A CE assay for the detection of agonist-stimulated adenylyl cyclase activity

A CE assay was a developed for the detection of adenylyl cyclase (AC) activity stimulated at the AC and G protein-coupled receptor (GPCR) level. In the assay, cell membranes over-expressing GPCR and/or AC were incubated with modulators and substrate ATP to produce cAMP in a dose-dependent manner. In both the CE-UV and a radiochemical assay, the addition of forskolin (FSK) resulted in a two- to three-fold maximum increase in AC activity with EC₅₀s of 4.2 ± 0.7 and 2.4 ± 0.7 μM, respectively, demonstrating that similar results were obtained by both assays. GPCR activation was also detected using cell membranes overexpressing AC and the β₂-adrenergic receptor (β₂AR) fused to the stimulatory G protein. Terbutaline (β₂AR agonist) increased the basal rate of cAMP formation 1.7 ± 0.1-fold resulting in an EC₅₀ of 62 ± 10 nM. The assay's ability to detect antagonists is demonstrated by the expected right-shifted EC₅₀ of terbutaline by the β₂AR antagonists propranolol The CE-UV assays offers advantages over the traditional radioactivity assay in terms of safety and labor.