Analysis of mechanism of functions mediated by receptors of arachodonate cascade

花生四烯酸级联受体介导的功能机制分析

• 批准号: 07407080
• 负责人: ICHIKAWA Atsushi
• 金额: $ 4.22万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07407080/
• 关键词: arachidonic acid; prostanoid; postaglandin receptor; 7th transmembrane receptor; GTP binding protein; signal transduction; gene targetting; structure-activity relationship; プロスタノイド受容体; G蛋白活性化ドメイン; 後根神経節; 成熟胸腺細胞; 動脈平滑筋

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.

为了阐明其分子作用，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表现。本课题取得了以下研究成果：（1）克隆了至少6种小鼠PG受体cDNA（EP 1、EP 2、EP 3、EP 4、FP、IP）。(2)在表达的CHO细胞中，我们检查了PG受体的结合位点和通过G蛋白偶联的信号传导的功能结构域。(3)在EP 3受体中，我们发现EP 3激动剂的羰基残基与EP 3受体第7跨膜结构域的309位精氨酸残基之间的氢键相互作用足以实现功能激活。(4)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。(5)我们已经制备了一些PGF（FP）受体和EP 2/EP 4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。(7)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。

项目成果

Manabu Negishi: "Selective coupling of prostaglandin E receptor EP3D to multiple G proteins depending on interaction of the carboxylic acid of agonist and arginine residue of seventh transmembrane domain" Biochemical and Biophysical Research Communication

Manabu Negishi：“前列腺素 E 受体 EP3D 与多种 G 蛋白的选择性偶联取决于激动剂的羧酸和第七跨膜结构域的精氨酸残基的相互作用”生物化学和生物物理研究通讯

Hironori Katoh: "Characterization of the signal transduction of prostaglandin E receptor EP1 subtype in cDNA-transfected Chinese hamster ovary cells" Biochimica et Biophysica Acta. 1244. 41-48 (1995)

Hironori Katoh：“cDNA 转染的中国仓鼠卵巢细胞中前列腺素 E 受体 EP1 亚型信号转导的表征”Biochimica et Biophysica Acta。

Atsushi Ichikawa: "Molecular aspects of the structtures and functions of the prostaglandin E receptors" J.Lipid Mediators Cell Signalling. 14(1-3). 83-87 (1996)

Atsushi Ichikawa：“前列腺素 E 受体结构和功能的分子方面”J.Lipid Mediators Cell Signalling。

Hiroshi Hasegawa: "Two isoforms of the prostaglandin E receptor EP3 subtype different in agonist-independent constitutive activity." J.Biol Chem.271-4. 1857-1860 (1996)

Hiroshi Hasekawa：“前列腺素 E 受体 EP3 亚型的两种亚型在不依赖激动剂的组成活性方面有所不同。”

Masato Katsuyama: "The mouse prostaglandin E receptor EP2 subtype : cloning, expression, and Northem blot analysis." FEBS Lett.372. 151-156 (1995)

Masato Katsuyama：“小鼠前列腺素 E 受体 EP2 亚型：克隆、表达和 Northem 印迹分析。”