Ransnäs L A, Svoboda P, Jasper J R, Insel P A
Department of Pharmacology, University of California, San Diego, La Jolla 92093.
Proc Natl Acad Sci U S A. 1989 Oct;86(20):7900-3. doi: 10.1073/pnas.86.20.7900.
The stimulatory guanine nucleotide-binding protein (Gs), which links cell-surface receptors to second-messenger effector systems, is assumed to be confined to plasma membranes. In the current studies we tested whether Gs redistributes within cells by treating S49 lymphoma cells with the beta-adrenergic agonist isoproterenol, then separating cytosol and crude membrane fractions (defined as pellet and supernatant, respectively, after centrifugation for 1 hr at 150,000 x g), and assaying fractions for the alpha subunit of Gs (alpha s) using a competitive ELISA and reconstitution techniques. Under basal conditions, a small (10%) pool of alpha s was identified in supernatant fractions of S49 cells. The size of this pool decreased in the first 15 min after agonist treatment of cells. This decrease was blocked by a beta-adrenergic receptor antagonist and did not occur in an S49 variant, UNC, which lacks functional interaction between receptors and Gs. The size of the alpha s pool in supernatant fractions increased to almost 50% of total cellular alpha s during a 1-hr incubation of cells with isoproterenol. Before isoproterenol treatment only the competitive ELISA was sensitive enough to detect cytosolic alpha s, whereas at later time points (greater than or equal to 30 min) the presence of alpha s in the cytosol was confirmed by both immunoblotting and by reconstitution of adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in Gs-deficient membranes derived from cyc-S49 cells. In contrast to membrane alpha s, cytosolic alpha s did not require activation (e.g., by AlF4-) in the reconstitution assay to stimulate adenylyl cyclase. Use of an antibody that selectively recognizes monomeric dissociated alpha s, but not heterotrimeric alpha s, indicated that cytosolic alpha s is monomeric. These data indicate that alpha s is not exclusively localized to the plasma membrane and that agonist treatment redistributes this protein within target cells.
刺激性鸟嘌呤核苷酸结合蛋白（Gs）可将细胞表面受体与第二信使效应系统相连，一般认为它局限于质膜。在当前研究中，我们通过用β-肾上腺素能激动剂异丙肾上腺素处理S49淋巴瘤细胞，然后分离胞质溶胶和粗膜组分（分别定义为在150,000×g离心1小时后的沉淀和上清液），并使用竞争性酶联免疫吸附测定法和重组技术检测各组分中Gs的α亚基（αs），来测试Gs是否在细胞内重新分布。在基础条件下，在S49细胞的上清液组分中鉴定出一小部分（10%）的αs。在激动剂处理细胞后的最初15分钟内，这部分αs的量减少。这种减少被β-肾上腺素能受体拮抗剂阻断，并且在缺乏受体与Gs功能相互作用的S49变体UNC中未发生。在用异丙肾上腺素孵育细胞1小时期间，上清液组分中αs的量增加到细胞总αs的近50%。在异丙肾上腺素处理之前，只有竞争性酶联免疫吸附测定法足够灵敏以检测胞质溶胶中的αs，而在稍后的时间点（大于或等于30分钟），通过免疫印迹以及在源自cyc-S49细胞的缺乏Gs的膜中重组腺苷酸环化酶[ATP焦磷酸裂解酶（环化），EC 4.6.1.1]证实了胞质溶胶中存在αs。与膜αs相反，胞质溶胶中的αs在重组测定中不需要激活（例如，通过AlF4-）来刺激腺苷酸环化酶。使用一种选择性识别单体解离的αs而不识别异三聚体αs的抗体表明，胞质溶胶中的αs是单体形式。这些数据表明，αs并非仅定位于质膜，并且激动剂处理可使该蛋白在靶细胞内重新分布。
