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JOINT STUDY ON STRUCTURE AND FUNCTION OF GABA_B RECEPTOR

GABA_B受体结构与功能的联合研究

基本信息

批准号：
06044193
负责人：
KURIYAMA Kinya
金额：
$ 11.26万
依托单位：
KYOTO PREFECTURAL UNIVERSITY OF MEDICINE
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

项目摘要

We have previously reported that a GABA_B receptor, approximately 80 kDa in size, purified from the bovine cerebral cortex, is characterized to be one of G protein-coupled receptors. Recently, we obtained a partial amino acid sequence of the purified receptor protein, and its deduced DNA sequences were utilized for synthesizing PCR primers in various combinations of antisense oligonucleotides. In addition, another set of PCR primers was designed to include one of sequences which have been found commonly in transmembrane domains of various G protein-coupled receptors. Using various combinations of these two types of primers, PCR amplification of mRNA from the rat cerebellum gave us a PCR product. Its partial sequence indicated that the cDNA of the product appeared to code a protein possessing hydrophobic, probably transmembrane, regions. We therefore tried to extend this cDNA product toward both 5'-and 3'-ends by using the method of rapid amplification of cDNA ends (RACE). Our analysis of the RACE-products indicated that the sequences of both nucleotide and their deduced amino acid possessed possible transmembrane domains which were highly homologous to those of certain G protein-coupled receptors. Our further analysis by RT-PCR revealed that the RACE-products were expressed not only in the cerebellum but also in the forebrain, midbrain, pons, medulla and spinal cord of the rat. The present study suggests that the PCR product reported here may represents the presence of a novel G protein-coupled protein.

我们曾报道从牛大脑皮层中分离纯化出一种GABA_B受体，其分子量约为80 kDa，是一种G蛋白偶联受体。最近，我们获得了纯化的受体蛋白的部分氨基酸序列，并利用其推导的DNA序列合成各种反义寡核苷酸组合的PCR引物。此外，另一套PCR引物被设计成包括在各种G蛋白偶联受体的跨膜结构域中常见的序列之一。使用这两种类型的引物的各种组合，从大鼠小脑的mRNA的PCR扩增给我们一个PCR产物。其部分序列表明，该产物的cDNA似乎编码具有疏水性，可能是跨膜区的蛋白质。因此，我们试图通过使用cDNA末端快速扩增（RACE）方法将该cDNA产物向5 '和3'末端延伸。我们对RACE产物的分析表明，这两个核苷酸及其推导的氨基酸序列具有可能的跨膜结构域，这些结构域与某些G蛋白偶联受体的结构域高度同源。进一步的RT-PCR分析表明RACE产物不仅在小脑中表达，而且在前脑、中脑、脑桥、延髓和脊髓中也有表达。本研究表明，PCR产物在这里报告可能代表一种新的G蛋白偶联蛋白的存在。