Molecular cloning of a human basic fibroblast growth factor receptor cDNA and expression of a biologically active extracellular domain in a baculovirus system.
Molecular cloning of a human basic fibroblast growth factor receptor cDNA and expression of a biologically active extracellular domain in a baculovirus system.
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DOI：
10.3109/08977199109000276
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发表时间：
1991-01-01
期刊：
影响因子：
--
通讯作者：
Barr, P J
中科院分区：
文献类型：
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作者：
Kiefer, M C;Baird, A;Barr, P J
从肝癌细胞系cDNA文库中分离到人成纤维细胞生长因子（FGF）受体cDNA克隆。该cDNA编码三免疫球蛋白样结构域FGF受体，与人类胎盘FGF受体cDNA相似，但缺少两个氨基酸。在这两种氨基酸上观察到的差异，也在两种免疫球蛋白样结构域fgf受体上观察到的差异，可以用另一种剪接机制来解释。我们使用杆状病毒表达系统产生高水平的可溶性细胞外结构域形式的FGF受体（EC-FGF受体）。用重组EC-FGF受体病毒感染狐尾蛾（Spodoptera frugiperda, Sf9）昆虫细胞，合成并分泌EC-FGF受体，表观Mr = 58,000。用扁豆凝集素亲和层析法从感染Sf9细胞的条件培养基中纯化EC-FGF受体，发现EC-FGF受体与碱性FGF具有高亲和力（Kd = 1 ~ 5 nM），抑制放射性碘化碱性FGF与其高亲和受体的结合，抑制内皮细胞增殖。此外，肝素显著增强了碱性FGF与EC-FGF受体的结合。生物活性FGF受体的可用性将允许分析它们与FGF蛋白家族成员和疱疹病毒家族成员的相互作用，这些成员已被证明使用FGF受体系统进入细胞。
A cDNA clone encoding a human fibroblast growth factor (FGF) receptor was isolated from a hepatoma cell line cDNA library. The cDNA encodes a three immunoglobulinlike-domain FGF receptor that is similar to a human placental FGF receptor cDNA but lacks two amino acids. The variation observed at these two amino acids, also seen in the two immunoglobulinlike-domain FGF-receptors, can be explained by an alternate splicing mechanism. We have used a baculovirus expression system to produce high levels of a soluble, extracellular domain form of the FGF receptor (EC-FGF receptor). Spodoptera frugiperda (Sf9) insect cells infected with recombinant EC-FGF receptor viruses synthesized and secreted an EC-FGF receptor of apparent Mr = 58,000. The EC-FGF receptor purified from conditioned media of infected Sf9 cells by lentil lectin affinity chromatography was shown to bind basic FGF with high affinity (Kd = 1-5 nM), to inhibit the binding of radioiodinated basic FGF to its high affinity receptor and to inhibit endothelial cell proliferation. Furthermore, binding of basic FGF to the EC-FGF receptor was shown to be significantly enhanced by heparin. The availability of biologically active FGF receptors will allow an analysis of their interaction with members of the FGF family of proteins and viruses of the herpes family that have been shown to use the FGF receptor system for cell entry.