CHARACTERIZATION AND ISOLATION OF A NOVEL VEGF RECEPTOR

血管内皮细胞生长因子(VEGF)是一种高度特异的 培养内皮细胞的趋化和促有丝分裂因子 和体内的血管生成因子。它结合到180 kDa和200 kDa高 亲和力酪氨酸激酶受体，分别为Flt-1和KDR/Flk-1， 主要在欧共体上发现。最近，亲和力相对较低的120-130 已在EC和肿瘤细胞系上发现KDA受体 结合VEGF165，但不结合VEGF121，并相应地 命名为VEGF165受体(VEGF165R)。VEGF165 R 似乎与KDR/Flk-1和Flt-1无关，因为它与 通过这些受体的抗体进行免疫沉淀。的约束性 VEGF165到VEGF165R是由编码的蛋白结构域介导的 外显子7，在VEGF121中缺失2×10(-9)Mg。外显子7- 编码融合蛋白抑制血管内皮生长因子165与EC和肿瘤的结合 细胞。这项建议的具体目标是：1.描述一种 与VEGF165结合但不结合的细胞表面受体(VEGF165R) VEGF121包括：a)表达以下内容的细胞类型的标识 受体；b)与血管内皮生长因子家族配体的结合；c)调节 VEGF165R的增殖、迁移和形态；和d) VEGF165R受体的磷酸化和信号转导；2.纯化和 VEGF165受体的克隆和抗体的产生包括： A)VEGF165R的纯化；b)VEGF165R的表达克隆；c) 在细胞中产生抗VEGF165R；3.外显子7- VEGF165结合EC和肿瘤的抑制因子编码蛋白 细胞包括：a)融合蛋白制备外显子7蛋白 方法：b)检测外显子7编码的蛋白对 血管内皮生长因子165与内皮细胞的结合及其促有丝分裂和趋化活性 C)抗外显子7中和蛋白的制备 抑制VEGF165与VEGF165R结合的抗体；d) 核心区抑制氨基酸序列的测定 外显子7蛋白结构域；和e)嵌合外显子7的产生 肿瘤细胞和肿瘤EC的蛋白质-白喉毒素融合蛋白 瞄准目标。

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.