title: Detection and analysis of agonist-induced formation of the complex of the stimulatory guanine nucleotide-binding protein with adenylate cyclase in intact wild-type and beta 2-adrenoceptor-expressing NG108-15 cells.
abstract: Neuroblastoma x glioma hybrid, NG108-15, cells appear to express the alpha-subunit of the guanine nucleotide-binding protein Gs in a substantial molar excess over its effector adenylate cyclase [Kim, Adie and Milligan (1994) Eur. J. Biochem. 219, 135-143]. Addition of the IP prostanoid receptor agonist iloprost to intact NG108-15 cells resulted in a dose-dependent increase in formation of the complex between Gs alpha and adenylate cyclase (GSAC) as measured by specific high-affinity binding of [3H]forskolin. NG108-15 cells transfected to express either relatively high (clone beta N22) or low (clone beta N17) levels of beta 2-adrenoceptor both showed dose-dependent increases in specific [3H]forskolin binding in response to the beta-adrenoceptor agonist isoprenaline, and maximally effective concentrations of isoprenaline resulted in the generation of similar numbers of GSAC complexes in both clones. The dose-effect curve for clone beta N22, however, was some 15-fold to the left of that for clone beta N17, which is similar to that noted for isoprenaline-mediated stimulation of adenylate cyclase activity [Adie and Milligan (1994) Biochem. J. 303, 803-808]. In contrast, dose-effect curves for iloprost stimulation of [3H]forskolin binding were not different in clones beta N22 and beta N17. Basal specific [3H]forskolin binding in the absence of agonist was significantly greater in cells of clone beta N22 than clone beta N17. This was not a reflection of higher immunological levels of adenylate cyclase, indicating that the higher basal formation of GSAC probably reflects empty-receptor activation of Gs. This higher basal specific [3H]forskolin binding was partially reversed by propranolol. The addition of the opioid peptide D-Ala-D-Leu-enkephalin to NG108-15 cells did not reduce iloprost-stimulated [3H]forskolin binding even though this peptide inhibits stimulated adenylate cyclase activity by activation of a delta opioid receptor.
authors.affiliation: Division of Biochemistry and Molecular Biology, University of Glasgow, Scotland, UK.
authors.forename: G D
authors.initials: GD
authors.lastname: Kim
authors.forename: I C
authors.initials: IC
authors.lastname: Carr
authors.forename: G
authors.initials: G
authors.lastname: Milligan
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D005576:Colforsin / Q000378:metabolism; D016308:Enkephalin, Leucine-2-Alanine / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D016285:Iloprost / Q000494:pharmacology; D007158:Immunologic Techniques; D007545:Isoproterenol / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D011433:Propranolol / Q000494:pharmacology; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D011994:Recombinant Proteins; D015398:Signal Transduction; D014162:Transfection
chemical_list: D010455:Peptides; D011943:Receptors, Adrenergic, beta; D011994:Recombinant Proteins; D005576:Colforsin; D016308:Enkephalin, Leucine-2-Alanine; D011433:Propranolol; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D016285:Iloprost; D007545:Isoproterenol