Zhang Sheng, Gao Xiaoping, Fu Wei, Li Shengwei, Yue Limin
Department of Physiology, West China School of Preclinical and Forensic Medicine, Sichuan University, No. 17, Section 3 Renmin South Road, Chengdu, 610064, Sichuan, People's Republic of China.
Sichuan Institute of Population and Family Planning, Chengdu University of Traditional Chinese Medicine, Chengdu, 610041, Sichuan, People's Republic of China.
Breast Cancer Res Treat. 2017 Jun;163(3):423-434. doi: 10.1007/s10549-017-4189-5. Epub 2017 Mar 16.
The extracellular region (EC) of the vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) contains seven immunoglobulin-like (Ig-like) domains that are required for specific ligand binding and receptor dimerization. Studies of domain 4-7 deletions and substitutions provided insights into the interaction between receptors in the absence of VEGF. In this study, we investigated the effect of domain 4 in ligand-independent VEGFR-2 dimerization and activation in human vascular endothelial cells and human breast cancer cells.
To confirm the role of domain 4 in ligand-independent receptor dimerization and activation, two VEGFR-2 fragments with and without domain 4, KFP1 and KFP2, were generated by recombinant DNA technology. We measured the affinity of KFP1 and KFP2 with VEGFR-2, and the roles of KFP1 and FKP2 in dimerization and phosphorylation of VEGFR-2. We also evaluated the effect of KFP1 and FKP2 on cell proliferation and migration in HUVECs and in human breast cancer cells.
We showed that KFP1 did not affect the interaction of VEGFR-2 and VEGF but bound VEGFR-2 in the absence of VEGF. Furthermore, cross-linking and cross-linking immunoblotting demonstrated that KFP1 could form a complex with VEGFR-2, which resulted in VEGFR-2 dimerization in the absence of VEGF. Importantly, we found that the KDR fragment with domain 4 induced phosphorylation of VEGFR-2, as well as phosphorylation of downstream receptor kinases in HUVECs and VEGFR-2-positive breast cancer cells. Consistent with these results, this ligand-independent activation of VEGFR-2 also promoted downstream signaling and cell proliferation and migration.
The domain 4 of VEGFR-2 plays an important role in the interaction between VEGFR receptors in the absence of VEGF.
血管内皮生长因子（VEGF）受体-2（VEGFR-2）的细胞外区域（EC）包含七个免疫球蛋白样（Ig样）结构域，这些结构域是特异性配体结合和受体二聚化所必需的。对结构域4-7缺失和替换的研究为在无VEGF情况下受体之间的相互作用提供了见解。在本研究中，我们调查了结构域4在人血管内皮细胞和人乳腺癌细胞中不依赖配体的VEGFR-2二聚化和激活中的作用。
为了证实结构域4在不依赖配体的受体二聚化和激活中的作用，通过重组DNA技术产生了有和没有结构域4的两个VEGFR-2片段，即KFP1和KFP2。我们测量了KFP1和KFP2与VEGFR-2的亲和力，以及KFP1和FKP2在VEGFR-2二聚化和磷酸化中的作用。我们还评估了KFP1和FKP2对人脐静脉内皮细胞（HUVECs）和人乳腺癌细胞中细胞增殖和迁移的影响。
我们表明KFP1不影响VEGFR-2与VEGF的相互作用，但在无VEGF时能结合VEGFR-2。此外，交联和交联免疫印迹表明KFP1可与VEGFR-2形成复合物，这导致在无VEGF时VEGFR-2二聚化。重要的是，我们发现带有结构域4的KDR片段可诱导VEGFR-2磷酸化，以及在HUVECs和VEGFR-2阳性乳腺癌细胞中诱导下游受体激酶磷酸化。与这些结果一致，这种不依赖配体的VEGFR-2激活也促进了下游信号传导以及细胞增殖和迁移。
VEGFR-2的结构域4在无VEGF情况下VEGFR受体之间的相互作用中起重要作用。
