Sugimoto Y, Namba T, Honda A, Hayashi Y, Negishi M, Ichikawa A, Narumiya S
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
J Biol Chem. 1992 Apr 5;267(10):6463-6.
A functional cDNA clone for mouse EP3 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library using polymerase chain reaction based on the sequence of the human thromboxane A2 receptor and cross-hybridization screening. The mouse EP3 receptor consists of 365 amino acid residues with putative seven-transmembrane domains. The sequence revealed significant homology to the human thromboxane A2 receptor. Ligand binding studies using membranes of COS cells transfected with the cDNA revealed specific [3H]PGE2 binding. The binding was displaced with unlabeled PGs in the order of PGE2 = PGE1 greater than iloprost greater than PGF2 alpha greater than PGD2. The EP3-selective agonists, M&B 28,767 or GR 63799X, potently competed for the [3H]PGE2 binding, but no competition was found with EP1- or EP2-selective ligands. PGE2 and M&B 28,767 decreased forskolin-induced cAMP formation in a concentration-dependent manner in Chinese hamster ovary cells permanently expressing the cDNA. Northern blot analysis demonstrated that the EP3 mRNA is expressed abundantly in kidney, uterus, and mastocytoma P-815 cells and in a lesser amount in brain, thymus, lung, heart, stomach, and spleen.
基于人血栓素A2受体序列并通过杂交筛选，利用聚合酶链反应从小鼠cDNA文库中分离出前列腺素（PG）E受体小鼠EP3亚型的功能性cDNA克隆。小鼠EP3受体由365个氨基酸残基组成，具有推定的七跨膜结构域。该序列与人血栓素A2受体具有显著同源性。使用转染了该cDNA的COS细胞膜进行的配体结合研究显示出特异性的[3H]PGE2结合。未标记的PG以PGE2 = PGE1大于伊洛前列素大于PGF2α大于PGD2的顺序取代该结合。EP3选择性激动剂M&B 28,767或GR 63799X强烈竞争[3H]PGE2结合，但未发现与EP1或EP2选择性配体存在竞争。PGE2和M&B 28,767以浓度依赖的方式降低了在永久表达该cDNA的中国仓鼠卵巢细胞中福斯高林诱导的cAMP形成。Northern印迹分析表明，EP3 mRNA在肾脏、子宫和肥大细胞瘤P-815细胞中大量表达，而在脑、胸腺、肺、心脏、胃和脾脏中的表达量较少。
