Molecular characterization of cardiac. beta. -adrenergic receptors
A rat cardiac cDNA clone and a rat genomic clone encoding the {beta}{sub 2}-adrenergic receptor were isolated and characterized by restriction mapping and automated DNA sequence analysis. The rat cardiac {beta}{sub 2}-receptor consists of 418 amino acids with a calculated molecular weight of 46,890 Da. In order to characterize the rat cardiac {beta}{sub 2}-receptor in more detail, B-82 cells, a murine fibroblast cell line lacking intrinsic {beta}-adrenergic receptors, were transfected with the gene encoding the rat {beta}{sub 2}-receptor, using a vector that allows stable, steroid-inducible expression of mammalian genes. Membranes prepared from transfected cells exhibited specific binding of the {beta}-receptor antagonist ({sup 125}I)iodocyanopindolol (ICYP) that was saturable and of high affinity. Displacement of ICYP binding to rat cardiac receptors by {beta}-adrenergic ligands was stereoselective and exhibited a rank order of potency for agonists expected for a {beta}{sub 2}-adrenergic receptor subtype. Expressed {beta}{sub 2}-receptors photoaffinity labeled with ({sup 125}I)iodocyanopindolol diazirine (ICYP-D) displayed as SDS-PAGE apparent molecular mass of 68,000 suggesting that the receptor is glycosylated 30% by weight. B-82 cells transfected with the rat {beta}{sub 2}-receptor gene exhibited a dose-dependent increase in intracellular levels of cyclic AMP in response to stimulation by isoproterenol.
- Research Organization:
- State Univ. of New York, Stony Brook, NY (USA). Coll. of Engineering
- OSTI ID:
- 7147208
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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MOLECULAR BIOLOGY
SYMPATHOMIMETICS
AMINO ACID SEQUENCE
AMP
BIOCHEMICAL REACTION KINETICS
CELL MEMBRANES
DNA SEQUENCING
DNA-CLONING
DOSE-RESPONSE RELATIONSHIPS
ELECTROPHORESIS
FIBROBLASTS
GENE REGULATION
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TRACER TECHNIQUES
ANIMAL CELLS
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BODY
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CELL CONSTITUENTS
CLONING
CONNECTIVE TISSUE CELLS
DAYS LIVING RADIOISOTOPES
DNA HYBRIDIZATION
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ELECTRON CAPTURE RADIOISOTOPES
HYBRIDIZATION
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550201* - Biochemistry- Tracer Techniques