Decreased levels of guanyl nucleotide-binding regulatory protein alpha-subunits in Y1 adrenocortical tumor cell mutants resistant to forskolin.
Forskolin-resistant mutants arise from Y1 mouse adrenocortical tumor cells with a frequency indicative of a mutational event at a single genetic locus and exhibit adenylyl cyclases that are resistant to activation by forskolin, corticotropin, and guanyl-5'-yl-imidodiphosphate. This study examined the levels of guanyl nucleotide-binding regulatory protein subunits (G) in plasma membranes from the forskolin-resistant mutants by Western blot immunoanalysis. In plasma membranes prepared from parental Y1 cells and from four forskolin-resistant mutants, 10r-2, 10r-3, 10r-6, and 10r-9, the levels of the alpha-subunits of Gs and Gi-2 were reduced by 70-80% relative to the levels in parental Y1 cells. The levels of the beta 36-subunit were much less affected, and the levels of the alpha i-3 and beta 35-subunits varied independently of the forskolin-resistant phenotype. As determined by slot blot hybridization analyses, the levels of Gs alpha and Gi alpha RNA in the forskolin-resistant mutants were equivalent to those in the Y1 parent. Therefore, the decreased levels of Gs alpha and Gi alpha-2 subunits observed in the forskolin-resistant mutants did not result from decreased expression of the genes encoding these proteins. Our observations suggest that the forskolin-resistant phenotype of Y1 mutants resulted from single mutations that affected the processing of specific G alpha subunits or their incorporation into the plasma membrane.
Schimmer
B P
BP
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Tsao
J
J
D000262:Adenylyl Cyclases / Q000235:genetics; D000310:Adrenal Gland Neoplasms / Q000188:drug therapy / Q000235:genetics*; D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002772:Cholera Toxin / Q000494:pharmacology; D015342:DNA Probes; D003720:Densitometry; D004351:Drug Resistance / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics*; D051379:Mice; D009154:Mutation; D010641:Phenotype; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D012313:RNA; D002772:Cholera Toxin; D011065:Poly(ADP-ribose) Polymerases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases