； iii) 分析 sNRP 抑制肿瘤进展的机制，例如通过诱导肿瘤细胞凋亡。 6. 研究NRPs在肿瘤血管生成和癌症中的作用。 NWli 的过度表达可增强啮齿动物模型中的肿瘤血管生成和肿瘤生长。未来的实验包括 i) 在人类肿瘤细胞系（例如黑色素瘤、鳞状细胞癌）中过表达-NRP2； ii) NRP拮抗剂的过度表达，例如人肿瘤细胞系中的sNRP和信号蛋白。 7. 开发蛋白质组学程序，包括优化磷蛋白分析以研究信号。最初提案的具体目标是： 1. 研究 VEGF165 和 Semaphorid Collapsin 与 NRP1 的相互作用，包括： a,) EC 中 VEGF1~S/~RPI/KDinRte 反应的机制； b) 信号蛋白通过 NRPI 作为 EC 和肿瘤细胞运动的拮抗剂； c) VEGF165 和信号蛋白是否是与 EC 和肿瘤细胞相互作用的竞争性抑制剂。 2.研究肿瘤细胞中的NRP1表达和功能，包括：a)VEGF1bj是否是通过NRP1作用的肿瘤细胞运动的直接刺激剂以及可能涉及哪些途径； b)肿瘤细胞中的NRP1表达以及NRP1表达对肿瘤细胞运动性和转移潜能的贡献。 3.表征天然存在的可溶性NRP1(sNRP1)受体的结构、功能和分布，包括：a)天然存在的可溶性NRPI的克隆和纯化； b)sNRPl作为VEGF1bj诱导的细胞运动、增殖和血管生成的拮抗剂； c)与完整的膜结合的NRPl相比，sNRP1的分布； d)用于分析VEGF1NRP1相互作用调节剂的体外可溶性受体测定。 4.研究NRP1基因表达和活性的调节，包括：a)克隆NRPI和NRP2启动子并鉴定NRP基因表达的调节子； b) 使用酵母二杂交系统鉴定与 NRPI 细胞质结构域相互作用的细胞质蛋白； c) 含有PDZ结构域的蛋白质的表征。