Schimmer B P, Tsao J
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Mol Endocrinol. 1990 Nov;4(11):1698-703. doi: 10.1210/mend-4-11-1698.
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.
福斯高林抗性突变体源自Y1小鼠肾上腺皮质肿瘤细胞，其出现频率表明在单个基因位点发生了突变事件，并且表现出对福斯高林、促肾上腺皮质激素和鸟苷-5'-基-亚氨基二磷酸激活具有抗性的腺苷酸环化酶。本研究通过蛋白质印迹免疫分析检测了福斯高林抗性突变体细胞膜中鸟苷酸结合调节蛋白亚基（G）的水平。在由亲代Y1细胞以及四个福斯高林抗性突变体10r-2、10r-3、10r-6和10r-9制备的细胞膜中，Gs和Gi-2的α亚基水平相对于亲代Y1细胞中的水平降低了70 - 80%。β36亚基的水平受影响较小，αi-3和β35亚基的水平变化与福斯高林抗性表型无关。通过狭缝印迹杂交分析确定，福斯高林抗性突变体中Gsα和GiαRNA的水平与Y1亲代中的水平相当。因此，在福斯高林抗性突变体中观察到的Gsα和Giα-2亚基水平降低并非由编码这些蛋白质的基因表达降低所致。我们的观察结果表明，Y1突变体的福斯高林抗性表型是由影响特定Gα亚基加工或其整合到细胞膜中的单个突变引起的。
