Kaplan J B, Sridharan L, Zaccardi J A, Dougher-Vermazen M, Terman B I
Oncology Section, Wyeth-Ayerst Research, Pearl River, New York 10965, USA.
Growth Factors. 1997;14(4):243-56. doi: 10.3109/08977199709021523.
To investigate the interaction between vascular endothelial growth factor (VEGF) and its receptor, we have constructed a chimeric protein consisting of the extracellular ligand-binding domain of the human VEGF receptor subtype KDR fused to a human IgG1 Fc domain (KDR-Fc). KDR-Fc was expressed in human 293 kidney epithelial cells as a 300-kDa secreted, dimeric glycoprotein that bound 125I-VEGF165 with high affinity (Kd = 150 pM). Unlike the full length cellular receptor, KDR-Fc did not require heparin for 125I-VEGF165 binding, although heparin did stimulate 125I-VEGF165 binding approximately 50 to 100%. Similar results were observed for KDR-Fc expressed in yeast cells. Since yeast do not synthesize heparan sulfate proteoglycans, we conclude that cellular heparan sulfates do not account for the lack of a heparin requirement for 125I-VEGF165 binding to KDR-Fc. The polycationic protein protamine, which inhibits (IC50 = 1 microgram/ml) 125I-VEGF165 binding to bovine aortic endothelial cells and other KDR-expressing cells by blocking heparin interactions, had no effect on the heparin independent component of 125I-VEGF165 binding to KDR-Fc. Protamine does inhibit (IC50 = 1 microgram/ml) the heparin dependent component of 125I-VEGF165 binding to KDR-Fc. KDR-Fc bound VEGF121 with the same affinity as VEGF165. Heparin had no effect on 125I-VEGF121 binding to KDR-Fc, indicating that heparin interaction with the 44 amino acids contained in VEGF165 but not VEGF121 allow for maximal VEGF165 binding. Deletion analysis of KDR-Fc demonstrated that the determinants required for high affinity VEGF binding are located in the three aminoterminal Ig-domains of the protein. Heparin had no effect on 125I-VEGF165 binding to the three Ig-domain receptor, suggesting that there are heparin binding determinants located in KDR Ig-domains 4 to 7.
为了研究血管内皮生长因子（VEGF）与其受体之间的相互作用，我们构建了一种嵌合蛋白，该蛋白由人VEGF受体亚型KDR的细胞外配体结合结构域与人IgG1 Fc结构域（KDR-Fc）融合而成。KDR-Fc在人293肾上皮细胞中表达为一种300 kDa的分泌型二聚体糖蛋白，它以高亲和力（Kd = 150 pM）结合125I-VEGF165。与全长细胞受体不同，KDR-Fc在结合125I-VEGF165时不需要肝素，尽管肝素确实能刺激125I-VEGF165的结合约50%至100%。在酵母细胞中表达的KDR-Fc也观察到了类似的结果。由于酵母不合成硫酸乙酰肝素蛋白聚糖，我们得出结论，细胞硫酸乙酰肝素并不能解释125I-VEGF165与KDR-Fc结合时对肝素需求的缺乏。多阳离子蛋白鱼精蛋白通过阻断肝素相互作用抑制（IC50 = 1微克/毫升）125I-VEGF165与牛主动脉内皮细胞及其他表达KDR的细胞的结合，但对125I-VEGF165与KDR-Fc结合的肝素非依赖成分没有影响。鱼精蛋白确实抑制（IC50 = 1微克/毫升）125I-VEGF165与KDR-Fc结合的肝素依赖成分。KDR-Fc以与VEGF165相同的亲和力结合VEGF121。肝素对125I-VEGF121与KDR-Fc的结合没有影响，这表明肝素与VEGF165中但VEGF121中不存在的44个氨基酸的相互作用允许最大程度的VEGF165结合。对KDR-Fc的缺失分析表明，高亲和力VEGF结合所需的决定簇位于该蛋白的三个氨基末端Ig结构域中。肝素对125I-VEGF165与三个Ig结构域受体的结合没有影响，这表明在KDR的Ig结构域4至7中存在肝素结合决定簇。
