Cell-based assay of nongenomic actions of progestins revealed inhibitory G protein coupling to membrane progestin receptor α (mPRα).

ABSTRACT

Previously, we established cell lines stably producing goldfish membrane progestin receptor α (goldfish mPRα) proteins, which mediate steroidal nongenomic actions. In this study, we transfected these cell lines (MDA-MD-231) with cDNAs encoding a recombinant luciferase gene (GloSensor). These cells can be used for monitoring the effects of ligands that bind to mPR by means of luminescence, the intensity of which reflects intracellular cyclic adenosine monophosphate (cAMP) levels. Luminescence intensity of the cells increased significantly when cells were treated with forskolin, strong activator of adenylyl cyclase. Then, we established a strategy to measure changes in luminescence that correlated with the actions of the ligands. The actions of ligands were measurable by the prevention of stimulation caused by forskolin after ligand stimulation. The studies using these cell lines indicated that cAMP concentrations were decreased specifically by the mPR ligands 17α,20ß-dihydroxy-4-pregnen-3-one, diethylstilbestrol and progesterone. Furthermore, pertussis toxin inhibited the decrease in cAMP levels caused by mPR ligands. These results support evidence from previous results that mPRα is coupled to an inhibitory G protein.