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

Abstract

A CE assay was 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 overexpressing 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 EC 50 s of 4.2 14± 140.7 and 2.4 14± 140.7 14ΜM, respectively, demonstrating that similar results were obtained by both assays. GPCR activation was also detected using cell membranes overexpressing AC and the Β 2 -adrenergic receptor (Β 2 AR) fused to the stimulatory G protein. Terbutaline (Β 2 AR agonist) increased the basal rate of cAMP formation 1.7 14± 140.1-fold resulting in an EC 50 of 62 14± 1410 14nM. The assay's ability to detect antagonists is demonstrated by the expected right-shifted EC 50 of terbutaline by the Β 2 AR antagonist propranolol. The CE-UV assay offers advantages over the traditional radioactivity assay in terms of safety and labor.