Basal activity (not normalized to expression)

Activity

Activity

normalized to WT

Luminescence

10 mM HEPES

25C

7.5

None

None

None

cAMP assay
24 hrs after transfection, HEK293 cells were detached and re-plated in a white 96-well plate (50,000
cells per well) and incubated for another 24 hrs. After the 24 hr incubation period, the medium was changed to 100 uL CO2-independent Opti-MEM I Reduced-Serum Medium (31985070, Life Technologies). Following a
30 min incubation period, 1 μL Glosensor substrate (Promega, PRE1290) and 11 μL FBS (Sigma-
Aldrich, F0926) were added to the cells. After pre-equilibration for 10-15 min, basal cAMP signal was
measured. Cells were then treated with either 100 μM agonist peptide (synthesized by GenScript) or
DMSO for 5 min, then activated with 50 nM isoproterenol (I650450, Sigma).
Measurements were taken using Synergy HTX BioTeck plate reader at 25°C, GAIN
200 in 5 min intervals. Data from the 15 min time point were analysed in Excel and normalized to WT