GloSensor cAMP Assay

To measure intracellular cAMP levels, we used the GloSensor cAMP assay. The cAMP GloSensor was obtained from Promega and the assay was performed following the manufacturer’s instructions (Promega, Madison, WI, USA). HEK293 cells were harvested (15,000 cells per individual well) in tissue culture-treated 96-well flat bottom plate. Cells were kept in 37°C tissue culture incubator 5%–10% CO₂ overnight. Cells were transfected with 50ng of pGloSensor-20F cAMP, 50ng α2-AR, 50ng RGS of interest, and pcDNA in DMEM serum free media. After 24 hours, the media is removed without disrupting the cell monolayer and 100 μl of the equilibrium medium is added (2% v/v dilution of the GloSensor cAMP Reagent stock solution). Incubation with the equilibration reagent is done for 2 hours and cells are kept in 37°C tissue culture incubator 5%–10% CO₂ in the dark. After 2 hours, basal luminescence intensity was measured at 0 and at 5 minutes using a luminometer (FLUOstar) in triplicates. To measure α2-AR-Gαi/o directed inhibition of cAMP, cells are preincubated with 100 μl of 100 μM UK14,304 (agonist) or DMSO (vehicle) in HBSS for 10 minutes at room temperature following basal readings. At 10 minutes, cAMP production is stimulated by adding 10 μM forskolin and luminescence is measured every 5 minutes for a total of 50 minutes. The data in relative light units (RLU) from triplicates wells were averaged and a response over time graph is generated. Normalization was done by dividing each time point following forskolin stimulation over basal luminescence, then each time point is divided by empty vector (50ng pcDNA alone) control. Area under the curve for each condition is calculated and the effect of the mutants is compared with the WT RGS effect in Gαi/o-coupled α2-AR stimulation of cAMP.