CHARACTERIZATION AND ISOLATION OF A NOVEL VEGF RECEPTOR
CHARACTERIZATION AND ISOLATION OF A NOVEL VEGF RECEPTOR
批准号:
6344719
负责人:
MICHAEL KLAGSBRUN
金额:
$20.9万
依托单位国家:
美国
项目类别:
财政年份:
2000
资助国家:
美国
项目状态:
已结题
起止时间:
2000-08-15 至 2001-03-31
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
中文摘要
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 165 R)。 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
VEGF 165转化为VEGF 165 R是由编码VEGF 165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)与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)
VEGF 165 R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 165 R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.
英文摘要
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.
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