van Unen Jakobus, Stumpf Anette D, Schmid Benedikt, Reinhard Nathalie R, Hordijk Peter L, Hoffmann Carsten, Gadella Theodorus W J, Goedhart Joachim
Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, P.O. Box 94215, NL-1090 GE, Amsterdam, The Netherlands.
Bio-Imaging-Center/Rudolf-Virchow-Zentrum and Department of Pharmacology and Toxicology, University of Wuerzburg, Versbacher Strasse 9, 97078, Wuerzburg, Germany.
PLoS One. 2016 Jan 22;11(1):e0146789. doi: 10.1371/journal.pone.0146789. eCollection 2016.
G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gαi1, Gαi2 and Gαi3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gαi subunit, and cp173Venus fused to the Gγ2 subunit as acceptor. The Gαi FRET biosensors constructs are expressed together with Gβ1 from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gαi FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gαi FRET sensor in single cells upon stimulation of several GPCRs, including the LPA2, M3 and BK2 receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gαi1, Gαi2 and Gαi3 activation will be valuable for live-cell measurements that probe Gαi activation.
G蛋白偶联受体（GPCRs）能够以亚秒级动力学激活异源三聚体G蛋白复合物。基于Förster共振能量转移（FRET）的基因编码生物传感器非常适合用于研究单个活细胞中的此类快速信号事件。在此，我们报告了三种用于测量Gαi1、Gαi2和Gαi3激活的FRET生物传感器的构建与表征。为了能够对具有高动态范围的FRET生物传感器进行定量长期成像，需要具有增强光物理性质的荧光蛋白。因此，我们使用目前最亮且最光稳定的CFP变体mTurquoise2作为与Gαi亚基融合的供体，并使用与Gγ2亚基融合的cp173Venus作为受体。Gαi FRET生物传感器构建体与来自单个质粒的Gβ1一起表达，在哺乳动物细胞中提供了优选的相对表达水平且变异减少。Gαi FRET传感器对内源性或过表达的α-2A-肾上腺素能受体的激活表现出强烈响应，该响应被百日咳毒素抑制。此外，我们观察到在刺激包括LPA2、M3和BK2受体在内的几种GPCR时，单个细胞中的Gαi FRET传感器被激活。此外，我们表明这些传感器非常适合从毫秒时间范围内的快速测量中提取动力学参数。这种用于Gαi1、Gαi2和Gαi3激活的新一代FRET生物传感器对于探测Gαi激活的活细胞测量将具有重要价值。
