Cell-based assay of nongenomic actions of progestins revealed inhibitory G protein coupling to membrane progestin receptor α (mPRα).
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,20β-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.
Biological Science Course, Graduate School of Science, National University Corporation Shizuoka University, Oya 836, Suruga-ku, Shizuoka 422-8529, Japan.
Mikiko
M
Nakashima
Department of Biology, Faculty of Science, National University Corporation Shizuoka University, Oya 836, Suruga-ku, Shizuoka 422-8529, Japan.
Manami
M
Suzuki
Spectrography and Bioimaging Facility, National Institute for Basic Biology, Myodaiji, Okazaki, Aichi 445-8585, Japan.
Misako
M
Saida
Spectrography and Bioimaging Facility, National Institute for Basic Biology, Myodaiji, Okazaki, Aichi 445-8585, Japan; Department of Basic Biology, School of Life Science, The Graduate University for Advanced Studies (SOKENDAI), Okazaki 444-8585, Japan.
Yasuhiro
Y
Kamei
Integrated Bioscience Section, Graduate School of Science and Technology, National University Corporation Shizuoka University, Oya 836, Suruga-ku, Shizuoka 422-8529, Japan.
Md Babul
MB
Hossain
Biological Science Course, Graduate School of Science, National University Corporation Shizuoka University, Oya 836, Suruga-ku, Shizuoka 422-8529, Japan; Integrated Bioscience Section, Graduate School of Science and Technology, National University Corporation Shizuoka University, Oya 836, Suruga-ku, Shizuoka 422-8529, Japan. Electronic address: sbttoku@ipc.shizuoka.ac.jp.
Toshinobu
T
Tokumoto
D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000378:metabolism; D029941:Fish Proteins / Q000502:physiology*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology*; D006801:Humans; D011372:Progestins / Q000502:physiology*; D011980:Receptors, Progesterone / Q000502:physiology*; D015290:Second Messenger Systems
D029941:Fish Proteins; D011372:Progestins; D011980:Receptors, Progesterone; D000242:Cyclic AMP; D019206:GTP-Binding Protein alpha Subunits, Gi-Go
Diethylstilbestrol (DES); Endocrine disruptor; Goldfish; Luciferin; Membrane progestin receptor; Nongenomic steroid action