Targeting Extracellular Domains D4 and D7 of Vascular Endothelial Growth Factor Receptor 2 Reveals Allosteric Receptor Regulatory Sites
Targeting Extracellular Domains D4 and D7 of Vascular Endothelial Growth Factor Receptor 2 Reveals Allosteric Receptor Regulatory Sites
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DOI：
10.1128/mcb.06787-11
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发表时间：
2012-10-01
影响因子：
5.3
通讯作者：
Ballmer-Hofer, Kurt
中科院分区：
文献类型：
--
作者：
Hyde, Caroline A. C.;Giese, Alexandra;Ballmer-Hofer, Kurt
血管内皮生长因子 (VEGF) 激活三种受体酪氨酸激酶 VEGFR-1、-2 和 -3，调节血管生成和淋巴管生成信号传导。 VEGFR-2 是 VEGF 配体的血管生成信号传导中最重要的受体。 VEGF 受体的胞外部分由七个免疫球蛋白同源结构域（Ig 结构域）组成。早期研究表明结构域 2 和结构域 3 (D23) 介导配体结合，而通过电子显微镜和小角溶液散射对二聚体配体/受体复合物进行的结构分析揭示了近膜 Ig 结构域 D4 和 D7 中的额外同型接触。在这里，我们证明 D4 和 D7 对于受体信号传导是不可或缺的。为了确认这些结构域在信号传导中的重要作用，我们分离了与 D23、D4 或 D7 相互作用的 VEGFR-2 抑制性“设计锚蛋白重复蛋白”(DARPins)。与 D23 相互作用的 DARPins 抑制配体结合、受体二聚化和受体激酶激活，而 D4 或 D7 特异性的 DARPins 不会阻止配体结合或受体二聚化，但有效阻断受体信号传导和功能输出。这些数据表明 D4 和 D7 变构调节 VEGFR-2 活性。我们认为这些细胞外结构域特异性 DARPins 代表了新一代受体抑制药物，可用于体内应用，例如在医学诊断中靶向 VEGFR 和治疗血管病变。
Vascular endothelial growth factors (VEGFs) activate three receptor tyrosine kinases, VEGFR-1, -2, and -3, which regulate angiogenic and lymphangiogenic signaling. VEGFR-2 is the most prominent receptor in angiogenic signaling by VEGF ligands. The extracellular part of VEGF receptors consists of seven immunoglobulin homology domains (Ig domains). Earlier studies showed that domains 2 and 3 (D23) mediate ligand binding, while structural analysis of dimeric ligand/receptor complexes by electron microscopy and small-angle solution scattering revealed additional homotypic contacts in membrane-proximal Ig domains D4 and D7. Here we show that D4 and D7 are indispensable for receptor signaling. To confirm the essential role of these domains in signaling, we isolated VEGFR-2-inhibitory "designed ankyrin repeat proteins" (DARPins) that interact with D23, D4, or D7. DARPins that interact with D23 inhibited ligand binding, receptor dimerization, and receptor kinase activation, while DARPins specific for D4 or D7 did not prevent ligand binding or receptor dimerization but effectively blocked receptor signaling and functional output. These data show that D4 and D7 allosterically regulate VEGFR-2 activity. We propose that these extracellular-domain-specific DARPins represent a novel generation of receptor-inhibitory drugs for in vivo applications such as targeting of VEGFRs in medical diagnostics and for treating vascular pathologies.