基于BRET的G蛋白生物传感器用于测量活细胞中的G蛋白结合受体活性
Adeline Cœugnet1, Hannes Schihada2, Estelle Rascol1
1CNRS, Bordeaux INP, CBMN, UMR, University of Bordeaux.
Journal of visualized experiments : JoVE
|November 24, 2025
概括
G蛋白结合受体 (GPCRs) 是细胞信号传递的关键. 新的G-CASE生物传感器使用生物发光共振能量转移 (BRET) 进行敏感的实时GPCR活动监测,帮助药物发现.
科学领域:
- 细胞信号传递和分子生物学.
- 受体药理学和药物发现.
背景情况:
- G蛋白结合受体 (GPCRs) 是关键的跨膜受体,调解细胞反应.
- GPCR激活涉及异构三分子G蛋白解离,这是一个关键的信号事件.
- 目前研究GPCR激活的现有方法存在局限性.
研究的目的:
- 引入和验证基于G蛋白的三性活性传感器 (G-CASE生物传感器) 以实时监测GPCR活动.
- 为了证明G-CASE生物传感器在使用BRET的活细胞测试中的实用性.
- 评估特定GPCRs的激活,包括β2-上腺素受体 (β2-AR) 和大麻素1型受体 (CB1R).
主要方法:
- 使用基于生物发光共振能量转移 (BRET) 的G-CASE生物传感器.
- 在96孔板上进行活细胞测试.
- 感染了带有β2-AR的HEK 293T细胞,并使用稳定表达CB1R的细胞来测试Gs和Gi3 G-CASE生物传感器.
主要成果:
- 在HEK 293T细胞中证明了Gs和Gi3 G-CASE生物传感器的稳定性.
- 通过G蛋白激活,成功地实时监测了GPCR活性.
- 证实了基于BRET的G-CASE生物传感器的灵敏度和非侵入性.
结论:
- G-CASE生物传感器为研究GPCR活性提供了一种敏感且高效的方法.
- 这种方法促进了药理学研究和GPCR配体的高通量选.
- 这些发现支持通过阐明有偏见的信号通路来发现新型治疗化合物.
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