Soker S, Fidder H, Neufeld G, Klagsbrun M
Department of Surgery, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1996 Mar 8;271(10):5761-7. doi: 10.1074/jbc.271.10.5761.
Vascular endothelial growth factor (VEGF), a potent angiogenic factor, uses two receptor tyrosine kinases, FLK/KDR and FLT, to mediate its activities. We have cross-linked 125I-VEGF165 to the cell surface of various tumor cell lines and of human umbilical vein endothelial cells. High molecular mass (220 and 240 kDa) and/or lower molecular mass (165 and 175 kDa) labeled complexes were detected depending on the cell type. The 220- and 240-kDa labeled complexes were shown to contain FLT and FLK/KDR receptors, respectively. On the other hand, the 165- and 175-kDa complexes did not seem to contain FLK/KDR or FLT but instead appeared to contain novel VEGF receptors with relatively low molecular masses of approximately 120 and 130 kDa. These receptors were further characterized in breast cancer MDA MB 231 cells (231), which did not form the high molecular mass complexes and which did not express detectable amounts of flk/kdr or flt mRNA. The 231 cells displayed one VEGF165 binding site, with a Kd of 2.8 x 10(-10) M and 0.95 1.1 x 10(5) binding sites per cell. By comparison, human umbilical vein endothelial cells had two binding sites, one with a Kd of 7.5 x 10(-12) M, presumably FLK/KDR, and the other with a Kd of 2 x 10(-10) M, a value similar to the VEGF binding sites on 231 cells. These lower affinity/molecular mass receptors on 231 cells cross-linked 125I-VEGF165 but not 125I-VEGF121. Accordingly, exon 7 of VEGF, which encodes the 44 amino acids present in VEGF165 that are absent in VEGF121, was fused to glutathione S-transferase (GST). The GST-VEGF-exon 7 fusion protein bound to heparin-Sepharose with a similar affinity as VEGF165 and inhibited the binding of 125I-VEGF165 to 231 cells. Cross-linking of 125I-GST-VEGF-exon 7 to 231 cells resulted in the formation of 150- and 160-kDa labeled complexes that presumably contained the 120- and 130-kDa lower affinity/molecular mass VEGF165 receptors. It was concluded that certain tumor-derived cell lines express novel surface-associated receptors that selectively bind VEGF165 via the exon 7-encoded domain, which is absent in VEGF121.
血管内皮生长因子（VEGF）是一种强效血管生成因子，它通过两种受体酪氨酸激酶FLK/KDR和FLT来介导其活性。我们已将¹²⁵I-VEGF165交联到各种肿瘤细胞系和人脐静脉内皮细胞的细胞表面。根据细胞类型，检测到高分子量（220和240 kDa）和/或低分子量（165和175 kDa）的标记复合物。已证明220 kDa和240 kDa的标记复合物分别含有FLT和FLK/KDR受体。另一方面，165 kDa和175 kDa的复合物似乎不含有FLK/KDR或FLT，而是似乎含有分子量相对较低、约为120 kDa和130 kDa的新型VEGF受体。这些受体在乳腺癌MDA MB 231细胞（231）中得到了进一步表征，该细胞系不形成高分子量复合物，且不表达可检测量的flk/kdr或flt mRNA。231细胞显示出一个VEGF165结合位点，解离常数（Kd）为2.8×10⁻¹⁰ M，每个细胞有0.95×10⁵至1.1×10⁵个结合位点。相比之下，人脐静脉内皮细胞有两个结合位点，一个Kd为7.5×10⁻¹² M，推测为FLK/KDR，另一个Kd为2×10⁻¹⁰ M，该值与231细胞上的VEGF结合位点相似。231细胞上这些低亲和力/低分子量受体可交联¹²⁵I-VEGF165，但不能交联¹²⁵I-VEGF121。因此，将VEGF的第7外显子（其编码VEGF165中存在而VEGF121中不存在的44个氨基酸）与谷胱甘肽S-转移酶（GST）融合。GST-VEGF-第7外显子融合蛋白与肝素-琼脂糖的结合亲和力与VEGF165相似，并抑制¹²⁵I-VEGF165与231细胞的结合。将¹²⁵I-GST-VEGF-第7外显子交联到231细胞上导致形成150 kDa和160 kDa的标记复合物，推测其含有120 kDa和130 kDa的低亲和力/低分子量VEGF165受体。得出的结论是，某些肿瘤来源的细胞系表达新型表面相关受体，这些受体通过VEGF121中不存在的第7外显子编码结构域选择性结合VEGF165。
