Olate J, Mattera R, Codina J, Birnbaumer L
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
J Biol Chem. 1988 Jul 25;263(21):10394-400.
We placed the cDNAs encoding one of the short types of alpha s (alpha s-1) with Asp-Ser in positions 70 and 71 and one of the long types of alpha s (alpha s-2) in which Asp-Ser are substituted with a string of 16 amino acids, into the pGEM-3 transcription vector downstream from its T7 RNA polymerase promoter, obtained transcripts and translated the mRNAs using a rabbit reticulocyte lysate system, to determine if the molecules would be synthesized and, if so, whether they would be active as assessed in cyc- reconstitution assays. The translation products obtained from both alpha s RNAs were a mixture of primarily three polypeptides of which one (approximately 40-50% of total) represented the complete translation product and the other two appeared to be due to internal translation starts at Met60, before the splice difference between the RNAs, and the other at the first Met after the splice difference. Lysates incubated with short or long alpha s RNA when added to cyc- membranes reconstituted fluoride and GTP[gamma S]-stimulated activities. Thus, in vitro synthesized alpha s subunits are active in interacting both with guanine nucleotides and the adenylyl cyclase enzyme. On incubation without and with the receptor agonist isoproterenol, using GTP as sole added guanine nucleotide, both types of alpha s subunits reconstituted the isoproterenol-stimulated adenylyl cyclase activity. Thus, the synthetic alpha s also interact with receptors, and by inference with beta-gamma dimers, shown previously to be needed for activation by receptor. Quantitative assays in which the activity of the synthetic alpha s-1 was compared to that of native purified human erythrocyte type-1 Gs, indicated that the two products are equipotent within a 2-fold margin of error. Thus, the lysate made fully active alpha s subunits, and alpha s subunits require no post-translational modifications dependent on microsomal processes. This approach may be useful in studying biological functions of other cloned alpha subunits of G proteins.
我们将编码短型αs（αs-1）（第70和71位为天冬氨酸-丝氨酸）和长型αs（αs-2，天冬氨酸-丝氨酸被16个氨基酸的序列取代）的cDNA，置于pGEM-3转录载体中T7 RNA聚合酶启动子下游，获得转录本，并使用兔网织红细胞裂解物系统翻译mRNA，以确定这些分子是否会被合成，以及如果被合成，在环化酶重构试验中评估它们是否具有活性。从两种αs RNA获得的翻译产物主要是三种多肽的混合物，其中一种（约占总量的40-50%）代表完整的翻译产物，另外两种似乎是由于在RNA剪接差异之前的Met60处内部翻译起始，以及在剪接差异之后的第一个Met处内部翻译起始。当添加到环化酶膜中时，用短型或长型αs RNA孵育的裂解物重构了氟化物和GTP[γS]刺激的活性。因此，体外合成的αs亚基在与鸟嘌呤核苷酸和腺苷酸环化酶相互作用方面具有活性。在不添加和添加受体激动剂异丙肾上腺素的情况下孵育，使用GTP作为唯一添加的鸟嘌呤核苷酸，两种类型的αs亚基都重构了异丙肾上腺素刺激的腺苷酸环化酶活性。因此，合成的αs也与受体相互作用，并由此推断与β-γ二聚体相互作用，此前已表明β-γ二聚体是受体激活所必需的。将合成的αs-1的活性与天然纯化的人红细胞1型Gs的活性进行比较的定量分析表明，这两种产物在2倍误差范围内具有等效性。因此，裂解物产生了完全有活性的αs亚基，并且αs亚基不需要依赖微粒体过程的翻译后修饰。这种方法可能有助于研究G蛋白其他克隆的α亚基的生物学功能。
