Quantifying GPCR-mediated cAMP Signaling

The cell lines, culture, and cAMP assay are all described in our previous work (Apostol et al., 2021 (link)). Briefly, human PAC1, VPAC1, and VPAC2 receptors expressed in CHO cells were used for all experiments. Cells were grown in 50:50 DMEM-F12 medium with 10% heat-inactivated fetal bovine serum, 1X penicillin-streptomycin supplement, and 500 μg/ml G418 (neomycin) to maintain selection (all from ThermoFisher) at 37°C with 5% CO₂ atmosphere. For the cAMP assay, cells were seeded at 20,000 cells/well in a 96 well plate, recovered overnight, then serum starved for 4 h. The cells were then incubated with 500 μM IBMX for 20 min, followed by agonist concentration curves in 500 μM IBMX media for 10 min. The reaction was terminated, boiled, then collected, followed by competition with 7 μg of bovine protein kinase A (Sigma-Aldrich) and ∼1 pmol of ³H-cAMP (PerkinElmer). The assay was incubated at room temp for 1 h, then collected onto GF/B filter plates and the data read using a MicroBeta2 scintillation counter (PerkinElmer). The resulting data was fit to a 3 variable non-linear regression curve using GraphPad Prism, which resulted in potency (EC₅₀) and efficacy (E_MAX) measurements. Native PACAP_1–27 was used as a positive control, and also used to define an E_MAX level of 100%.