Mapping of the Sites for Ligand Binding and Receptor Dimerization at the Extracellular Domain of the Vascular Endothelial Growth Factor Receptor FLT-1*
Mapping of the Sites for Ligand Binding and Receptor Dimerization at the Extracellular Domain of the Vascular Endothelial Growth Factor Receptor FLT-1*
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血管内皮生长因子受体 FLT-1* 胞外域的配体结合和受体二聚化位点图谱
DOI：
10.1074/jbc.272.16.10382
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
G. Martiny
中科院分区：
文献类型：
--
作者：
B. Barleon;F. Totzke;Christel Herzog;Stephen Blanke;E. Kremmer;G. Siemeister;D. Marmé;G. Martiny
血管内皮生长因子（VEGF）受体FLT-1已被证明参与血管发生和血管新生。该受体的特点是在细胞外区域有7个类似igg的环。VEGF结合后，FLT-1被磷酸化，这被认为是在受体二聚化之前发生的。为了进一步研究VEGF与FLT-1的高亲和力结合和配体诱导的受体二聚化，我们在Sf9细胞中表达了包含所有7个igg样环的整个细胞外结构域：sFLT-1(7)和几个由环1、1和2、1到3、1到4和1到5组成的截断突变体。纯化相应的蛋白，命名为sFLT-1(1)、(2)、(3)、(4)和(5)。只有突变体sFLT-1(3)至(7)能够高亲和力地结合125i - vegf。未观察到VEGF与sFLT-1(1)和sFLT-1(2)结合，说明前三个igg样环参与了VEGF的高亲和力结合。VEGF与sFLT-1的结合(3)可能与VEGF相关配体胎盘生长因子（PlGF）竞争，表明VEGF与PlGF的高亲和力结合是由sFLT-1上相同或密切相关的接触位点介导的。sFLT-1的去糖基化(3)、(4)、(5)和(7)并没有消除VEGF的结合。此外，在大肠杆菌中表达的未糖基化的sFLT-1(3)能够以与Sf9细胞中表达的sFLT-1(3)或sFLT-1(7)相似的亲和力结合VEGF。这表明受体糖基化不是高亲和力结合所必需的。所有含有igg样环4的突变体在添加VEGF后检测到FLT-1细胞外结构域的二聚化。尽管sFLT-1(3)能够结合VEGF，但该突变体的二聚化效率较低，这表明igg样环4上的位点对于稳定受体二聚体至关重要。
The vascular endothelial growth factor (VEGF) receptor FLT-1 has been shown to be involved in vasculogenesis and angiogenesis. The receptor is characterized by seven Ig-like loops within the extracellular domain. Upon VEGF binding FLT-1 becomes phosphorylated, which has been thought to be preceded by receptor dimerization. To further investigate high affinity binding of VEGF to FLT-1 and ligand-induced receptor dimerization, we expressed in Sf9 cells the entire extracellular domain comprising all seven Ig-like loops: sFLT-1(7) and several truncated mutants consisting of loop one, one and two, one to three, one to four, and one to five. The corresponding proteins, named sFLT-1(1), (2), (3), (4), and (5) were purified. Only mutants sFLT-1(3) to (7) were able to bind125I-VEGF with high affinity. No binding of VEGF was observed with sFLT-1(1) and sFLT-1(2), indicating that the first three Ig-like loops are involved in high affinity binding of VEGF. The binding of VEGF to sFLT-1(3) could be competed with placenta growth factor (PlGF), a VEGF-related ligand, suggesting that high affinity binding of VEGF and PlGF is mediated by the same or closely related contact sites on sFLT-1. Deglycosylation of the sFLT-1(3), (4), (5), and (7) did not abolish VEGF binding. Furthermore, unglycosylated sFLT-1(3), expressed in Escherichia coli, was able to bind VEGF with similar affinity as sFLT-1(3) or sFLT-1(7), both expressed in Sf9 cells. This indicates that receptor glycosylation is not essential for high affinity binding. Dimerization of the extracellular domains of FLT-1 upon addition of VEGF was detected with all mutants containing the Ig-like loop four. Although sFLT-1(3) was able to bind VEGF, dimerization of this mutant was inefficient, indicating that sites on Ig-like loop four are essential to stabilize receptor dimers.
影响因子：
56.9
作者：
DEVRIES, C;ESCOBEDO, JA;WILLIAMS, LT
通讯作者：
WILLIAMS, LT
可溶性 c-kit 蛋白和抗受体单克隆抗体限制了干细胞因子的结合位点。
DOI：
--
发表时间：
1993
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Blechman,JM;Lev,S;Brizzi,MF;Leitner,O;Pegoraro,L;Givol,D;Yarden,Y
通讯作者：
Yarden,Y