Molecular cloning and expression of the rat beta 1-adrenergic receptor gene.
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.
Machida
C A
CA
Division of Neuroscience, Oregon Regional Primate Research Center, Beaverton 97006.
Bunzow
J R
JR
Searles
R P
RP
Van Tol
H
H
Tester
B
B
Neve
K A
KA
Teal
P
P
Nipper
V
V
Civelli
O
O
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D015342:DNA Probes; D004837:Epinephrine / Q000494:pharmacology; D015870:Gene Expression; D005796:Genes; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D007739:L Cells / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009638:Norepinephrine / Q000494:pharmacology; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D015342:DNA Probes; D011943:Receptors, Adrenergic, beta; D012313:RNA; D000262:Adenylyl Cyclases; D007545:Isoproterenol; D009638:Norepinephrine; D004837:Epinephrine