Comparison of the ligand-binding properties of fluorescent VEGF-A isoforms to VEGF receptor 2 in living cells and membrane preparations using NanoBRET.
Comparison of the ligand-binding properties of fluorescent VEGF-A isoforms to VEGF receptor 2 in living cells and membrane preparations using NanoBRET.
复制标题
使用 NanoBRET 比较活细胞和膜制剂中荧光 VEGF-A 亚型与 VEGF 受体 2 的配体结合特性。
DOI：
10.1111/bph.14755
复制
发表时间：
2019
影响因子：
7.3
通讯作者：
Peach CJ
中科院分区：
文献类型：
--
作者：
Peach CJ
背景和目的血管内皮生长因子A （VEGF‐A）是血管生成的关键介质。在活细胞中，VEGF165a的荧光类似物与纳米荧光素酶标记的VEGF受体2 （VEGFR2）结合的一个显著特征是BRET信号不持续，并随着时间的推移而下降。这可能继发于受体内化。在这里，我们比较了三种荧光VEGF‐A亚型在细胞和分离膜制剂中与VEGFR2的结合。实验方法：在完整的HEK293T细胞和膜（表达纳米荧光素酶标记的VEGFR2）中，使用标记受体和VEGF165a， VEGF165b和VEGF121a的荧光类似物之间的BRET来监测配体结合动力学。通过使用自动定量成像跟踪细胞内体中荧光配体相关受体的出现，监测表达VEGFR2的完整细胞中的VEGFR2内吞作用。荧光VEGF - A异构体对细胞中VEGFR2内噬作用的影响的定量分析表明，它们可以将配体结合的VEGFR2快速有效地易位到细胞内体中。NanoBRET可用于监测荧光VEGF - A异构体与VEGFR2结合的动力学。在分离膜制备中，配体结合结合曲线在90分钟的实验时间内保持不变。当pH值为6.0时，配体的停留时间比pH值为7.4时短。这些研究表明，当VEGFR2从质膜转移到细胞内体时，快速的VEGF - A异构体诱导受体内吞作用缩短了激动剂在受体上的停留时间（1/koff）。
Background and PurposeVascular endothelial growth factor A (VEGF‐A) is a key mediator of angiogenesis. A striking feature of the binding of a fluorescent analogue of VEGF165a to nanoluciferase‐tagged VEGF receptor 2 (VEGFR2) in living cells is that the BRET signal is not sustained and declines over time. This may be secondary to receptor internalisation. Here, we have compared the binding of three fluorescent VEGF‐A isoforms to VEGFR2 in cells and isolated membrane preparations.Experimental ApproachLigand‐binding kinetics were monitored in both intact HEK293T cells and membranes (expressing nanoluciferase‐tagged VEGFR2) using BRET between tagged receptor and fluorescent analogues of VEGF165a, VEGF165b, and VEGF121a. VEGFR2 endocytosis in intact cells expressing VEGFR2 was monitored by following the appearance of fluorescent ligand‐associated receptors in intracellular endosomes using automated quantitative imaging.Key ResultsQuantitative analysis of the effect of fluorescent VEGF‐A isoforms on VEGFR2 endocytosis in cells demonstrated that they produce a rapid and potent translocation of ligand‐bound VEGFR2 into intracellular endosomes. NanoBRET can be used to monitor the kinetics of the binding of fluorescent VEGF‐A isoforms to VEGFR2. In isolated membrane preparations, ligand‐binding association curves were maintained for the duration of the 90‐min experiment. Measurement of thekoffat pH 6.0 in membrane preparations indicated shorter ligand residence times than those obtained at pH 7.4.Conclusions and ImplicationsThese studies suggest that rapid VEGF‐A isoform‐induced receptor endocytosis shortens agonist residence times on the receptor (1/koff) as VEGFR2 moves from the plasma membrane to the intracellular endosomes.