Molecular basis for the phenotype of forskolin-resistant Y1 adrenal cells

grantor: University of TorontoA family of forskolin-resistant mutants was isolated from the Y1 mouse adrenocortical tumor cell line on the basis of their resistance to the growth-inhibiting effects of forskolin. In these mutants, the adenylyl cyclase was partially resistant to forskolin and completely resistant to ACTH. The mutant phenotype was accompanied by decreased levels of Gsà and Già subunits in plasma membrane and decreased Gßã activity. These observations led to the hypothesis that a decreased Gßã activity resulted in decreased levels of membrane-associated GsÃ, which in turn, caused ACTH and forskolin-resistance. The present study was undertaken to further characterize the forskolin-resistant mutant phenotype. The ACTH resistance in the mutants is accompanied by markedly decreased levels of ACTH receptor transcript, suggesting that the loss of the ACTH-responsive adenylyl cyclase activity resulted from impaired expression of ACTH receptor gene in these cells. Transfection of mutant cells with genes encoding the ß2-adrenergic receptor or ACTH receptor restored hormone-responsive adenylyl cyclase activity and the transformants showed normal receptor-G protein coupling. In addition, the levels of Gsà and Già were increased in mutant transformants and approached the levels seen in parental Y1 cells. These results suggested that the decreased levels of Gà resulted from impaired expression of the ACTH receptor and that a functional G protein-coupled receptor seems to be required to maintain the normal levels of G protein à subunit in the plasma membrane. Transfection of mutant cells, with genes encoding an epitope-tagged Gsà also restored the Gsà levels associated with the plasma membrane. Both the receptor and Gsà transformants, however, retained forskolin-resistant adenylyl cyclase activity and were not rescued with respect to expression of endogenous ACTH-R transcripts, suggesting that the phenotypic change in forskolin-resistance mutants cells is not a direct result of low levels of membrane-associated GsÃ. To investigate the contribution of decreased Gßã activity to the mutant phenotype, forskolin-resistant mutants were transfected with expression vectors encoding G protein ß1 or ß2, and ã2 subunits. The transformants, expressed ACTH receptor transcripts and functional ACTH receptors as evidenced by the recovery of ACTH-stimulated cAMP accumulation and cell rounding. These results demonstrated a role for Gßã in the regulation of ACTH receptor gene expression. The results also indicated that a defect in Gß or Gã may be responsible for the ACTH receptor deficiency in forskolin-resistant mutants, and that the impaired expression of ACTH receptor, in turn, results in decreased levels of Gsà and GiÃ. The impaired Gßã function may also accounts for forskolin-resistant adenylyl cyclase and cell growth in these mutants.Ph.D