Fig. 6.

Assessment of functional impact of wild-type versus mutant forms of RGS14, RGS10 and RGS4 on α2A-AR-G protein directed inhibition of cAMP levels. 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) or agonist (● 100 μM of UK 14,304). 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 up to 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 (Area under the curve). (A) Schematic representation of the GloSensor cAMP reporter and (B) schematic representation of the assay design. (C) In the absence of RGS proteins, FSK alone increases cAMP levels while agonist-Gai/o stimulation leads to a significant decrease in cAMP. Bar graph shows comparative data values of AUC. (D) RGS14 WT compared with mutants resulted in significantly different cAMP levels over time. Bar graph shows comparative data values of AUC. (E) Comparison between RGS10 and mutants did not lead to any significant differences. Bar graph shows comparative data values of AUC. (F) Comparison between RGS4 and mutants did not lead to any significant differences. Bar graph shows comparative data values of AUC. Statistical analysis was performed measuring the AUC, using unpaired t test for C, and one-way ANOVA and Dunnett’s multiple comparison test for D–F (*P < 0.05, **P < 0.005).