| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2005: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2004: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
The beta-type of progestin membrane receptor of medaka was newly cloned and registered as olGPCRβ with the accession number of AB239928. With this cloning we have collected all supposed three types of G-protein coupled membrane receptors for maturation-inducing steroid hormone of medaka (Oryzias latipes). Northern blot analyses showed that olGPCRα was expressed in brain, head kidney, muscle, ovary and testis, and olGPCRγ was expressed in ovary and testis. The expression of olGPCRβ was not detected in any tissues. We have never succeeded to generate recombinant receptor proteins although we had tried to express by using E.coli and HEK293 cell line.
In this project, we have been trying to generate specific polyclonal antibodies against synthetic peptides designed with predicted peptide sequences of these three receptor genes. All generated antibodies with acceptable titers against antigen peptides have been examined to detect receptor proteins by Western blotting analyses. However, we hav
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e never succeeded to get usable antibody for native receptor proteins.
We cloned medaka homologs (olPGRMC1 ; AB240046, olPGRMC2 ; AB23991) of human progesterone binding proteins, Hpr6.6 and Dg6. We have also found medaka sex hormone-binding protein from TIGR database (TC41349). One possible nuclear progestin receptor has been identified from medaka genome database (scaffold 5161) although it still needs detailed verification to distinguish from corticoid receptor.
At the end of this project we have shown successfully that medaka has 4groups/7 proteins as the receptor or the binding-protein of progestin as follows ;
1.G-protein coupled receptor : olGPCRa, olGPCRb, olGPCRg
2.nuclear receptor medaka PR
3.human membrane progesterone receptor homologs : olPGRMC1, olPGRMC2
4.Sex-hormone binding protein : medaka SHBG
We will analyze the expression levels and activities of all seven proteins along the development of medaka from fertilized egg to adult fish to find some new functions and correlations among these proteins. Less
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