Cloning and expression of a prostaglandin E receptor EP3 subtype from human erythroleukaemia cells.
Cloning and expression of a prostaglandin E receptor EP3 subtype from human erythroleukaemia cells.
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从人红白血病细胞中克隆和表达前列腺素 E 受体 EP3 亚型。
DOI：
10.1042/bj2980263
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Ashby,B
中科院分区：
文献类型：
--
作者：
Kunapuli,SP;FenMao,G;Bastepe,M;Liu-Chen,LY;Li,S;Cheung,PP;DeRiel,JK;Ashby,B
前列腺素通过刺激细胞内环AMP的形成抑制血小板活化。我们推测血小板细胞内的环腺苷酸水平是由一种独特的前列腺素受体介导的环腺苷酸形成抑制缓冲。为了给该模型提供证据，我们从巨核细胞系人红白血病（HEL）细胞中克隆了编码前列腺素受体EP 3亚型的cDNA，该亚型与腺苷酸环化酶的抑制作用相结合。使用基于由Sugimoto，Namba，本田，Hayashi，Negishi，Ichikawa和Narumiya [（1992）J.Biol.Chem.267，6463-6466]公开的小鼠前列腺素EP 3受体序列的引物产生的PCR产生的杂交探针，用于筛选λ gt 11 HEL细胞cDNA文库。由两个部分克隆pHEP 3 -7和pHEP 3 -5产生的复合全长cDNA克隆HEP 3长1.6kb，具有编码390个氨基酸的开放阅读框架。该克隆与小鼠EP 3受体的α亚型具有83%的同一性。将全长构建体转染到COS-1细胞中。克隆的受体表现出前列腺素EP 3亚型的特性，抑制毛喉素刺激的环AMP的形成响应前列腺素E2（PGE 2）和结合PGE 2具有高特异性和3.2 nM的Kd。放射性标记的PGE 2可被伊洛前列素置换，顺序为PGE 2 = PGE 1>伊洛前列素= PGD 2。北方印迹分析表明该受体也存在于人肾中。
Prostaglandins inhibit platelet activation by stimulating intracellular cyclic AMP formation. We have postulated that intracellular cyclic AMP levels in platelets are buffered by a distinct prostaglandin receptor that mediates inhibition of cyclic AMP formation. In order to provide evidence for the model, we have cloned the cDNA coding for a prostaglandin receptor EP3 subtype, which is coupled to inhibition of adenylate cyclase, from the megakaryocytic cell line human erythroleukaemia (HEL) cells. A PCR-generated hybridization probe, produced using primers based on the sequence of the mouse prostaglandin EP3 receptor published by Sugimoto, Namba, Honda, Hayashi, Negishi, Ichikawa and Narumiya [(1992) J. Biol. Chem. 267, 6463-6466], was used to screen a lambda gt11 HEL cell cDNA library. The composite full-length cDNA clone HEP3, generated from the two partial clones pHEP3-7 and pHEP3-5, is 1.6 kb long with an open reading frame coding for 390 amino acids. This clone is 83% identical to the alpha subtype of the mouse EP3 receptor. The full-length construct was transfected into COS-1 cells. The cloned receptor exhibited the properties of a prostaglandin EP3 subtype, inhibiting forskolin-stimulated cyclic AMP formation in response to prostaglandin E2 (PGE2) and binding PGE2 with high specificity and a Kd of 3.2 nM. Radiolabelled PGE2 could be displaced by prostaglandins in the order PGE2 = PGE1 > iloprost = PGD2. Northern blot analysis revealed that the receptor is also present in human kidney.