Molecular cloning and expression of the rat β1-adrenergic receptor gene.
Molecular cloning and expression of the rat β1-adrenergic receptor gene.
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大鼠β1-肾上腺素能受体基因的分子克隆和表达。
DOI：
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发表时间：
1990
期刊：
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通讯作者：
Olivier CivellilI
中科院分区：
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作者：
C. Machida;James R. Bunzowll;Robert P. SearlesS;Hubert Van Tolll;Barbara Testerg;K. Neve;P. Teal;V. Nipper;Olivier CivellilI
摘要利用序列同源性方法克隆G蛋白偶联受体基因家族中的相关基因，我们克隆了大鼠β 1-肾上腺素能受体（β 1-AR）基因。在低严格杂交条件下，使用仓鼠β 2-肾上腺素能受体（β 2-AR）编码序列作为探针，从λ EMBL 3大鼠基因组DNA文库中分离大鼠β 1-肾上腺素能受体基因。大鼠β 1-AR基因编码466个氨基酸的蛋白质，其含有一个N-连接糖基化的共有位点（Asn-15）和三个cAMP依赖性蛋白激酶磷酸化的共有位点（Ser-296、Ser-301和Ser-401）。编码的大鼠β 1-AR在跨膜结构域和整个序列中与人β 1-AR在氨基酸水平上分别具有98%和91%的相似性。基因组Southern杂交和基因剂量分析表明，大鼠β 1-AR基因是一个单拷贝基因。大鼠β 1-AR mRNA的组织分布在松果体中最高，其他脑区和外周组织（包括心脏）以中等水平表达mRNA。将含有大鼠β 1-AR基因及其天然启动子的噬菌体克隆与赋予新霉素抗性的选择性标记（pRSVneo）共转染到β 1-AR缺陷型小鼠L细胞中。对所选转染子的分析表明β 1-AR基因和功能性受体的有效表达。125 I标记的碘氰基吲哚酚结合转染子膜，亲和力KD = 24 pm; β 1-AR选择性拮抗剂ICI 89，406取代碘氰基吲哚酚结合，Ki比β 2-AR选择性拮抗剂ICI 118，551低约140倍。此外，在转染细胞系中，腺苷酸环化酶被β-肾上腺素能受体激动剂刺激，异丙肾上腺素的效力等级顺序大于去甲肾上腺素=肾上腺素，与β 1-AR亚型的预期性质一致。
Abstract Using the sequence homology approach for cloning related genes within the G-protein-coupled receptor gene family, we have cloned the gene for the rat beta 1-adrenergic receptor (beta 1-AR). The rat beta 1-adrenergic receptor gene was isolated from a lambda EMBL3 rat genomic DNA library using the hamster beta 2-adrenergic receptor (beta 2-AR) coding sequence as a probe under low stringency hybridization conditions. The rat beta 1-AR gene encodes a protein of 466 amino acids that contains one consensus site for N-linked glycosylation (Asn-15) and three consensus sites for cAMP-dependent protein kinase phosphorylation (Ser-296, Ser-301, and Ser-401). The encoded rat beta 1-AR is 98 and 91% similar at the amino acid level with the human beta 1-AR in the transmembrane domains and in the overall sequence, respectively. Genomic Southern blot and gene dosage analyses indicate that the rat beta 1-AR gene is a single copy gene. The tissue distribution of the rat beta 1-AR mRNA was highest in the pineal gland with other brain regions and peripheral tissues, including the heart, expressing the mRNA at moderate levels. The bacteriophage clone containing the rat beta 1-AR gene with its natural promoter was co-transfected with the selectable marker (pRSVneo) conferring neomycin resistance into beta 1-AR-deficient mouse L cells. Analyses of the selected transfectant demonstrates efficient expression of the beta 1-AR gene and functional receptor. 125I-Labeled iodocyanopindolol bound transfectant membranes with an affinity of KD = 24 pm; the beta 1-AR-selective antagonist ICI 89,406 displaced iodocyanopindolol binding with a Ki approximately 140 times lower than that for the beta 2-AR-selective antagonist ICI 118,551. In addition, in the transfectant cell line, adenylylcyclase was stimulated by beta-adrenergic receptor agonists with the rank order of potency of isoproterenol greater than norepinephrine = epinephrine, consistent with properties expected of the beta 1-AR subtype.