A FRET-based biosensor for measuring Gα13 activation in single cells

Förster Resonance Energy Transfer (FRET) provides a way to directly observe the activation of heterotrimeric G-proteins by G-protein coupled receptors (GPCRs). To this end, FRET based biosensors are made, employing heterotrimeric G-protein subunits tagged with fluorescent proteins. These FRET based biosensors complement existing, indirect, ways to observe GPCR activation. Here we report on the insertion of mTurquoise2 at several sites in the human Gα13 subunit, aiming to develop a FRET-based Gα13 activation biosensor. Three fluorescently tagged Gα13 variants were found to be functional based on i) plasma membrane localization and ii) ability to recruit p115-RhoGEF upon activation of the LPA2 receptor. The tagged Gα13 subunits were used as FRET donor and combined with cp173Venus fused to the Gγ2 subunit, as the acceptor. We constructed Gα13 biosensors by generating a single plasmid that produces Gα13-mTurquoise2, Gβ1 and cp173Venus-Gγ2. The Gα13 activation biosensors showed a rapid and robust response when used in primary human endothelial cells that were exposed to thrombin, triggering endogenous protease activated receptors (PARs). This response was efficiently inhibited by the RGS domain of p115-RhoGEF and from the biosensor data we inferred that this is due to GAP activity. Finally, we demonstrated that the Gα13 sensor can be used to dissect heterotrimeric G-protein coupling efficiency in single living cells. We conclude that the Gα13 biosensor is a valuable tool for live-cell measurements that probe spatiotemporal aspects of Gα13 activation.福斯特共振能量转移（Förster Resonance Energy Transfer，FRET）为直接观测G蛋白偶联受体（G-protein coupled receptors，GPCRs）激活异三聚体G蛋白（heterotrimeric G-proteins）提供了可行手段。为此，研究人员构建了基于FRET的生物传感器，采用标记有荧光蛋白（fluorescent proteins）的异三聚体G蛋白亚基。此类基于FRET的生物传感器可弥补现有间接观测GPCR激活方法的不足。本研究报道了将mTurquoise2插入人源Gα13亚基多个位点的工作，旨在开发基于FRET的Gα13激活生物传感器。基于以下两点，我们筛选得到3个具备功能活性的荧光标记Gα13变体：①质膜定位特性；②在溶血磷脂酸2受体（LPA2 receptor）激活后能够募集p115-RhoGEF的能力。我们将该标记Gα13亚基用作FRET供体，并与融合至Gγ2亚基的cp173Venus作为受体配对。通过构建可同时表达Gα13-mTurquoise2、Gβ1与cp173Venus-Gγ2的单质粒载体，我们获得了Gα13生物传感器。当该生物传感器应用于暴露于凝血酶（可激活内源性蛋白酶激活受体（protease activated receptors，PARs））的原代人内皮细胞时，可观测到快速且稳健的响应。该响应可被p115-RhoGEF的RGS结构域（RGS domain）有效抑制；通过生物传感器数据分析，我们推断该抑制效应源于GAP活性（GAP activity）。最后，我们证实该Gα13传感器可用于解析单个活细胞内异三聚体G蛋白的偶联效率。综上，本研究开发的Gα13生物传感器是一款适用于活细胞检测的宝贵工具，可用于探究Gα13激活过程的时空特征。