FIGURE 1.
D2-A4-mediated inhibition of cyclic AMP accumulation and G protein activation. A, HEK293T cells transfected with D2-WT or D2-A4 and CAMYEL, a BRET-based cAMP sensor. The inhibition of forskolin (10 μm)-stimulated cAMP by quinpirole at the indicated concentrations was measured after 10 min. Dose-response curves are representative of three independent experiments performed with triplicate samples (mean ± S.E.). B, maximal inhibition of forskolin-stimulated cAMP by D2-A4 in response to quinpirole or dopamine stimulation determined from concentration-response curves and expressed as a percentage of the activation by D2-WT. Each bar represents the mean ± S.E. (error bars) of three independent experiments. *, p < 0.05 compared with D2-WT. C, schematic of the heterotrimeric G protein BRET biosensor. G protein activation is detected as a decrease in BRET between the Gαi1-Rluc8 donor and complemented mVenus-Gβ1γ2 acceptor. D, HEK293T cells transfected with the components of the G protein biosensor depicted in C and D2-WT or D2-A4 receptors were incubated with quinpirole at the indicated concentrations for 2 min before measuring the G protein BRET response. Dose-response curves are representative of three independent experiments performed with triplicate samples (mean ± S.E.). E, maximal G protein activation by D2-A4 in response to quinpirole or dopamine stimulation determined from concentration response curves and expressed as a percentage of the activation by D2-WT. Each bar represents the mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01 compared with D2-WT within the same treatment group, Student's t test. For the cylic AMP experiments, D2-A4 expression was 104 ± 18% that of the wild-type receptor, whereas for the G protein activation experiments, D2-A4 expression was 80 ± 3% that of the wild type receptor.