CHARACTERIZATION AND ISOLATION OF A NOVEL VEGF RECEPTOR

新型 VEGF 受体的表征和分离

• 批准号: 6344719
• 负责人: MICHAEL KLAGSBRUN
• 金额: $ 20.9万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-15 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6344719
• 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 内皮细胞趋化和促有丝分裂因子 和血管生成因子。 它结合180和200 kDa高 亲和酪氨酸激酶受体，分别为Flt-1和KDR/Flk-1， 主要是在EC上。 最近，相对较低的亲和力120 - 130 kDa受体已在EC和肿瘤细胞系上被鉴定， 结合VEGF 165，但不结合VEGF 121，因此， 称为VEGF 165受体（VEGF 165R）。 VEGF165 R 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免疫沉淀。 的结合 VEGF165转化为VEGF165R是由编码VEGF165的蛋白质结构域介导的。 第7外显子缺失2 × 10~（-9）M g。 外显子7- 编码的融合蛋白抑制VEGF 165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（VEGF 165 R），结合VEGF 165但不 a）鉴定表达VEGF 121的细胞类型， 受体; B）VEGF家族配体的结合; c）调节 VEGF 165 R的增殖、迁移和形态学;和d） VEGF165R受体磷酸化和信号传导; 2.纯化及 VEGF 165受体的克隆和抗体生产，包括： a）VEGF 165 R的纯化; B）VEGF 165 R的表达克隆; c） 在细胞中产生抗VEGF 165 R; 3.外显子7的特征- 编码的蛋白，VEGF 165与EC和肿瘤结合的抑制剂 a）通过融合蛋白制备外显子7蛋白 方法; B）测量外显子7编码的蛋白对 VEGF 165与EC的结合以及对EC的促有丝分裂和趋化活性 c）抗外显子7蛋白中和的比率 抑制VEGF 165与VEGF 165R结合的抗体; d） 确定所述多肽内的核心抑制性氨基酸序列， 外显子7蛋白结构域;和e）产生嵌合外显子7 用于肿瘤细胞和肿瘤EC的蛋白-白喉毒素融合蛋白 面向.