Analysis of mechanism of functions mediated by receptors of arachodonate cascade

为了阐明前列腺素的分子作用，需要克隆其受体cdna，研究其受体在表达细胞中的结构和功能，并检测其受体敲除小鼠的表型外观。在这个项目中，我们得到了以下结果：(1)克隆了至少6种小鼠PG受体cdna （EP1、EP2、EP3、EP4、FP、IP）。(2)在表达的CHO细胞中，我们检测了PG受体的结合位点和G蛋白偶联信号的功能域。(3)在EP3受体中，我们发现EP3激动剂羰基残基与EP3受体第7跨膜结构域309残基精氨酸的氢键相互作用足以激活EP3受体的功能。(4)这种相互作用对EP3D受体中Gs和Gq的激活至关重要，而不是Gi的激活。(5)制备了PGF （FP）受体和EP2/EP4受体缺失的敲除小鼠。(6) FP敲除小鼠显示生长中的胎儿无法分娩。其机制可能与黄体退化过程中PGF作用缺失有关。(7)我们一直在制作EP2和EP4敲除小鼠。EP4基因敲除小鼠具有严重的表型表现，由于血管循环功能缺陷，它们在出生后1-2天内死亡。

In order to clarify the molecular actions of prostaglandins, it needs to clone their receptor cDNAs and studied the structures and functions of their receptors in expressed cells, and to examine the phenotypic appearance of their receptor knock-out mice. In this project, we have obtained the following results ; (1) we cloned at least 6 kinds (EP1, EP2, EP3, EP4, FP,IP) of mouse PG receptor cDNAs. (2) In expressed CHO cells, we examined the functional domains of PG receptor for binding sites and signaling via G protein coupling. (3) In EP3 receptor, we showed the hydrogen bonding interation of carbonyl residue of EP3 agonists and residue 309 arginine in 7th transmembrane domain of EP3 receptor is sufficient for the functional activation. (4) This interaction is essential for the activation of Gs and Gq but not Gi in EP3D receptor. (5) we have prepared some knock-out mice which are deficient in PGF (FP) receptor and EP2/EP4 receptors. (6) FP knock-out mice revealed the loss of delivery of growing fetus. The mechanism is supposed to be involved in the deficient of PGF action in the regression of corpus luteum. (7) We have been making EP2 and EP4 knock-out mice. EP4 knock-out mice has a severe phenotypic appearance, since they die during 1-2 days after birth because of the deficient function of vascular circulation.