Extended Data Fig. 3: Conformational fingerprint of a positive allosteric modulator.
From: Ligand-specific activation trajectories dictate GPCR signalling in cells
(a) Representative fluorescence intensity changes (ΔF/F0) recorded in real-time from single HEK293T cells expressing indicated M2R biosensors superfused with 30 µM LY2119620 (LY), followed by 1 mM ACh after wash out with buffer. Ligand application is indicated by shaded areas in different colours. Non-shaded areas indicate buffer wash-out. (b) Mean fluorescence intensity changes (ΔF/F0) at all seven biosensors after addition of LY and ACh. Positive values indicate an increase in fluorescence, negative values indicate a fluorescence decrease. Bars indicate means ± sem, each data point represents a single cell. M2RXXX (n = X number of individual cells examined over Y number of independent experiments): M2R84 (12; 3), M2R175 (8; 3), M2R181 (23; 3), M2R188 (10; 3), M2R414 (35; 8), M2R415 (13; 4), M2R419 (11; 4). ****p < 0.0001, ***p < 0.001 (T84: 0.0006, F188: 0.0002), **p < 0.01 (F181: 0.0041), *p < 0.05 (E175: 0.0147, N419: 0.0166), according to a two-tailed paired t-test. (c) Radar plot of mean fluorescence intensity changes ΔF/F0 in response to LY, normalized to ΔF/F0 of 1 mM ACh in the same cell. The bold line indicates ACh (set to 100%). The direction of ΔF/F0 is indicated with arrows (decrease, down). The change of direction in fluorescence emission for M2R84 and M2R175 upon LY stimulation compared to ACh stimulation is highlighted in red.
