title: Regulation of cellular Gs alpha levels and basal adenylyl cyclase activity by expression of the beta 2-adrenoceptor in neuroblastoma cell lines.
abstract: Mouse neuroblastoma x rat glioma hybrid NG 108-15 and mouse neuroblastoma x embryonic hamster brain NCB20 cells were transfected with a construct containing a human beta 2 adrenoceptor cDNA under the control of the beta actin promoter. Clones were selected on the basis of resistance to geneticin sulphate and those expressing a range of levels of the receptor expanded for further study. Membranes from a clone of NG108-15 cells expressing high levels of the receptor (beta N22) but not one expressing only low levels of the receptor (beta N17) exhibited a markedly elevated adenylyl cyclase activity when measured in the presence of Mg2+ compared to wild type cells. This was not due to elevated levels of the adenylyl cyclase catalytic moiety however as there was no difference in these membranes when the adenylyl cyclase activity was measured in the presence of Mn2+. The elevated basal activity was partially reversed by addition of the beta-adrenoceptor antagonist propranolol. Agonist activation of beta N22 but not beta N17 cells led to a large selective down-regulation of cellular Gs alpha levels which was independent of the generation of cyclic AMP. Isoprenaline stimulation of adenylyl cyclase activity and of the specific high affinity binding of [3H] forskolin was achieved with substantially greater potency (some 30 fold) in beta N22 cell membranes than in beta N17. By contrast agonist activation of the endogenously expressed IP prostanoid receptor caused stimulation of adenylyl cyclase and stimulation of high affinity [3H] forskolin binding which was equipotent in each of beta N22, beta N17 and wild type NG108-15 cells. Agonist activation of the IP prostanoid receptor caused an equivalent degree of Gs alpha down-regulation in each cell type. Expression of an epitope tagged variant of Gs alpha in NG108-15 cells resulted in prostanoid agonist-induced down-regulation of this polypeptide in a manner indistinguishable from that of wild type Gs alpha. Isolation of clones of NCB20 cells expressing high levels of the beta 2 adrenoceptor also resulted in a specific agonist-induced down-regulation of Gs alpha.
authors.affiliation: Department of Biochemistry, University of Glasgow, Scotland, UK.
authors.forename: G
authors.initials: G
authors.lastname: Milligan
authors.forename: G D
authors.initials: GD
authors.lastname: Kim
authors.forename: I
authors.initials: I
authors.lastname: Mullaney
authors.forename: E J
authors.initials: EJ
authors.lastname: Adie
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D006224:Cricetinae; D015536:Down-Regulation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D006822:Hybrid Cells; D007545:Isoproterenol / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D051379:Mice; D009447:Neuroblastoma / Q000201:enzymology*; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D011994:Recombinant Proteins
chemical_list: D000318:Adrenergic beta-Agonists; D011943:Receptors, Adrenergic, beta; D011994:Recombinant Proteins; D008345:Manganese; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D008274:Magnesium; D007545:Isoproterenol