Molecular cloning and characterization of the four rat prostaglandin E2 prostanoid receptor subtypes
Molecular cloning and characterization of the four rat prostaglandin E2 prostanoid receptor subtypes
复制标题
四种大鼠前列腺素 E2 前列腺素受体亚型的分子克隆和表征
DOI：
10.1016/s0014-2999(97)01383-6
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发表时间：
1997-12-11
影响因子：
5
通讯作者：
Abramovitz, M
中科院分区：
文献类型：
--
作者：
Boie, Y;Stocco, R;Abramovitz, M
我们已经从大鼠脾、肝细胞和/或肾脏的cDNA文库中克隆了大鼠前列腺素EP1、EP2、EP3α和EP4受体亚型。大鼠EP受体的推导氨基酸序列与其在小鼠和人中的同源性分别为91%~98%和82%，与的同源性分别为91%~98%和82%。对表达EP受体的人胚胎肾(HEK)293细胞进行放射受体结合实验和功能实验。前列腺素E-2对前列腺素受体EP1、EP2、EP3α和EP4亚型的K-D值分别约为24、5、1和1 nM。前列腺素受体EP2、EP3α和EP4受体亚型与前列腺素的亲和力排序为：前列腺素E-2=前列腺素E-1；前列腺素F-2α；前列腺素D-2；U46619。前列腺素EP1受体的排列顺序基本相同，只是伊洛前列素与被测前列腺素的亲和力最高。在选择性配体中，丁前列素对前列腺素受体EP2有选择性，M&B28767和舒前列酮对EP3α有选择性，恩前列醇对前列腺素受体EP1和EP3α都有双重选择性。在HEK 293细胞中，所有四种受体都连接到各自的主要信号转导通路。值得注意的是，使用一种新的水飞蓟素发光分析来监测前列腺素EP1介导的细胞内钙的增加，伊洛前列酮和舒前列酮都被鉴定为部分激动剂。Northern印迹分析表明，EP3转录本在肝脏和肾脏中含量最高，前列腺素EP2受体在脾、肺和睾丸中表达最丰富，前列腺素EP1受体转录本主要在肾脏中表达。大鼠前列腺素EP1探针还检测到在所有被检查的组织中存在额外的和丰富的转录本。这些都被发现与一种新的蛋白激酶基因的表达有关，而不是前列腺素EP1基因的表达[Batshke，B.，Sundelin，J.，1996。小鼠的EP1前列腺素受体基因和新的蛋白激酶基因重叠。生物化学。生物群落。[中英文摘要]Re.Commun.227、1329-1333]。(C)1997年爱思唯尔科学公司。
We have characterized the rat prostanoid EP1, EP2, EP3 alpha and EP4 receptor subtypes cloned from spleen, hepatocyte and/or kidney cDNA libraries. Comparison of the deduced amino acid sequences of the rat EP receptors with their respective homologues from mouse and human showed 91% to 98% and 82% to 89% identity, respectively. Radioreceptor binding assays and functional assays were performed on EP receptor expressing human embryonic kidney (HEK) 293 cells. The K-D values obtained with prostaglandin E-2 for the prostanoid receptor subtypes EP1, EP2, EP3 alpha and EP4 were approximately 24, 5, 1 and 1 nM, respectively. The rank order of affinities for various prostanoids at the prostanoid receptor subtypes EP2, EP3 alpha and EP4 receptor subtypes was prostaglandin E-2 = prostaglandin E-1 > iloprost > prostaglandin F-2 alpha > prostaglandin D-2 > U46619. The rank order at the prostanoid EP1 receptor was essentially the same except that iloprost had the highest affinity of the prostanoids tested. Of the selective ligands, butaprost was selective for prostanoid EP2, M&B28767 and sulprostone were selective for EP3 alpha and enprostil displayed dual selectivity, interacting with both prostanoid receptor subtypes EP1 and EP3 alpha. All four receptors coupled to their predominant signal transduction pathways in HEK 293 cells. Notably, using a novel aequorin luminescence assay to monitor prostanoid EP1 mediated increases in intracellular calcium, both iloprost and sulprostone were identified as partial agonists. Finally, by Northern blot analysis EP3 transcripts were most abundant in liver and kidney whereas prostanoid EP2 receptor mRNA was expressed in spleen, lung and testis and prostanoid EP1 receptor mRNA transcripts were predominantly expressed in the kidney. The rat prostanoid EP1 probes also detected additional and abundant transcripts present in all the tissues examined. These were found to be related to the expression of a novel protein kinase gene and not the prostanoid EP1 gene [Batshake, B., Sundelin, J., 1996. The mouse genes for the EP1 prostanoid receptor and the novel protein kinase overlap. Biochem. Biophys. Res. Commun. 227, 1329-1333]. (C) 1997 Elsevier Science B.V.