Biochemical Characterization and Cloning of Prostaglanoin D_2 Recoptor

虽然前列腺素（PG） d2的许多生物学作用如诱导睡眠和抑制血小板聚集已被报道，但目前关于PGD_2受体与Ca^<2+>动员的偶联模式和受体的分子结构的信息很少。这主要是由于PGD_2在除血小板外的其他组织中的结合活性检测失败，以及该受体在组织中的浓度非常有限。近年来，我们发现牛肾上腺髓质非染色质细胞和牛胚胎气管细胞（EBTr）细胞系可通过PGD_2特异性提高cAMP水平。在本研究中，我们尝试克隆编码PGD_2受体的cDNA (I)，利用培养细胞研究Ca^<2+>动员机制（II），并报道了PGD_2新的生物学效应（III）。(I)根据Okayama-Berg方法在表达载体pcD2上生成了非染色质细胞cDNA文库。琼脂糖凝胶电泳分离cDNA后，在同一质粒载体上重新克隆出较大的cDNA群体（小于等于> kb），并通过Ca^<2+>-磷酸沉淀法转染到FRTL5细胞中。通过G418筛选，筛选出约1500个稳定的转化子，最终获得了PGD_2可重复提高cAMP水平1.4 ~ 1.8倍的克隆A4-7。PGD_2拮抗剂BWA868C和AH6809阻断了PGD_2受体的表达，提示该克隆可能在细胞上表达PGD_2受体。采用Hirt法回收含有15种不同插入物的质粒dna。将A4-7亚克隆转染到FRTL5细胞中，用PGD_2筛选cAMP升高情况。获得了全长1.3 kb的pgd_2敏感克隆A4-7-30，目前正在进行序列分析。（II）我们证明了PGD_2对Ca^<2+>的动员有两种机制。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_<2alpha>/PGD_2受体刺激磷酸肌苷代谢，增加[Ca^<2+>]_i。这两种信号转导途径可能解释了先前报道的PGD_2敏感组织和细胞中PGD_2受体的明显亚型。（III）虽然PGD_2对细胞生长的体内外抑制作用归因于其脱水产物DELTA^<12>-PGJ_2，但本研究首次证明PGD_2通过cAMP对EBTr细胞的细胞生长和c-myc表达具有抑制作用。鞘内注射PGD_2和PGF_<2alpha>可引起小鼠异常性疼痛，这是一种由无害刺激引起的不适和疼痛状态。这是由自然物质引起的异常性疼痛的第一个例子，可能是慢性疼痛患者的一个模型。少

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