Molecular cloning and expression of the rat β1-adrenergic receptor gene

Curtis Machida, James R. Bunzow, Robert Searles, Hubert Van Tol, Barbara Tester, Kim Neve, Peter Teal, Valerie Nipper, Olivier Civelli

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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 β1-adrenergic receptor (β1-AR). The rat β1-adrenergic receptor gene was isolated from a λ EMBL3 rat genomic DNA library using the hamster β2-adrenergic receptor (β2-AR) coding sequence as a probe under low stringency hybridization conditions. The rat β1-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 β1-AR is 98 and 91% similar at the amino acid level with the human β1-AR in the transmembrane domains and in the overall sequence, respectively. Genomic Southern blot and gene dosage analyses indicate that the rat β1-AR gene is a single copy gene. The tissue distribution of the rat β1-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 β1-AR gene with its natural promoter was co-transfected with the selectable marker (pRSVneo) conferring neomycin resistance into β1-AR-deficient mouse L cells. Analyses of the selected transfectant demonstrates efficient expression of the β1-AR gene and functional receptor. 125I-Labeled iodocyanopindolol bound transfectant membranes with an affinity of KD = 24 pm; the β1-AR-selective antagonist ICI 89,406 displaced iodocyanopindolol binding with a Ki ∼ 140 times lower than that for the β2-AR-selective antagonist ICI 118,551. In addition, in the transfectant cell line, adenylylcyclase was stimulated by β-adrenergic receptor agonists with the rank order of potency of isoproterenol > norepinephrine = epinephrine, consistent with properties expected of the β1-AR subtype.

Original languageEnglish (US)
Pages (from-to)12960-12965
Number of pages6
JournalJournal of Biological Chemistry
Volume265
Issue number22
StatePublished - Aug 5 1990

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Cloning
Molecular Cloning
Adrenergic Receptors
Rats
Genes
Iodocyanopindolol
Adrenergic Antagonists
Amino Acids
Adrenergic Agonists
Messenger RNA
Tissue
Pineal Gland
Neomycin
Gene Dosage
Genomic Library
Glycosylation
Tissue Distribution
Phosphorylation
Bacteriophages
Sequence Homology

ASJC Scopus subject areas

  • Biochemistry

Cite this

Machida, C., Bunzow, J. R., Searles, R., Van Tol, H., Tester, B., Neve, K., ... Civelli, O. (1990). Molecular cloning and expression of the rat β1-adrenergic receptor gene. Journal of Biological Chemistry, 265(22), 12960-12965.

Molecular cloning and expression of the rat β1-adrenergic receptor gene. / Machida, Curtis; Bunzow, James R.; Searles, Robert; Van Tol, Hubert; Tester, Barbara; Neve, Kim; Teal, Peter; Nipper, Valerie; Civelli, Olivier.

In: Journal of Biological Chemistry, Vol. 265, No. 22, 05.08.1990, p. 12960-12965.

Research output: Contribution to journalArticle

Machida, C, Bunzow, JR, Searles, R, Van Tol, H, Tester, B, Neve, K, Teal, P, Nipper, V & Civelli, O 1990, 'Molecular cloning and expression of the rat β1-adrenergic receptor gene', Journal of Biological Chemistry, vol. 265, no. 22, pp. 12960-12965.
Machida, Curtis ; Bunzow, James R. ; Searles, Robert ; Van Tol, Hubert ; Tester, Barbara ; Neve, Kim ; Teal, Peter ; Nipper, Valerie ; Civelli, Olivier. / Molecular cloning and expression of the rat β1-adrenergic receptor gene. In: Journal of Biological Chemistry. 1990 ; Vol. 265, No. 22. pp. 12960-12965.
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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 β1-adrenergic receptor (β1-AR). The rat β1-adrenergic receptor gene was isolated from a λ EMBL3 rat genomic DNA library using the hamster β2-adrenergic receptor (β2-AR) coding sequence as a probe under low stringency hybridization conditions. The rat β1-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 β1-AR is 98 and 91{\%} similar at the amino acid level with the human β1-AR in the transmembrane domains and in the overall sequence, respectively. Genomic Southern blot and gene dosage analyses indicate that the rat β1-AR gene is a single copy gene. The tissue distribution of the rat β1-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 β1-AR gene with its natural promoter was co-transfected with the selectable marker (pRSVneo) conferring neomycin resistance into β1-AR-deficient mouse L cells. Analyses of the selected transfectant demonstrates efficient expression of the β1-AR gene and functional receptor. 125I-Labeled iodocyanopindolol bound transfectant membranes with an affinity of KD = 24 pm; the β1-AR-selective antagonist ICI 89,406 displaced iodocyanopindolol binding with a Ki ∼ 140 times lower than that for the β2-AR-selective antagonist ICI 118,551. In addition, in the transfectant cell line, adenylylcyclase was stimulated by β-adrenergic receptor agonists with the rank order of potency of isoproterenol > norepinephrine = epinephrine, consistent with properties expected of the β1-AR subtype.",
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