Molecular Cloning and Expression of a cDNA of the Bovine Prostaglandin F_<2alpha> Receptor

为了阐明前列腺素F_2α&gt；受体的结构和功能，我们用聚合酶链式反应方法从牛黄体的cDNA文库中克隆了编码前列腺素F_2α&gt；受体的基因。用多对简并引物通过聚合酶链式反应方法分离得到的SN463克隆含有血栓素A_2受体跨膜基序IV~VI的同源序列。将该扩增产物作为探针DNA进行杂交，从牛黄体cDNA文库中分离到一个含有2.2kbp插入DNA的克隆BC2211。该克隆编码362个氨基酸残基(Mr为40,983)，具有7个潜在的跨膜区。将体外合成的基因注入非洲爪哇卵母细胞中，对代表BC2211的PGF_2α&gt；产生电生理反应，编码有功能的全长cDNA。在哺乳动物COS-7细胞膜上的配体结合位移表明，受体与血栓素A_2激动剂PGs：PGF_&lt；2alpha&gt；&gt；PGD_2&gt；PGE_2&gt；STA_2，的亲和力顺序为。Northern印迹分析和原位杂交结果表明，在性周期和妊娠过程中，PGF_2α&gt；受体的mRNA在黄体中高度表达和积聚，PGF_2α&gt；受体的mRNA水平在黄体中受到调节。这些数据将有助于进一步了解受体功能的分子机制。

In order to elucidate the structure and function of prostaglandine F_<2alpha> receptor, we have used a polymerase chain reaction (PCR) approach to isolate a cDNA encoding the PGF_<2alpha> receptor from a bovine corpus luteal cDNA library. The clone SN463 which was isolated by PCR method by using several pairs of degenerated primers carried the homologous sequence which covered transmembrane motif IV to VI of thromboxane (TX) A_2 receptor. This PCR product was used as a probe DNA for following cross hybridization and a clone BC2211 carrying a 2.2-kbp insert DNA was isolated form bovine corpus luteal cDNA library. This clone encodes a protein of 362 amino acid residues (Mr 40,983) with seven potential transmembrane domains. Injection of the mRNA synthesized in vitro from the cloned cDNA into a Xenopus oocyte elicited electrophysiological response to PGF_<2alpha>, representing BC2211 encodes functional full-length cDNA.Ligand-binding displacement in membranes of mammalian COS-7 cells transfected with the cDNA indicated the rank order of affinity of the receptor to PGs : PGF_<2alpha>>PGD_2>PGE_2>STA_2, a TXA_2 agonist. PGF_<2alpha> activated InsP formation in COS-7 cells transfected with receptor cDNA.Northern blot analysis and in situ hybridization indicated that the PGF_<2alpha> receptor mRNA is highly expressed and accumulated in corpus luteum and mRNA level of PGF_<2alpha> receptor was regulated in corpus luteum during the sexual cycle and pregnancy. These data should help further understanding of molecular mechanism of the receptor function.