Biochemical Characterization and Cloning of Prostaglanoin D_2 Recoptor

前列腺素 D_2 受体的生化表征和克隆

• 批准号: 02670118
• 负责人: ITO Seiji
• 金额: $ 1.6万
• 依托单位: Osaka Bioscience Institute
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02670118/
• 关键词: Prostaglandin D_2; cAMP; onchromaffin cells; Receptor; Cloning; FRTL5 cells; EBTr cells; Astrocytes; プロスタグランジンD_2(PGD_2); アデニレ-トシクラ-ゼ

Although many biological actions of prostaglandin (PG) D_2 such as sleep induction and inhibition of platelet aggregatio, have been reported, little information is currently available concerning the mode of coupling of PGD_2 receptors to Ca^<2+> mobilization and the molecular structure of the receptor. This is mainly due to failure in detection of PGD_2 binding activity in tissues other than and platelets and the concentration of the receptor in tissues being very limited. Recently we found that nonchromaffin cells of bovine adrenal medulla and a cell line derived from bovine embryonic tracheal (EBTr) cells elevated cAMP level specifically by PGD_2. In this study, we tried to clone cDNA encoding PGD_2 receptors (I), characterized mechanisms linked to Ca^<2+> mobilization using cuitured cells (II), and reported new biological effects of PGD_2 (III).(I) A cDNA library of nonchromaffin cells was generated in the expression vector pcD2 according to the method of Okayama-Berg. After the siz … More e fractionation of cDNAs by the agarose gel electrophoresis, large size cDNA population (<greater than or equal> kb) was recloned in the same plasmid vector and transfected into FRTL5 cells by the Ca^<2+>-phosphate precipitation method. About 1500 stable transformants were screened by G418 selection and a clone A4-7 which reproducibly elevated the cAMP level 1.4-1.8-fold by PGD_2 was finally obtained. This elevation was blocked by PGD_2 antagonists BWA868C and AH6809, suggesting that this clone may express the PGD_2 receptor on the cell. Plasmid DNAs containing 15 different inserts were recovered by the Hirt method. Subclones of A4-7 were transfected into FRTL5 cells and screened for cAMP elevation by PGD_2. A PGD_2-resnsive clone A4-7-30 with a 1.3 kb insert was obtained and sequence analysis of this clone is in progress.(II) We demonstrate that there are two mechanisms of Ca^<2+> mobilization by PGD_2. In EBTr cells, PGD_2 increases intracellular Ca^<2+> concentration ([Ca^<2+>]_i) mediated by cAMP through PGD_2 receptor. In rat astrocytes, PGD_2 increases [Ca^<2+>]_i by stimulating phosphoinositide metabolism through the PGF_<2alpha>/PGD_2 receptor. These two signal transduction pathways may account for apparent subrypes of PGD_2 receptors among PGD_2-sensitive tissues and cells reported previously.(III) Although the in vivo and in vitro inhibitory effects of PGD_2 on cell growth have been attributed to the dehydrate product DELTA^<12>-PGJ_2, this study is the first to demonstrate that PGD_2 exerts an inhibitory effect on cell growth and c-myc expression in EBTr cells via cAMP. Intrathecal administration of PGD_2 and PGF_<2alpha> into mice is shown to evoke allodynia, a state of discomfort and pain induced by innocuous stimuli. This is the first exarnple of allodynia induced by natural substances and could be a model of patients with chronic pain. Less

