Stable overexpression of human beta 2-adrenergic receptors in mammalian cells.
Human beta 2-adrenergic receptors were overexpressed in chinese hamster ovary (CHO) and human epitheloid carcinoma (HeLa) cells. Stable expression in these cells was achieved by transfection of a vector containing the cDNAs for the human beta 2-adrenergic receptor as well as for dihydrofolate reductase. By stepwise increases of the concentration of methotrexate - an inhibitor of dihydrofolate reductase - the expression in CHO cells could be increased to levels of almost 200 pmol/mg membrane protein, which is more than 1% of the total membrane protein. In contrast, overexpression of the receptors in HeLa cells by the same technique led to cell death. The receptors produced in overexpressing CHO cells were correctly processed and were fully functional with respect to their ligand binding and signalling properties. The adenylyl cyclase activity of membranes from these cells responded with extremely high sensitivity to the beta-adrenergic receptor agonist isoproterenol. The receptors could be purified from these membranes to apparent homogeneity by solubilization and chromatography on a single affinity column. Thus, the expression system described here allows the preparation of human beta 2-adrenergic receptors in quantities sufficient for pharmacological and biochemical investigations.
Lohse
M J
MJ
Laboratorium für Molekulare Biologie, Universität München, Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D015972:Gene Expression Regulation, Neoplastic; D006168:Guinea Pigs; D006367:HeLa Cells; D006801:Humans; D007457:Iodine Radioisotopes; D020120:Iodocyanopindolol; D010869:Pindolol / Q000031:analogs & derivatives; D011943:Receptors, Adrenergic, beta / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000378:metabolism
D007457:Iodine Radioisotopes; D011943:Receptors, Adrenergic, beta; D020120:Iodocyanopindolol; D010869:Pindolol; D000262:Adenylyl Cyclases