Real-Time Ligand Binding of Fluorescent VEGF-A Isoforms that Discriminate between VEGFR2 and NRP1 in Living Cells.
Real-Time Ligand Binding of Fluorescent VEGF-A Isoforms that Discriminate between VEGFR2 and NRP1 in Living Cells.
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DOI：
10.1016/j.chembiol.2018.06.012
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发表时间：
2018-10-18
影响因子：
8.6
通讯作者：
Hill SJ
中科院分区：
文献类型：
--
作者：
Peach CJ;Kilpatrick LE;Friedman-Ohana R;Zimmerman K;Robers MB;Wood KV;Woolard J;Hill SJ
在使用BRET的活细胞实时配体结合研究中，已经评估了荧光VEGF-A异构体区分VEGFR2和NRP1的能力。为此，我们合成了单位点（n端半胱氨酸）标记版本的VEGF165a、VEGF165b和VEGF121a。这些与n端nanoluc标记的VEGFR2或NRP1结合使用，以评估VEGF同种异构体对这两种膜蛋白的选择性。所有VEGF-A荧光异构体对VEGFR2均表现出高亲和力。只有VEGF165a-TMR以相似的高亲和力（4.4 nM）结合到nanolac - nrp1上。竞争NRP1结合实验得到VEGF165a > VEGF189a > VEGF145a的效价等级。VEGF165b、VEGF-Ax、VEGF121a和VEGF111a无法与NRP1结合。VEGF165a-TMR对NRP1和VEGFR2的动力学结合谱存在显著差异。这些数据强调了配体与VEGFR2及其共受体结合的动力学方面在VEGF信号传导动力学中的重要性。VEGF165a、VEGF121a和VEGF165b用四甲基罗丹明单位点标记，NanoBRET定量证实VEGF-A异构体与VEGFR2具有相似的结合特性，活细胞中表达的NRP1不结合VEGF165b、VEGF121a、VEGF-Ax、VEGFR2和NRP1结合VEGF165a-TMR具有明显不同的动力学特征。Peach等人利用荧光VEGF-A异构体证明，在活细胞的实时配体结合研究中，它们可以区分VEGFR2及其共受体NRP1。这种精确的化学生物学方法表明，荧光VEGF165a比VEGFR2更快地结合NRP1。
Fluorescent VEGF-A isoforms have been evaluated for their ability to discriminate between VEGFR2 and NRP1 in real-time ligand binding studies in live cells using BRET. To enable this, we synthesized single-site (N-terminal cysteine) labeled versions of VEGF165a, VEGF165b, and VEGF121a. These were used in combination with N-terminal NanoLuc-tagged VEGFR2 or NRP1 to evaluate the selectivity of VEGF isoforms for these two membrane proteins. All fluorescent VEGF-A isoforms displayed high affinity for VEGFR2. Only VEGF165a-TMR bound to NanoLuc-NRP1 with a similar high affinity (4.4 nM). Competition NRP1 binding experiments yielded a rank order of potency of VEGF165a > VEGF189a > VEGF145a. VEGF165b, VEGF-Ax, VEGF121a, and VEGF111a were unable to bind to NRP1. There were marked differences in the kinetic binding profiles of VEGF165a-TMR for NRP1 and VEGFR2. These data emphasize the importance of the kinetic aspects of ligand binding to VEGFR2 and its co-receptors in the dynamics of VEGF signaling. VEGF165a, VEGF121a, and VEGF165b were single-site labeled with tetramethylrhodamine NanoBRET quantified that VEGF-A isoforms have similar binding properties at VEGFR2 NRP1 expressed in live cells does not bind VEGF165b, VEGF121a, VEGF-Ax, or VEGF111a VEGFR2 and NRP1 have markedly distinct kinetic profiles binding VEGF165a-TMR Peach et al. have used fluorescent VEGF-A isoforms to demonstrate that they can discriminate between VEGFR2 and its co-receptor NRP1 in real-time ligand binding studies in live cells. This precision chemical biology approach showed that fluorescent VEGF165a binds more rapidly to NRP1 than VEGFR2.
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