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Establishment of procedures for studying mPR-interacting agents and physiological roles of mPR
Toshinobu Tokumoto1, Md Babul Hossain2, Jun Wang2
1Department of Biology, Faculty of Science, National University Corporation Shizuoka University, Ohya 836, Suruga-ku, Shizuoka 422-8529, Japan; Integrated Bioscience Section, Graduate School of Science and Technology, National University Corporation Shizuoka University, Ohya 836, Suruga-ku, Shizuoka 422-8529, Japan.
Abstract:
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.
Insights
Membrane progestin receptors (mPRs) have unclear physiological roles, but new research uses cell lines and medaka fish models to investigate their functions and potential as pharmaceutical targets.
Area of Science:
- Endocrinology and reproductive biology
- Molecular and cellular biology
- Genetics and genomics
Background:
- Membrane progestin receptors (mPRs) were discovered over a decade ago, but their precise physiological roles remain largely unknown, except for their function in fish reproduction.
- The widespread distribution of mPRs suggests diverse roles in various tissues, with emerging evidence linking them to breast cancer progression and T-cell proliferation.
- These findings highlight mPRs as potential targets for novel pharmaceutical development.
Purpose of the Study:
- To develop and utilize novel tools for investigating the physiological functions of membrane progestin receptors (mPRs).
- To screen for ligands that interact with mPRs and to understand the signaling pathways involved.
- To generate genetically modified medaka fish models to elucidate the in vivo roles of mPR subtypes.
Main Methods:
- Established a GloSensor cell line expressing mPRα and a recombinant luciferase gene to monitor intracellular cyclic adenosine monophosphate (cAMP) levels in response to ligand binding.
- Developed screening methods for identifying mPR ligands and successfully expressed and purified recombinant mPR protein from Pichia pastoris for use as a molecular probe.
- Generated medaka fish strains with induced point mutations in mPR subtypes (α, β, γ, α2) and are currently producing double and triple mutants to overcome genetic redundancy.
Main Results:
- Ligand binding to mPRα in the GloSensor cells resulted in decreased cAMP concentration, supporting its coupling to inhibitory G protein (Gi).
- Recombinant mPR protein purified from yeast demonstrated hormonal binding activity, suitable for developing molecular probes.
- Homozygous single-gene knockout medaka mutants exhibited no discernible phenotype, suggesting functional redundancy among mPR subtypes.
Conclusions:
- The developed cell lines and recombinant protein provide valuable tools for studying mPRα signaling and for screening mPR-interacting agents.
- Medaka fish models, particularly multi-gene knockout strains, are crucial for definitively establishing the physiological roles of mPRs.
- Further investigation using double and triple medaka mutants is expected to reveal the complex physiological functions of membrane progestin receptors.
