Continuous high density expression of human beta 2-adrenergic receptors in a mouse cell line previously lacking beta-receptors.
The PvuII fragment of human genomic clone LCV-517 which contains the entire coding region of a beta-adrenergic receptor gene was cloned into the SmaI site of the expression vector pMSG. The recombinant DNA was cotransfected with pRSVneo into mouse B-82 cells using the CaPO4 precipitation method. B-82 cells do not possess beta-adrenergic receptors but do contain prostaglandin E1 receptors that stimulate adenylate cyclase. Following transfection, several colonies expressing beta-adrenergic receptors were isolated. Analysis of ligand binding to expressed beta-receptors indicated that the protein encoded by the gene in clone LCV-517 was a beta 2-adrenergic subtype. Human beta 2-adrenergic receptors photoaffinity labeled with [125I]iodocyanopindolol diazirine migrated on sodium dodecyl sulfate-polyacrylamide gels consistent with a molecular mass of 68,000, demonstrating that the receptor is glycosylated to an extent of 25-30% by weight. Addition of isoproterenol to cultures of transfected cells resulted in a 3-4-fold stimulation of adenylate cyclase, an effect similar to that seen in control B-82 cells with prostaglandin E1. These data describe the production of stable murine clonal cell lines expressing human beta 2-adrenergic receptors and illustrate the utility of such lines in the biochemical and pharmacological characterization of receptor proteins.
Fraser
C M
CM
Section of Receptor Biochemistry and Molecular Biology, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, Maryland 20892.
Chung
F Z
FZ
Venter
J C
JC
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D011943:Receptors, Adrenergic, beta / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D011943:Receptors, Adrenergic, beta; D004247:DNA; D000262:Adenylyl Cyclases