Kvapil P, Novotny J, Svoboda P, Ransnäs L A
Wallenberg Laboratory for Cardiovascular Research, Gothenburg University, Sweden.
Eur J Biochem. 1994 Nov 15;226(1):193-9. doi: 10.1111/j.1432-1033.1994.tb20041.x.
We report here that desensitization of the beta-adrenergic receptor-triggered transmembrane signalling in S49 wild-type lymphoma cells, induced by (-)-isoproterenol (1 microM), results in unequal intracellular redistribution of the splicing variants of the alpha subunit of the stimulatory guanine-nucleotide-binding regulatory (Gs alpha) protein (Gs alpha-short and Gs alpha-long) and alters the functional characteristics of the membrane-associated signal transduction complex. We found that two cellular pools of membranes, light-density membranes and plasma membranes prepared by sucrose-density-gradient centrifugation of cell homogenates differed in their content of Gs alpha splicing subforms and, moreover, that prolonged activation of the beta-adrenergic pathway induced intermembrane redistribution of the splicing variants of Gs alpha. Short (10 min) as well as prolonged (1 h) (-)-isoproterenol treatment of the cells shifted Gs alpha-short from light-density membranes to plasma membranes and increased the total amount of light-density membrane-bound Gs alpha-long; in parallel, the maximal (-)-isoproterenol-stimulated or AlF4(-)-stimulated adenylyl cyclase activities measured in the plasma membrane pools prepared from treated cells decreased. The functional characteristics of the membrane-bound Gs alpha pools were examined by a cyc(-)-reconstitutive adenylyl cyclase assay where extracts of the plasma membrane and light-density-membrane pools, respectively, were mixed with plasma membranes derived from the mutant S49 cell line, cyc-, lacking Gs alpha. The maximal cyc(-)-reconstitutive activities of the extracts prepared from light-density membranes of short-term as well as long-term desensitized cells increased compared to control cells. These findings may indicate differences in the functioning of the splicing variants of Gs alpha.
我们在此报告，（-）-异丙肾上腺素（1微摩尔）诱导的S49野生型淋巴瘤细胞中β-肾上腺素能受体触发的跨膜信号脱敏，导致刺激性鸟嘌呤核苷酸结合调节蛋白（Gsα）α亚基剪接变体在细胞内分布不均（Gsα-短和Gsα-长），并改变膜相关信号转导复合物的功能特性。我们发现，通过细胞匀浆的蔗糖密度梯度离心制备的两个细胞膜池，即低密度膜和质膜，其Gsα剪接亚型的含量不同，此外，β-肾上腺素能途径的长期激活诱导了Gsα剪接变体的膜间重新分布。对细胞进行短时间（10分钟）以及长时间（1小时）的（-）-异丙肾上腺素处理，可使Gsα-短从低密度膜转移至质膜，并增加低密度膜结合的Gsα-长的总量；与此同时，在处理细胞制备的质膜池中测得的最大（-）-异丙肾上腺素刺激或AlF4（-）刺激的腺苷酸环化酶活性降低。通过cyc（-）-重组腺苷酸环化酶测定法检测膜结合的Gsα池的功能特性，即将质膜和低密度膜池的提取物分别与来自缺乏Gsα的突变S49细胞系cyc-的质膜混合。与对照细胞相比，短期和长期脱敏细胞的低密度膜提取物的最大cyc（-）-重组活性增加。这些发现可能表明Gsα剪接变体在功能上存在差异。
