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Analysis of the signal transduction pathway through the thromboxane A_2 receptor using the receptor-G-protein reconstitution system

使用受体-G-蛋白重建系统分析血栓素 A_2 受体的信号转导途径

基本信息

批准号：
05670121
负责人：
USHIKUBI Fumitaka
金额：
$ 1.28万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05670121/
关键词：
Thromboxane Receptor GTP-binding protein Reconstitution

项目摘要

Thromboxane (TX) A_2 is a metabolite of arachidonic acid. It induces activation of platelets and constriction of vascular- and bronchial smooth muscles. It is well known that TXA_2 has physiologically and path-physologically important roles in these tissues. On the other hand, we have recently cloned a cDNA of the mouse TXA_2 receptor and have revealed its primary structure. We have also examined the distribution of the mouse TXA_2 receptor mRNA and found its broad and specific expressions in various mouse organs. In this study, we intended to construct the TXA_2 receptor-G-protein reconstitution system and to clarify the signal transduction mechanisms through the receptor using this system.We have purified Gi and Gq proteins and partially purified the TXA_2 receptor from human platelets. We also succeeded in reconstituting these proteins in phospholipid vesicles last year.This year, we examined the ligand binding characteristics of the reconstituted receptor and the agonist-induced activation of the reconstituted G-proteins. We found that the TXA_2 receptor couples to both Gq and Gi proteins. We also evaluated the efficacies and the extent of the receptor-G-protein coupling. These results explained at least partially the multiplicity of signal transduction pathways through the TAX_2 receptor and suggested the participation of the Gi protein in the signal transduction, with which TXA_2 induces cell proliferation.

血栓素A_2是花生四烯酸的代谢物。它能引起血小板的激活以及血管和支气管的平滑肌收缩。众所周知，TXA2在这些组织中具有重要的生理和路径生理作用。另一方面，我们最近克隆了小鼠血栓素A_2受体的cDNA，并揭示了其一级结构。我们还研究了小鼠TXA2受体mRNA的分布，发现其在小鼠不同器官中广泛而特异地表达。在本研究中，我们试图构建TXA2受体-G蛋白重组系统，并利用该系统阐明受体的信号转导机制。我们从人血小板中纯化了GI和GQ蛋白，并部分纯化了TXA2受体。去年，我们也成功地在磷脂囊泡中重组了这些蛋白质。今年，我们研究了重组受体的配体结合特性和激动剂诱导的重组G蛋白的激活。我们发现TXA2受体与Gq和Gi蛋白偶联。我们还评估了受体-G蛋白偶联的效果和程度。这些结果至少部分解释了TAX_2受体信号转导途径的多样性，提示Gi蛋白参与了TXA_2诱导细胞增殖的信号转导过程。