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

C. A. Machida, J. R. Bunzow, R. P. Searles, H. Van Tol, B. Tester, K. A. Neve, P. Teal, V. Nipper, O. Civelli

Research output: Contribution to journalArticle

110 Scopus citations

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-p15) and three consensus sites for cAMP-dependent protein kinase phosphorylation (der-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 K(D) = 24 pm; the β1-AR-selective antagonist ICI 89,406 displaced iodocyanopindolol binding with a K(i) ~ 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 - 1990

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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