A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 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 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.

Fig 1. Development and characterization of the new Gα_i1-sensor.

(A) Representative image showing the plasma membrane localization of Gα_i1 fused to mTurquoise2-Δ9, expressed in HeLa cells. (B) Schematic overview of the plasmid containing pGβ-2A-YFP-Gγ₂-IRES-Gα_i1-CFP, driven by a CMV promoter. The inset shows the DNA sequence encoding the end of the IRES sequence and the start of the Gα_i1 sequence. The proposed protein translation is shown in the line below the DNA sequence (single letter abbreviations of the amino acids). (C) Confocal images of the localization of Gα_i1-mTurquoise2-Δ9 (top row) and cp173Venus-Gγ₂ (bottom row) in HeLa cells, for variant 1.0 (left column) and variant 2.0 (right column) of the Gα_i1-sensor. (D) Quantitative co-expression analysis of the CFP and YFP channels of the cp173Venus-Gγ₂ and Gα_i1-mTurquoise2-Δ9 transfections in HeLa cells. Single plasmid transfection (left) versus the transfection of the separate plasmids (right). The dots depict the CFP and YFP intensity, quantified from individual single cells. The r² is the coefficient of determination. Width of the individual images in A and C is 143μm.

Fig 2. Performance of the Gα_i-sensors in single cell GPCR signaling assays.

(A) FRET ratio-imaging experiments in HeLa cells transfected with the Gα_i1-sensor. Rapid loss of FRET, observed by a decreased YFP/CFP ratio, after stimulation of the cells with 10μM UK-14,304, an α₂AR specific agonist, addition of 60μM Yohimbine returns the FRET ratio towards baseline levels. Overnight treatment with (100ng/mL) PTX abolishes the response on the Gα_i1-sensor in UK-14304 stimulated cells. (B) FRET ratio-imaging experiments in Huvecs transfected with the Gα_i1-sensor. A sustained loss of FRET is observed after stimulation with 500nM S1P (Sphingosine-1-phosphate). Overnight treatment with (100ng/mL) PTX abolishes the response on the Gα_i1-sensor in S1P stimulated cells. (C) FRET ratio-imaging experiments of HeLa cells transfected with the Gα_i1-sensor and BK_2B (top-right), LPA₂ (top-left), M₃ (bottom-right) and β₂AR-2A2-mCherry (bottom-left) were stimulated with 1μM bradykinin (BK_2B), 1μM lysophosphatidic acid (LPA₂), 100μM carbachol and 10μM atropine (M₃) or 10μM isoproterenol and 10μM propranolol (β₂AR). HeLa cells transfected with BK_2B, LPA₂ and M₃ receptors show a clear change in YFP/CFP FRET ratio upon addition of their respective agonists, whereas stimulation of the β₂AR does not alter the FRET ratio of the Gα_i1-sensor. In the control conditions, HeLa cells with only the Gα_i1-sensor transfected received identical stimulations. (D) FRET ratio-imaging experiments in HeLa cells transfected with the Gα_i2-sensor or Gα_i3-sensor. Rapid loss of FRET is observed after stimulation of the cells with 10μM UK-14,304, subsequent addition of 60μM Yohimbine returns the FRET ratio towards baseline levels. Overnight treatment with (100ng/mL) pertussis toxin (PTX) abolishes the response on the Gα_i2 and Gα_i3 biosensors in UK-14304 stimulated cells. HeLa cells were stimulated with an agonist at t = 32s and an antagonist was added at t = 152s where indicated. Huvec cells were stimulated with S1P at t = 55s. Time traces show the average ratio change of YFP/CFP fluorescence (±s.e.m). Average curves consist of data from at least 3 independent experiments, conducted on different days, with the indicated number of cells (n) per condition.

Fig 3. Performance of the Gα_i1-sensor in kinetic measurements.

(A) HEK293 cells transfected with the Gα_i1-sensor and the α₂AR were repeatedly stimulated with 20μM norepinephrine during intervals that are indicated by short horizontal lines. The presented data is representative for at least six different transfections performed on six experimental days. Top panel: YFP emission, center panel: CFP emission, bottom panel: corrected and normalized FRET ratio. (B) HEK293 cells transfected with the Gα_i1-sensor and the Adenosine A1-receptor were stimulated with 30μM adenosine, indicated by the short horizontal line. The presented data is representative for at least six different transfections performed on six experimental days. Top panel: YFP emission, center panel: CFP emission, bottom panel: corrected and normalized FRET ratio. (C) A close-up of the on-kinetics of Gα_i1 activation, showing the normalized FRET ratio during the first stimulation of the experiment in (A), fitted to a one component exponential decay function with tau = 1160ms and amplitude = 0.18 (R = 0.99). (D) Scatter plot showing the average exponential time constants (tau) of pooled data from (n = 10) individual fits of HEK293 cells transfected with the Gα_i1-sensor and the α₂AR stimulated with 100μM norepinephrine or pooled data (n = 14) from individual fits of the Gα_i1-sensor and the Adenosine A1-receptor stimulated with 30μM adenosine, respectively. Error bars indicate 95% CI.