A New Generation of FRET Sensors for Robust Measurement of G?i1, G?i2 and G?i3 Activation Kinetics in Single Cells.

ABSTRACT: 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 G?i1, G?i2 and G?i3 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 G?i subunit, and cp173Venus fused to the G?2 subunit as acceptor. The G?i FRET biosensors constructs are expressed together with G?1 from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The G?i 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 G?i 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 G?i1, G?i2 and G?i3 activation will be valuable for live-cell measurements that probe G?i activation.

SUBMITTER: van Unen J 

PROVIDER: S-EPMC4723041 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature