Molecular cloning and expression of the rat beta 1-adrenergic receptor gene.

摘要: Using the sequence homology approach for cloning related genes within G-protein-coupled receptor gene family, we have cloned rat beta 1-adrenergic (beta 1-AR). The was isolated from a lambda EMBL3 genomic DNA library using hamster 2-adrenergic 2-AR) coding as probe under low stringency hybridization conditions. 1-AR encodes protein of 466 amino acids that contains one consensus site N-linked glycosylation (Asn-15) and three sites cAMP-dependent kinase phosphorylation (Ser-296, Ser-301, Ser-401). encoded is 98 91% similar at acid level with human in transmembrane domains overall sequence, respectively. Genomic Southern blot dosage analyses indicate single copy gene. tissue distribution mRNA highest pineal gland other brain regions peripheral tissues, including heart, expressing moderate levels. bacteriophage clone containing its natural promoter co-transfected selectable marker (pRSVneo) conferring neomycin resistance into 1-AR-deficient mouse L cells. Analyses selected transfectant demonstrates efficient expression functional receptor. 125I-Labeled iodocyanopindolol bound membranes an affinity KD = 24 pm; 1-AR-selective antagonist ICI 89,406 displaced binding Ki approximately 140 times lower than 2-AR-selective 118,551. In addition, cell line, adenylylcyclase stimulated by beta-adrenergic agonists rank order potency isoproterenol greater norepinephrine epinephrine, consistent properties expected subtype.