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CLONING OF HISTAMINE H1-RECEPTOR cDNA

组胺 H1 受体 cDNA 的克隆

基本信息

批准号：
03670102
负责人：
FUKUI Hiroyuki
金额：
$ 1.28万
依托单位：
OSAKA 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：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

(a) Histamine H1 receptors in bovine adrenal medulla were characterized by [^3H]mepyramine binding assay and by using an affinity labeling reagent (1). When mRNA extracted from bovine adrenal medulla was injected into Xenopus oocytes, expression of H1 receptors were electrophysiologically detected (2). A cDNA library was built from mRNA of bovine adrenal medulla using an expression vector, lambdaZAPII. A histamine H1 receptor cDNA clone was isolated from 65 pools of the library containing 20,000 clones by a combination of cloning in an in vitro transcription of mRNA and electrophysiological assay of H1 receptors expressed in Xenopus oocytes which were injected with mRNA by in vitro transcription (3). The H1 receptor cDNA encoded 491 amino acid residues with molecular weight of 55,954 and seven hydrophobic amino acid sequences which are characteristic to G protein-coupled receptors. Expressed H1 receptors in mammalian cells using the H1 receptor cDNA showed common characteristics of H1 … More receptors. Antidepressants, neuroleptics and antiserotonins which have affinities to H1 receptors also showed affinities to the H1 receptor. The expressed H1 receptor mediated histamine-induced inositol phosphates accumulation and Ca^<2+> mobilization.(b) Rat histamine H1 receptor gene was cloned (4). The clone did not have an intron and encoded 486 amino acid residues. In the promotor region, a glucocorticoid response element and an AP-2 element were observed. A band of H1 receptor mRNA form rat brain was visualized by Northern blot analysis. However, that from intestine was faint and those from lung and heart were not detected, and this results suggested the low sensitivity to histamine.(c) Another [^3H]mepyramine binding protein than H1 receptors was purified from rat liver and revealed to be a relating protein of debrisoquine 4-hydroxylase, cytochrome P450. The affinity of [^3H]mepyramine binding protein in liver was 1,000 times higher than that of the H1 receptor, and [^3H]mepyramine labeled the H1 receptor in the presence of 10 muM quinine, an inhibitor of debrisoquine 4-hydroxylase. Less

(a)牛肾上腺髓质中的组胺H1受体通过[^3H]甲苯胺结合试验和亲和标记试剂进行表征（1）。当将从牛肾上腺髓质提取的mRNA注射到爪蟾卵母细胞中时，电生理检测到H1受体的表达（2）。利用表达载体pZAPII构建了牛肾上腺髓质mRNA的cDNA文库。组胺H1受体cDNA克隆是从含有20，000个克隆的文库的65个库中分离的，这是通过将mRNA体外转录克隆和非洲爪蟾卵母细胞中表达的H1受体的电生理学测定相结合进行的，非洲爪蟾卵母细胞通过体外转录注射了mRNA（3）。H1受体cDNA编码491个氨基酸残基，分子量为55，954和7个疏水氨基酸序列，这是G蛋白偶联受体的特征。使用H1受体cDNA在哺乳动物细胞中表达的H1受体显示出H1受体的共同特征。 ...更多信息受体。与H1受体有亲和力的抗抑郁药、神经安定药和抗抑郁素也显示出与H1受体的亲和力。表达的H1受体介导组胺诱导的磷酸肌醇积累和Ca^2+动员。(b)克隆大鼠组胺H1受体基因（4）。该克隆不含内含子，编码486个氨基酸残基。在启动子区域，观察到糖皮质激素反应元件和AP-2元件。用北方印迹分析显示大鼠脑H1受体mRNA有一条带。而肠组织中的组胺含量较低，肺和心组织中未检测到，提示对组胺的敏感性较低。(c)从大鼠肝脏中纯化了除H1受体外的另一种[^3H]美托咪胺结合蛋白，并证实它是异喹胍4-羟化酶细胞色素P450的相关蛋白。在肝脏中，[^3H]美托咪胺结合蛋白的亲和力比H1受体的亲和力高1,000倍，在存在10 μ M奎宁（一种异喹胍4-羟化酶抑制剂）的情况下，[^3H]美托咪胺标记H1受体。少