A FRET-based biosensor for measuring G alpha 13 activation in single cells

作者：Mastop Marieke; Reinhard Nathalie R; Zuconelli Cristiane R; Terwey Fenna; Gadella Theodorus W J Jr; van Unen Jakobus; Adjobo Hermans Merel J W; Goedhart Joachim^*

来源：PLos One, 2018, 13(3): e0193705.

DOI：10.1371/journal.pone.0193705

Fo E rster 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 G alpha 13 subunit, aiming to develop a FRET-based G alpha 13 activation biosensor. Three fluorescently tagged G alpha 13 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 G alpha 13 subunits were used as FRET donor and combined with cp173Venus fused to the G gamma 2 subunit, as the acceptor. We constructed G alpha 13 biosensors by generating a single plasmid that produces G alpha 13-mTurquoise2, G ss 1 and cp173Venus-G gamma 2. The G alpha 13 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 G alpha 13 sensor can be used to dissect heterotrimeric G-protein coupling efficiency in single living cells. We conclude that the G alpha 13 biosensor is a valuable tool for live-cell measurements that probe spatiotemporal aspects of G alpha 13 activation.

• 出版日期2018-3-5