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

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

SUBMITTER: Mastop M 

PROVIDER: S-EPMC5837189 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature