Hadcock J R, Ros M, Watkins D C, Malbon C C
Department of Pharmacology, School of Medicine, State University of New York, Stony Brook 11794-8651.
J Biol Chem. 1990 Sep 5;265(25):14784-90.
The hormone-sensitive adenylyl cyclase system is under dual control, receiving both stimulatory and inhibitory inputs. Guanine nucleotide-binding regulatory proteins (G-proteins) transduce signals from cell surface receptors to effectors such as adenylyl cyclase. Hormonal stimulation is propagated via Gs, inhibition by Gi. Persistent (24-h) activation of the stimulatory pathway of adenylyl cyclase by the diterpene forskolin or the beta-adrenergic agonist isoproterenol in S49 mouse lymphoma cells enhanced the effects of somatostatin mediated via the inhibitory pathway of adenylyl cyclase. Stimulating cells with forskolin or isoproterenol for 24 h resulted in a 3-fold increase in the steady-state levels of Gi alpha 2 and a 25% decline in Gs alpha, as quantified by immunoblotting. Within 12 h of stimulation of adenylyl cyclase, Gi alpha 2 mRNA levels increased 4-fold, measured by DNA-excess solution hybridization. Gs alpha mRNA levels, in contrast, increased initially (25%), but then declined to 75% of control. In S49 variants that lack functional protein kinase A (kin-), stimulation by isoproterenol failed to alter Gi alpha 2 expression at either the protein or the mRNA levels. A 3-fold increase in relative synthesis rate and no change in the half-life (approximately 80 h) of Gi alpha 2 was observed in response to forskolin stimulation. Although Gs alpha synthesis increased (70%) modestly in response to forskolin stimulation, the half-life of Gs alpha actually decreased from 55 h in naive cells to 34 h in treated cells. Thus, the two G-protein-mediated pathways controlling adenylyl cyclase display "cross-regulation." Persistent activation of the stimulatory pathway increases Gi alpha 2 mRNA and expression. Transiently elevated Gs alpha mRNA levels are counterbalanced by a reduction in the half-life of the protein.
激素敏感型腺苷酸环化酶系统受双重控制，可接收刺激性和抑制性输入信号。鸟嘌呤核苷酸结合调节蛋白（G蛋白）将信号从细胞表面受体传导至效应器，如腺苷酸环化酶。激素刺激通过Gs蛋白进行传导，而抑制作用则通过Gi蛋白。在S49小鼠淋巴瘤细胞中，二萜类化合物福斯高林或β-肾上腺素能激动剂异丙肾上腺素对腺苷酸环化酶刺激途径的持续（24小时）激活增强了通过腺苷酸环化酶抑制途径介导的生长抑素的作用。用福斯高林或异丙肾上腺素刺激细胞24小时后，通过免疫印迹法测定，Giα2的稳态水平增加了3倍，而Gsα则下降了25%。在刺激腺苷酸环化酶12小时内，通过DNA过量溶液杂交法测定，Giα2 mRNA水平增加了4倍。相比之下，Gsα mRNA水平最初有所增加（25%），但随后降至对照的75%。在缺乏功能性蛋白激酶A的S49变体（kin-）中，异丙肾上腺素刺激未能改变Giα2在蛋白质或mRNA水平上的表达。在福斯高林刺激下，观察到Giα2的相对合成速率增加了3倍，半衰期（约80小时）没有变化。尽管福斯高林刺激后Gsα的合成适度增加（70%），但其半衰期实际上从未处理细胞中的55小时降至处理细胞中的34小时。因此，控制腺苷酸环化酶的两条G蛋白介导途径表现出“交叉调节”。刺激途径的持续激活增加了Giα2 mRNA和表达。Gsα mRNA水平的短暂升高被蛋白质半衰期的缩短所抵消。
