CLONING OF HISTAMINE H1-RECEPTOR cDNA

(a)牛肾上腺髓质组胺H1受体通过[^3H]甲皮拉米结合试验和亲和标记试剂进行了表征(1)。将提取自牛肾上腺髓质的mRNA注射到爪蟾卵母细胞中，电生理检测H1受体的表达(2)。以lambdaZAPII为表达载体，从牛肾上腺髓质mRNA中构建cDNA文库。采用体外转录mRNA克隆和体外转录注入爪蟾卵母细胞中表达的H1受体电生理检测相结合的方法，从包含2万个克隆文库的65个池中分离出一个组胺H1受体cDNA克隆(3)。H1受体cDNA编码491个分子量为55,954的氨基酸残基和7个G蛋白偶联受体特有的疏水氨基酸序列。利用H1受体cDNA在哺乳动物细胞中表达H1受体，显示了H1受体的共同特征。与H1受体有亲和关系的抗抑郁药、神经抑制剂和抗血清素也显示出与H1受体的亲和关系。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。Northern blot分析显示大鼠脑H1受体mRNA条带。但肠组胺反应微弱，肺、心组胺反应未见，提示组胺敏感性较低。(c)从大鼠肝脏中纯化出另一种与H1受体不同的[^3H]甲锥胺结合蛋白，该蛋白与碎片喹4-羟化酶细胞色素P450相关。[^3H]新皮拉明结合蛋白在肝脏中的亲和力比H1受体高1000倍，并且[^3H]新皮拉明在10 muM奎宁存在下标记H1受体，奎宁是一种羟化酶抑制剂。少

(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