Partial characterization of endothelial FGF receptor functional domain by monoclonal antibody VBS-1
Partial characterization of endothelial FGF receptor functional domain by monoclonal antibody VBS-1
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DOI：
10.1089/153685902760173863
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发表时间：
2002-06-01
期刊：
影响因子：
--
通讯作者：
Schelling, ME
中科院分区：
文献类型：
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作者：
Blanckaert, VD;Venkateswaran, S;Schelling, ME
多肽生长因子通过结合并激活特定的细胞表面受体来介导其细胞反应。根据 I-125-FGF-2 Scatchard 分析和 [H-3] 胸苷摄取测定 (ED50 = 80 ng/mL) 测定，针对天然成纤维细胞生长因子受体 1 (FGFR-1) 产生的单克隆抗体 (MAb) VBS-1 抑制成纤维细胞生长因子 2 (FGF-2) 与其在冠状静脉内皮细胞 (CVEC) 上的受体的结合。酶研究表明 MAb VBS-1 与蛋白质表位结合。 CVEC-FGFR 的蛋白水解图谱确定 52 kDa 双联体包含 FGF 结合位点和 MAb VBS-1 抗原表位。 N-聚糖酶消化表明存在 CVEC-FGFR 的 50 kDa 核心蛋白。衣霉素处理导致核心蛋白和成熟受体表达丧失，表明 CVEC-FGFR n 连接糖基化的重要性。通过Northern印迹分析，确定CVEC表达fgfr-1而不是fgfr-2。 VBS-1 识别 FGFR-1 (140 kDa) 并与 FGFR-2 (135 kDa) 发生微弱交叉反应。通过结合亲和交联、蛋白水解作图和 Mab VBS-1 结合研究，我们将 FGF 结合位点定位在靠近高酸性盒的受体 NH2 末端结构域附近。
Polypeptide growth factors mediate their cellular responses by binding to and activating specific cell surface receptors. Monoclonal antibody (MAb) VBS-1, produced against native fibroblast growth factor receptor-1 (FGFR-1), inhibited the binding of fibroblast growth factor-2 (FGF-2) to its receptor on coronary venular endothelial cells (CVECs) as determined by I-125-FGF-2 Scatchard analysis and [H-3]thymidine uptake assays (ED50 = 80 ng/mL). Enzyme studies demonstrated that MAb VBS-1 binds to a protein epitope. Proteolytic mapping of the CVEC-FGFR established that a 52 kDa doublet contained the FGF binding site and the MAb VBS-1 antigenic epitope. N-glycanase digestion suggested the presence of a 50 kDa core protein for the CVEC-FGFR. Tunicamycin treatment resulted in the loss of expression of the core protein and the mature receptor, indicating the importance of CVEC-FGFR n-linked glycosylation. By Northern blot analysis, it was determined that CVECs express fgfr-1 and not fgfr-2. VBS-1 recognized FGFR-1 (140 kDa) and crossreacted weakly with FGFR-2 (135 kDa). Using a combination of affinity crosslinking, proteolytic mapping and Mab VBS-1 binding studies, we have located the FGF binding site near the NH2-terminal domain of the receptor close to the highly acidic box.