title: Mutations to forskolin resistance result in loss of adrenocorticotropin receptors and consequent reductions in levels of G protein alpha-subunits.
abstract: A family of mutants isolated from the Y1 mouse adrenal cell line on the basis of their resistance to the growth inhibitory effects of forskolin have an underlying mutation that affects the activity of adenylyl cyclase. As part of the mutant phenotype, adenylyl cyclase is partially resistant to activation by forskolin, completely insensitive to ACTH, and fully responsive to NaF; the levels of Gs alpha and G1 alpha in plasma membrane fractions are decreased; and the activity of G beta/gamma is impaired. In the present study, we examine the basis for the complex phenotype associated with forskolin resistance to better understand the factors that contribute to the regulation of adenylyl cyclase activity. We demonstrate that the resistance of these mutants to ACTH results from the failure to express ACTH receptor transcripts. Transfection of these mutants with a gene encoding the mouse beta 2-adrenergic receptor led to the recovery of transformants with normal receptor-G protein coupling and with increased levels of Gs alpha and G1 alpha that approached those in parental Y1 cells. These beta 2-adrenergic receptor transformants, nonetheless, remained resistant to forskolin and ACTH. Two spontaneous Y1 mutants, Y6 and OS3, previously characterized as ACTH-resistant clones that failed to accumulate ACTH receptor transcripts, were shown to be forskolin resistant and to contain less Gs alpha in membrane fractions, indicating that forskolin resistance, failure to express the ACTH receptor, and the consequent reduction in Gs alpha are closely linked. Expression of the human ACTH receptor in Y6 and OS3 cells restored ACTH-responsive adenylyl cyclase activity and increased the level of Gs alpha, but did not otherwise reverse the forskolin-resistant phenotype. Together, these results demonstrate that mutations to forskolin resistance have downstream consequences that result in the loss of ACTH receptor expression and the consequent reduction in levels of membrane-associated G alpha subunits. The results further suggest that G protein-coupled receptors may have a stabilizing influence on G alpha subunits associated with the cell membrane. According to current models, forskolin activates adenylyl cyclase by forming a ternary complex with adenylyl cyclase and Gs alpha. Our results suggest that this model may be incomplete and that an additional component, acting directly or indirectly, is required for optimal activation of adenylyl cyclase by forskolin.
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto Ontario, Canada.
authors.forename: R
authors.initials: R
authors.lastname: Qiu
authors.forename: J
authors.initials: J
authors.lastname: Tsao
authors.forename: W K
authors.initials: WK
authors.lastname: Kwan
authors.forename: B P
authors.initials: BP
authors.lastname: Schimmer
mesh_terms: D000262:Adenylyl Cyclases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D000306:Adrenal Cortex Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D000324:Adrenocorticotropic Hormone / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002999:Clone Cells; D005576:Colforsin / Q000494:pharmacology*; D004351:Drug Resistance / Q000235:genetics*; D004789:Enzyme Activation; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009154:Mutation; D010641:Phenotype; D011943:Receptors, Adrenergic, beta / Q000235:genetics / Q000378:metabolism; D018043:Receptors, Corticotropin / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D011943:Receptors, Adrenergic, beta; D018043:Receptors, Corticotropin; D005576:Colforsin; D000324:Adrenocorticotropic Hormone; D019204:GTP-Binding Proteins; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases