Fig. 1
Development of R-GenGAR-DA1.1, which showed a positive response to dopamine (DA). a Strategy to develop R-GenGAR-DA1.0. Left panels show a schematic illustration of human DRD1 and the red fluorescent protein ‘cpmApple’ insertion site. Right panels show screening flow chart. Linker sequences connecting DRD1 and cpmApple were randomly mutated using saturation PCR. Mutation sites in the linker are shown in red boxes. The plasmids expressing each linker mutant were isolated, followed by the transfection into HeLa cells by lipofection. Changes in fluorescence intensity following 10 μM DA stimulation was monitored by live-cell imaging of HeLa cells expressing each mutant. ICL, intracellular loop; SP, signal peptide (hemagglutinin secretory sequence). b Summary of screening results. The fluorescence changes (ΔF/F0) of the HeLa cells expressing each mutant in response to 10 μM DA stimulation are shown. Each bar represents the average of 1–3 independent experiments. We selected a mutant “R-GenGAR-DA1.1” that showed a maximum response to 10 μM DA stimulation. c Representative images of HeLa cells expressing DA1.1 stimulated with 10 µM DA. The fluorescence change (ΔF/F0) before and after DA stimulation are shown in the pseudocolor intensity-modulated display mode. d The fluorescence change (ΔF/F0) of DA1.1 in HeLa cells in panel (c). DA (10 µM) and SCH 23,390 (SCH, 10 µM) were treated at the time points indicated by pink and blue bars, respectively (Additional file 1: Fig. S5a). Mean ΔF/F0 values of 10 cells from 1 experiment are shown with SD (shaded area). e Dose–response curves, with temperature equilibration (Additional file 1: Fig. S4d), of DA (pink) and NE (green) in HeLa cells expressing DA1.1. DA: max ΔF/F0 = 0.23 ± 0.02 and EC50 = 0.45 ± 0.21 µM; NE: max ΔF/F0 = 0.17 ± 0.03 and EC50 = 5.68 ± 1.19 µM (DA and NE, n = 3 experiments in both cases, 10 cells per experiment). Experimental data (dots) were fitted with the Hill equation (lines). DA1.1 has 12.6-fold selectivity for DA over NE