title: Forskolin-resistant Y1 mutants harbor defects associated with the guanyl nucleotide-binding regulatory protein, Gs.
abstract: The properties of the adenylate cyclase from forskolin-resistant mutants of Y1 adrenocortical tumor cells was compared with the properties of the enzyme from parental Y1 cells in order to localize the site of mutation. In parental Y1 cells, forskolin stimulated adenylate cyclase activity with kinetics suggestive of an interaction at two sites; in mutant cells, forskolin resistance was characterized by a decrease in enzymatic activity at both sites. Forskolin potentiated the enzyme's responses to NaF and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) in parent and mutant clones, and the mutant enzyme showed the same requirements for Mg2+ and Mn2+ as did the parent enzyme. The adenylate cyclase associated with forskolin-resistant mutants was insensitive to ACTH and was less responsive to Gpp(NH)p than was the parent enzyme. In parental Y1 cells and in the forskolin-resistant mutants, cholera toxin catalyzed the transfer of [32P]ADP-ribose from [32P]NAD+ into three membrane proteins associated with the alpha subunit of Gs; however, the amount of labeled ADP-ribose incorporated into mutant membranes was reduced by as much as 70%. Both parent and mutant membranes were labeled by pertussis toxin to the same extent. The insensitivity of the mutant adenylate cyclase to ACTH and Gpp(NH)p and the selective resistance of the mutant membranes to cholera toxin-catalyzed ADP-ribosylation suggest that a specific defect associated with Gs is involved in the mutation to forskolin resistance in Y1 cells.
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
authors.forename: B P
authors.initials: BP
authors.lastname: Schimmer
authors.forename: J
authors.initials: J
authors.lastname: Tsao
authors.forename: R
authors.initials: R
authors.lastname: Borenstein
authors.forename: L
authors.initials: L
authors.lastname: Endrenyi
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism; D000306:Adrenal Cortex Neoplasms / Q000235:genetics; D000324:Adrenocorticotropic Hormone / Q000494:pharmacology; D000818:Animals; D005576:Colforsin / Q000494:pharmacology*; D004351:Drug Resistance; D019204:GTP-Binding Proteins / Q000235:genetics*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D051379:Mice; D009154:Mutation; D009243:NAD / Q000378:metabolism; D010641:Phenotype; D012969:Sodium Fluoride / Q000494:pharmacology
chemical_list: D009243:NAD; D005576:Colforsin; D000246:Adenosine Diphosphate Ribose; D006165:Guanylyl Imidodiphosphate; D008345:Manganese; D012969:Sodium Fluoride; D000324:Adrenocorticotropic Hormone; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D008274:Magnesium