STUDIES OF STRUCTURE AND FUNCTION ON CEREBRAL GABA_B RECEPTOR

从牛大脑皮层中纯化gtp结合蛋白，发现该制剂含有Gi蛋白，而不含Gs和Go蛋白。将其与另外两种功能蛋白、纯化的GABA_B受体和纯化的腺苷酸环化酶重组成磷脂囊泡。这些重构实验表明，GABA_B受体通过Gi蛋白直接抑制腺苷酸环化酶活性。另一方面，分析了GABA_B受体的部分氨基酸序列。这一序列可能是G蛋白偶联受体的一部分。因此，聚合酶链反应（PCR）已经尝试使用跨膜结构域（TM），它在各种G蛋白偶联受体之间具有高度同源的序列。结果表明，该PCR产物的氨基酸序列可能为TM III ~ TM v区。并以该PCR产物为探针，筛选了大鼠胚胎脑cDNA文库。作为这些筛选的结果，获得了几个阳性克隆，这些克隆可能包含一个新的G蛋白偶联受体的编码区。这些cDNA的大小为1.5-3.0kb。

The GTP-binding protein was purified from the bovine cerebral cortex and this preparation was found to contain the Gi protein but not Gs and Go proteins. It was reconstituted into the phospholipid vesicles with another two functional proteins, purified GABA_B receptor and purified adenylyl cyclase. These reconstitutionexperimentshave indicated that the GABA_B receptor directly inhibits the adenylyl cyclase activity through the Gi protein. On the other hand, a partial amino acid sequence of the GABA_B receptor was analyzed. It has been suggested a possibility that this sequence may be a part of G protein-coupled receptor. Therefore, the polymerase-chain-reaction (PCR) has been attempted using the transmembrane domains (TM), which have highly homologous sequences among various G protein-coupled receptors. A PCR product (278 bp) was obtained and its deduced amino acid sequences indicated that it might be a region from TM III to TM V.Furthermore, a cDNA library from the rat embryo brain was screened using this PCR product as a probe. As the results of these screenings, several positive clones which contained possible coding regions of a novel G protein-coupled receptor were obtained. The size of these cDNA was found to be 1.5-3.0kb.