Pharmaceutical approaches using prostanoid receptor knockout mice

为了阐明甘草素的分子作用，并在此基础上开发新药，需要研究以下两点：1。前列腺素受体的结构和功能，特别是与G蛋白活化相关的结合域和功能域的研究。分析其受体的组织特异性表达及其受体敲除小鼠的表型外观。在第一个项目中，我们获得了以下结果：（1）分析了前列腺素受体与配体的相互作用，发现第七跨膜区的Arg残基是前列腺素羧酸的结合位点。(2)在EP 3受体的点突变中，我们发现激动剂和EP 3激动剂的羰基残基之间的氢键相互作用足以实现功能激活。(3)这种相互作用对于激活EP 3D受体中的Gs和Gq而不是Gi是必需的。在第二个项目中，（1）制备了PGF（FP）受体和EP 2/EP 4受体基因敲除小鼠。(2)FP基因敲除小鼠表现为生长中胎儿的分娩丢失。其作用机制可能与PGF在黄体退化过程中的作用不足有关。(3)我们一直在制造EP 2和EP 4基因敲除小鼠。EP 4基因敲除小鼠具有严重的表型外观，因为它们在出生后1-2天内由于血管循环功能缺陷而死亡。今后我们希望把实验重点放在揭示局部组织环境的特殊作用上。

In order to clarify the molecular actions of prostaglandins and the development of new drug on the basis of such molecular information, it needs to study the following two points ; 1. the structures and functions of prostanoid receptors, especially on the focus of the binding domain and functional domain relating to G protein activation, 2. analyzes of tissue specific expression of their receptors and of the phenotypic appearance of their receptor knock-out mice. In the first project, we have obtained the following results ; (1) we analyzed the prostanoid receptor-ligand interaction, and found that the Arg residue within 7th transmembrane domain is the binding site for the carboxylic acid of prostanoid. (2) In the point mutation of the EP3 receptor we obtained that the hydrogen bonding interation of agonists and carbonyl residue of EP3 agonists is sufficient for the functional activation. (3) This interaction is essential for the activation of Gs and Gq but not Gi in EP3D receptor. In the second project, (1) we have prepared some knock-out mice which are deficient in PGF (FP) receptor and EP2/EP4 receptors. (2) 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. (3) 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. In future we like to focus our experimental point to reveal the specific action of local tissue circumstances.