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.

中文翻译：

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在活细胞中区分 VEGFR2 和 NRP1 的荧光 VEGF-A 同种型的实时配体结合

在使用 BRET 的活细胞中实时配体结合研究中，已经评估了荧光 VEGF-A 同种型区分 VEGFR2 和 NRP1 的能力。为了实现这一点，我们合成了 VEGF165a、VEGF165b 和 VEGF121a 的单位点（N 端半胱氨酸）标记版本。它们与 N 端 NanoLuc 标记的 VEGFR2 或 NRP1 结合使用，以评估 VEGF 同种型对这两种膜蛋白的选择性。所有荧光 VEGF-A 同种型都显示出对 VEGFR2 的高亲和力。只有 VEGF165a-TMR 以类似的高亲和力 (4.4 nM) 与 NanoLuc-NRP1 结合。竞争性 NRP1 结合实验产生了 VEGF165a > VEGF189a > VEGF145a 的效力等级顺序。VEGF165b、VEGF-Ax、VEGF121a 和 VEGF111a 不能与 NRP1 结合。VEGF165a-TMR 对 NRP1 和 VEGFR2 的动力学结合谱存在显着差异。这些数据强调了配体结合 VEGFR2 及其共同受体在 VEGF 信号动力学方面的重要性。