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(230) IDENTIFYING A FAMILY OF PUTATIVE MEMBRANE PROGESTIN RECEPTORS IN VERTEBRATES IN THE NEW GENOMIC ERA. Zhu, Yong1,2, Rice, Charles3, Thomas, Peter1, 1 Marine Science Institute, Port Aransas, TX2 Department of Biology, Greenville, NC3 Department of ENTOX & Bioscience, Pendleton, SC ABSTRACT- Despite current intensive research efforts, there is no consensus for the existence of genes encoding membrane steroid receptors in vertebrates. Using a novel approach, we have discovered a new protein with characteristics of the membrane progestin receptor (mPR), which controls meiotic maturation of fish oocytes. Following characterization of the putative mPR in fish, we have successfully identified or cloned cognate cDNAs from other vertebrates including human, mouse, swine, Xenopus, zebrafish and pufferfish using the unfinished human genomic database, other genomic resources, cDNA library screening, RT-PCR, and 5′ and 3′RACE. Sequence and phylogenetic analyses indicate that these cDNAs belong to a new family composing three distinct groups. Structural analyses of translated cDNAs suggest that the proteins possess 6-7 transmembrane domains. Western blotting and immunocytochemistry also clearly localized the protein (40 kDa) on the cell plasma membrane of fish oocytes. Transcripts of three putative vertebrate mPR genes showed different expression patterns in the following fish and human tissues: gonads, brain, pituitary, placenta, kidney, adrenal gland and intestine. In addition, the steroid binding properties of several of them have been confirmed using recombinant expression systems. The recombinant proteins displayed highest binding affinities for progesterone and also bound other progestins, but did not bind corticosteroids, estrogens and androgens. Scatchard and saturation analyses of progesterone binding to the recombinant proteins revealed a single class of high affinity binding sites (KD=28-37 nM). Association and dissociation kinetics of progesterone binding to the recombinant proteins were rapid with t1/2s of 5-8 min and 2-3 min, respectively. Furthermore, progestins activated a signal transduction pathway (MAP kinase) in a mPR transfected mammalian cell line. Finally, the levels of the putative fish mPR protein changed according to hormonal stimulation or stage of oocyte maturation, which was consistent with a role for the mPR as an intermediary in progestin induction of oocyte meiotic maturation by a nongenomic mechanism. In conclusion, the available evidence indicates that this new family of cDNAs may encode membrane progestin receptors (mPR) in vertebrates. KEY WORDS: mPR, transmembrane, nongenomic, RACE |
