Analysis of mechanism of functions mediated by receptors of arachodonate cascade

为了阐明前列腺素的分子作用，需要克隆它们的受体cDNA，研究它们在表达细胞中的结构和功能，并检测它们的受体敲除小鼠的表型。在本项目中，我们获得了以下结果：(1)克隆了至少6种(EP1、EP2、EP3、EP4、FP、IP)小鼠PG受体基因。(2)在表达的CHO细胞中，我们通过G蛋白偶联检测了PG受体的结合部位和信号转导的功能域。(3)在EP3受体中，EP3激动剂的羰基残基与EP3受体第7跨膜区的309个精氨酸残基的氢键相互作用足以满足功能激活的需要。(4)这种相互作用是EP3D受体激活Gs和GQ所必需的，而不是激活Gi所必需的。(5)制备了PGF(FP)受体和EP2/EP4受体缺陷的基因敲除小鼠。(6)Fp基因敲除小鼠表现为生长中胎儿流产。其作用机制可能与PGF在黄体退行性变中的作用不足有关。(7)制作了EP2和EP4基因敲除小鼠。EP4基因敲除小鼠由于血管循环功能缺陷，在出生后1-2天内死亡，具有严重的表型特征。

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.