Fig. 7.

Assessment of the functional impact of wild-type versus mutant forms of RGS14, RGS10 and RGS4 on FSK-stimulated cAMP production by adenylyl cyclase (AC). HEK293 cells were transfected with constructs encoding GloSensor cAMP reporter, α2A-AR, and RGS proteins of interest. At time 0, cells were treated with vehicle (DMSO) and after a 10-minute incubation at RT, cells were stimulated with FSK (10 μM). Luminescence intensity indicative of cAMP production was measured every 5 minutes for 50 minutes at room temperature. Values shown in each time-course panel are means of triplicates from individual experiments, representative of three to four independent experiments. Data shown as ±S.D.; n = 3 to 4 independent experiments represent the relative luminescence intensity AUC. (A) Schematic representation of the assay measuring the effects of RGS proteins in AC stimulated cAMP. (B) RGS14-D137Y led to a significant increase in AC stimulated cAMP levels compared with WT and other mutants. Bar graph shows comparative data values of AUC. (C) RGS10-S64T led to a significant increase in AC stimulated cAMP levels compared with WT and other mutants. Bar graph shows comparative data values of AUC. (D) There was no significant difference between RGS4 WT and mutants. Bar graph shows comparative data values of AUC. Statistical analysis was performed measuring the AUC and analyzing the difference between the conditions using one-way ANOVA and Dunnett’s multiple comparison test (*P < 0.05).