尽管前列腺素D_2（PGD_2）的许多生物学作用如诱导睡眠、抑制血小板聚集等已被报道，但目前关于PGD_2受体与Ca^2+动员的偶联方式及受体的分子结构的资料很少。这主要是由于PGD_2在血小板以外的组织中不能检测到结合活性，而且组织中PGD_2受体的浓度非常有限。近年来，我们发现牛肾上腺髓质非嗜铬细胞和牛胚胎气管细胞系（EBTr）能特异性地通过PGD_2升高cAMP水平。在本研究中，我们试图克隆PGD_2受体的cDNA（Ⅰ），用培养的细胞研究与Ca^2+动员有关的机制（Ⅱ），并报道PGD_2的新的生物学效应（Ⅲ）。(I)根据Okayama-Berg的方法，在表达载体pcD 2中构建了非嗜铬细胞的cDNA文库。大小调整后 ...更多信息 通过琼脂糖凝胶电泳分离cDNA，将大片段cDNA群体（<greater than or equal>kb）重新克隆到同一质粒载体中，并通过Ca^2+-磷酸盐沉淀法转染FRTL 5细胞。经G418筛选，获得了1500个稳定的转化子，最终获得了一个经PGD_2处理后cAMP水平提高1.4-1.8倍的克隆A4-7。PGD_2受体拮抗剂BWA 868 C和AH 6809可阻断此作用，提示此克隆可能表达PGD_2受体。通过Hirt方法回收含有15种不同插入片段的质粒DNA。将A4-7的亚克隆转染入FRTL 5细胞，并通过PGD_2筛选cAMP升高。获得了一个PGD_2抗性克隆A4-7-30，其插入片段大小为1.3kb。(II)我们证明PGD_2的Ca^&lt;2+&gt;动员有两种机制。在EBTr细胞中，PGD_2通过PGD_2受体增加cAMP介导的细胞内Ca^2+浓度（[Ca^2+] i）。在大鼠星形胶质细胞中，PGD_2通过PGF_2/PGD_2受体刺激磷酸肌醇代谢，增加[Ca^&lt;2+&gt;]_i<2alpha>。这两条信号转导通路可能是导致PGD_2敏感组织和细胞中PGD_2受体明显亚基化的原因。(III)虽然PGD_2在体内和体外对细胞生长的抑制作用已被归因于其代谢产物DELTA^<12>-PGJ_2，但本研究首次证实PGD_2通过cAMP对EBTr细胞的生长和c-myc表达产生抑制作用。小鼠鞘内注射PGD_2和PGF_2<2alpha>可引起异常性疼痛，即由无害刺激引起的不适和疼痛状态。这是第一个由天然物质引起的异常性疼痛的例子，可以作为慢性疼痛患者的模型。少

项目成果

E.OkudaーAshitaka: "Cyclic AMPーmediated inhibition of cell growth by prostaglandin D_2 in a fibroblastic cell line(EBTr)" Eicosanoids. 3. 213-218 (1990)

E. Okuda Ashitaka：“成纤维细胞系 (EBTr) 中前列腺素 D_2 对细胞生长的环 AMP 介导的抑制”，类二十烷酸。

Toshiaki Minami: "Allodynia evoked by intrathecal administration of prostaglandin F_<2α> to conscious mice" Pain. (1992)

Toshiaki Minami：“对有意识的小鼠鞘内注射前列腺素 F_<2α> 引起的异常疼痛”(1992)。

Minami, T., Uda, R., Horiguchi, S., Ito, S., Hyodo, M., and Hayaishi, O.: "Allodynia evoked by intrathecal administration of prostaglandin F_2alpha to conscious mice." Pain. (1992)

Minami, T.、Uda, R.、Horiguchi, S.、Ito, S.、Hyodo, M. 和 Hayaishi, O.：“对清醒小鼠鞘内注射前列腺素 F_2α 引起的异常疼痛。”

Toshiaki Minami: "Allodynia evoked by intrathecal administration of prostaglandin F_<2a> to conscious mice" Pain. (1992)

Toshiaki Minami：“对有意识的小鼠鞘内施用前列腺素F_<2a>引起的异常疼痛”疼痛。

Ito, S., Narumiya, S., and Hayaishi, O.: "Prostaglandin D_2 : a biochemical perspective." Prostaglandins Leukotrienes and Essential Fatty Acids. 37. 219-234 (1989)

Ito, S.、Narumiya, S. 和 Hayaishi, O.：“前列腺素 D_2：生化视角。”