Ahmadova Zhala, Yagublu Vugar, Förg Tassilo, Hajiyeva Yegana, Jesenofsky Ralf, Hafner Mathias, Keese Michael
Clinic for Vascular and Endovascular Surgery, University Hospital Frankfurt, Frankfurt, Germany.
Anticancer Res. 2014 May;34(5):2123-33.
Activation of vascular endothelial growth factor receptor 1 (VEGFR1/FLT1) and 2 (VEGFR2/KDR) involves receptor dimerization. Formation of VEGFR dimer has so far not been visualized in single intact cells. In the present study we describe different optical assays which can be used to observe dimerization of VEGFR1 and VEGFR2. Bimolecular fluorescence complementation (BIFC) assays confirmed homo,- and heterodimerization of transfected receptors. Fluorescence resonance energy transfer (FRET) techniques in living and fixed CHO-K1 cells allowed observation of VEGFR1 homodimer,- and VEGFR1 and VEGFR2 heterodimer formation after ligand stimulation. After inhibition of ligand binding by the VEGFA JH121 antibody VEGFR1 homodimerization was completely abolished. Under the same conditions, cells transfected by VEGFR1 and VEGFR2 maintained relevant receptor heterodimerization. These techniques to monitor VEGFR1 and VEGFR2 homo- and heterodimerization in living and fixed cells may help in the search for new angiogenesis-directed inhibitors of VEGFR dimerization.
血管内皮生长因子受体1（VEGFR1/FLT1）和2（VEGFR2/KDR）的激活涉及受体二聚化。迄今为止，尚未在单个完整细胞中观察到VEGFR二聚体的形成。在本研究中，我们描述了可用于观察VEGFR1和VEGFR2二聚化的不同光学检测方法。双分子荧光互补（BIFC）检测证实了转染受体的同源二聚化和异源二聚化。在活细胞和固定的CHO-K1细胞中使用荧光共振能量转移（FRET）技术，可以观察到配体刺激后VEGFR1同源二聚体以及VEGFR1和VEGFR2异源二聚体的形成。用VEGFA JH121抗体抑制配体结合后，VEGFR1同源二聚化完全被消除。在相同条件下，转染VEGFR1和VEGFR2的细胞维持相关的受体异源二聚化。这些在活细胞和固定细胞中监测VEGFR1和VEGFR2同源二聚化和异源二聚化的技术，可能有助于寻找新的针对血管生成的VEGFR二聚化抑制剂。
