Fig. 2
Effect of S81L mutation on GAP activity of RGS9-2 complex. a Schematic of the assay design. Stimulation of dopamine D2 receptor (D2R) by dopamine results in the dissociation of GαoA from the heterotrimer. Released Venus-tagged Gβγ subunits become available for interaction with Nluc-tagged GRK3ct reporter, producing the BRET signal, which is determined by the change in the emission ratio at 535 nm and 480 nm. RGS9-2/Gβ5 complexes exert GTPase Activating Protein (GAP) activity and accelerate deactivation of G proteins. b Representative BRET response of cells reconstituted D2R-GoA signaling. Responses to sequential application of dopamine (100 μM) and haloperidol (100 μM) were recorded. Data are means of six replicates. c Trace lines represent the deactivation phase of D2R-GoA signaling after haloperidol application to cells transfected with different condition (left without R7BP and right with R7BP). Data are means of six replicates. d k GAP rate constants were calculated as an enzymatic activity of RGS9-2 complexes (for further details, see “Methods”) and plotted as a bar graph. The same color code was used in panel c and d. A single asterisk (*) indicates P <0.0001. One-way ANOVA followed by Dunett’s post-hoc test was conducted with GraphPad Prism Ver. 6. Results shown are representative of two independent experiments each performed with six replicates. Values represent means ± SEM