Expression of the human beta 2-adrenoceptor in NCB20 cells results in agonist activation of adenylyl cyclase and agonist-mediated selective down-regulation of Gs alpha.
Murine neuroblastoma x embryonic Chinese hamster brain NCB20 cells were transfected with a construct containing the human beta 2-adrenoceptor under the control of a beta-actin promoter. Two clones were selected for detailed analysis: D1, which expressed some 12.7 pmol/mg of membrane protein, and L9, which expressed 1.2 pmol/mg of membrane protein of the receptor. Incubation with the beta-adrenoceptor agonist isoprenaline resulted in stimulation of adenylyl cyclase activity in both of the clones, whereas no such activation was observed in wild-type NCB20 cells. The EC50 for isoprenaline stimulation of adenylyl cyclase activity in membranes of clone D1 (0.8 nM) was significantly lower, however, than in membranes of clone L9 (10.4 nM). Although the maximal adenylyl cyclase stimulation by isoprenaline was similar in both clones, D1 had a higher basal activity. Immunoblotting studies with specific antipeptide antisera directed against various G protein alpha subunits showed that treatment of the cells with isoprenaline resulted in a 35% reduction in the membrane-associated levels of Gs alpha in membranes of clone L9 cells and a 50% reduction in Gs alpha levels in membranes prepared from clone D1. Isoprenaline treatment had no effect on the levels of Gs alpha in wild-type NCB20 cells, and such treatment had no effect on the levels of other G protein alpha subunits such as Gq/G11 and Gi2 in any of the cell lines investigated. Time course analysis revealed that half-maximal loss of Gs alpha in clone D1 was achieved within 1-2 h of addition of agonist.(ABSTRACT TRUNCATED AT 250 WORDS)
Mullaney
I
I
Division of Biochemistry and Molecular Biology, University of Glasgow, Scotland.
Shah
B H
BH
Wise
A
A
Milligan
G
G
D000262:Adenylyl Cyclases / Q000819:agonists* / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D006224:Cricetinae / Q000196:embryology; D003412:Cricetulus; D015536:Down-Regulation / Q000187:drug effects*; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006822:Hybrid Cells; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D009447:Neuroblastoma; D012333:RNA, Messenger / Q000378:metabolism; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D015335:Molecular Probes; D012333:RNA, Messenger; D011943:Receptors, Adrenergic, beta; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol