CHARACTERIZATION AND ISOLATION OF A NOVEL VEGF RECEPTOR

新型 VEGF 受体的表征和分离

• 批准号: 6443843
• 负责人: MICHAEL KLAGSBRUN
• 金额: $ 9.16万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6443843
• 关键词: angiogenesis; angiogenesis inhibitors; cell growth regulation; cell line; cell type; chimeric proteins; expression cloning; growth factor receptors; immunological substance; northern blottings; phosphorylation; protein isoforms; protein purification; protein structure function; receptor binding; receptor expression; tissue /cell culture; vascular endothelial growth factors

Vascular endothelial cell growth factor (VEGF) is a highly specific chemotactic and mitogenic factor for cultured endothelial cells (EC) and an angiogenesis factor in vivo. It binds to 180 and 200 kDa high affinity tyrosine kinase receptors, Flt-1 and KDR/Flk-1, respectively, found primarily on EC. Recently, a relatively lower affinity 120-130 kDa receptor has been identified on EC and tumor cell lines that binds VEGF165, but not VEGF121, and accordingly has been designated as the VEGF165 receptor (VEGF165R). VEGF165 R does not appear to be related to KDR/Flk-1 and Flt-1 since it is not immunoprecipitated by antibodies to these receptors. The binding of VEGF165 to VEGF165R is mediated by the protein domain encoded by exon 7, which is lackin2 x 10(-9) M g in VEGF121. An exon 7- encoded fusion protein inhibits VEGF165 binding to EC and tumor cells. The Specific Aims of this proposal are: 1. Characterization of a cell surface receptor (VEGF165R) that binds VEGF165 but not VEGF121 including: a) identification of a cell types that express this receptor; b) binding of VEGF family ligands; c) modulation of proliferation, migration and morphology by VEGF165R; and d) VEGF165R receptor phosphorylation and signaling; 2. Purification and cloning of the VEGF165 receptor and antibody production including: a)purification of VEGF165R; b)expression cloning of VEGF165R; c) production of anti-VEGF165R in cells; 3. Characterization of exon 7- encoded protein, an inhibitor of VEGF165 binding to EC and tumor cells including: a) Preparation of exon 7 protein by fusion protein methods; b) measuring inhibitory effects of exon 7-encoded protein on VEGF165 binding to, and mitogenic and chemotactic activity for, EC and tumor cells; c) preparatio of anti exon 7 protein neutralizing antibodies to inhibit VEGF165 binding to VEGF165R; d) determination of the core inhibitory amino acid sequence within the exon 7 protein domain; and e) production of a chimeric exon 7 protein-diphtheria toxin fusion protein for tumor cell and tumor EC targeting.

血管内皮细胞生长因子（VEGF）是一种高度特异性的 培养内皮细胞 (EC) 的趋化和促有丝分裂因子 和体内血管生成因子。 它结合 180 和 200 kDa 高 亲和性酪氨酸激酶受体，分别为 Flt-1 和 KDR/Flk-1， 主要发现于 EC。 最近亲和力比较低120-130 kDa 受体已在 EC 和肿瘤细胞系中被发现 结合 VEGF165，但不结合 VEGF121，因此已被 命名为 VEGF165 受体 (VEGF165R)。 VEGF165R 似乎与 KDR/Flk-1 和 Flt-1 无关，因为它不是 通过这些受体的抗体进行免疫沉淀。 的绑定 VEGF165 到 VEGF165R 由编码的蛋白质结构域介导 由外显子 7 决定，VEGF121 中缺少 2 x 10(-9) M g。 外显子 7- 编码的融合蛋白抑制 VEGF165 与 EC 和肿瘤的结合 细胞。 该提案的具体目标是： 1. 表征 细胞表面受体 (VEGF165R) 结合 VEGF165，但不结合 VEGF121 包括： a) 表达该蛋白的细胞类型的鉴定 受体； b) VEGF家族配体的结合； c) 调制 VEGF165R 的增殖、迁移和形态学；和 d) VEGF165R 受体磷酸化和信号转导； 2. 纯化与 VEGF165 受体的克隆和抗体的生产包括： a) VEGF165R的纯化； b) VEGF165R的表达克隆； c) 细胞中抗 VEGF165R 的产生； 3. 外显子 7- 的表征 编码蛋白，VEGF165 与 EC 和肿瘤结合的抑制剂 细胞包括： a) 融合蛋白制备外显子7蛋白 方法; b) 测量外显子 7 编码蛋白对 VEGF165 与 EC 结合，并具有促有丝分裂和趋化活性 和肿瘤细胞； c) 抗外显子7蛋白中和剂的制备 抑制 VEGF165 与 VEGF165R 结合的抗体； d) 确定内的核心抑制氨基酸序列 外显子 7 蛋白结构域； e) 嵌合外显子 7 的产生 肿瘤细胞蛋白-白喉毒素融合蛋白及肿瘤EC 瞄准。