Establishment of procedures for studying mPR-interacting agents and physiological roles of mPR.

More than 10years have passed since the discovery of membrane progestin receptors (mPRs). Although the identification of mPR genes in various organisms and mPR expression patterns have been described since then, the precise physiological roles of mPRs are still unclear, except their function as a receptor for maturation-inducing steroid in fish. The wide distribution of mPRs suggests variable actions for progestins through mPRs in the tissues. Information about the physiological roles of mPRs, such as roles in the progression of breast cancer and T-cell proliferation, has gradually accumulated recently. These results suggest that mPRs are possible targets for new pharmaceuticals. We established a cell line that was transformed with cDNAs for mPRα and a recombinant luciferase gene named GloSensor. The cells can be used for monitoring the effects of ligands on mPRα based on intracellular cyclic adenosine monophosphate (cAMP) levels. Studies using these cell lines indicated that the cAMP concentration is decreased by ligands for mPRα. The results provide support for previous results suggesting that mPRα is coupled to inhibitory G protein (Gi). We also established screening methods that make it possible to screen ligands for mPR. Recently, we succeeded in expressing and purifying recombinant mPR protein in the yeast Pichia pastoris. Relatively large amounts of mPR protein with hormonal binding activity can be purified by our method. The recombinant protein will be applicable to establishing a molecular probe to detect mPR-interacting agents. To obtain decisive evidence for the roles of mPRs, we are establishing strains of medaka fish that are deficient in mPRs. In medaka, four subtypes of mPR genes (α, β, γ, and α2) have been identified. By reverse genetic screening, we have selected three to four strains in which a point mutation has been induced in the coding sequence of the mPR subtypes. However, homozygous mutants of each mPR gene showed no phenotype. The results suggested that mPR genes share redundancy. We are currently producing double and triple mutants of the mPR subtypes. The physiological roles of mPRs will be demonstrated using the mutant medaka strains.