Quantification of signalling components and amplification in the beta-adrenergic-receptor-adenylate cyclase pathway in isolated adult rat ventricular myocytes.
Quantification of signalling components and amplification in the beta-adrenergic-receptor-adenylate cyclase pathway in isolated adult rat ventricular myocytes.
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分离成年大鼠心室肌细胞中 β-肾上腺素能受体-腺苷酸环化酶途径信号成分的定量和放大。
DOI：
10.1042/bj3110075
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Insel,PA
中科院分区：
文献类型：
--
作者：
Post,SR;Hilal-Dandan,R;Urasawa,K;Brunton,LL;Insel,PA
我们研究了在离体大鼠心肌细胞中参与β-肾上腺素能受体介导的信号转导的蛋白质的化学计量关系。这些细胞含有约2.1 × 10（5）β-肾上腺素能受体每个细胞，通过放射性配体结合试验确定。我们已经评估了目前在肌细胞膜中的Gs α的量通过免疫印迹使用纯化的谷胱甘肽S-转移酶-Gs α融合蛋白作为定量标准。通过这种方法，我们确定了心肌细胞分别含有约35 × 10（6）和12 × 10（6）分子/细胞的45和52 kDa形式的Gs α。[3 H]毛喉素结合试验用于评估高亲和力毛喉素结合位点的形成，该结合位点代表响应于Gs α活化而发生的Gs α-腺苷酸环化酶复合物。腺苷酸环化酶复合物的定量通过用皂苷透化细胞来促进。将异丙肾上腺素（异丙肾上腺素）和鸟苷5′-[γ-硫代]三磷酸加入到皂化透化的肌细胞中，导致形成6 × 10（5）Gs α-腺苷酸环化酶复合物。总之，这里提供的数据表明，在生理相关的设置，G蛋白是存在于大的化学计量过量相对于受体和效应。此外，我们表明，总体而言，只有适度的信号放大发生在受体和腺苷酸环化酶之间。因此，腺苷酸环化酶（而不是Gs）是受体远端的组分，限制了激动剂介导的环AMP产生的增加。虽然有限的数据是尚未获得其他G蛋白调节效应，我们假设，化学计量的信号成分和β-肾上腺素能反应途径所描述的信号放大的程度将适用于其他G蛋白偶联激素受体系统。
We have investigated the stoichiometric relationship of proteins involved in beta-adrenergic-receptor-mediated signal transduction in isolated rat cardiac myocytes. These cells contain about 2.1 x 10(5) beta-adrenergic receptors per cell, as determined by radio-ligand-binding assays. We have assessed the amount of Gs alpha present in myocyte membranes by immunoblotting using a purified glutathione S-transferase-Gs alpha fusion protein as a standard for quantification. By this method, we determined that cardiac myocytes contain about 35 x 10(6) and 12 x 10(6) molecules per cell of the 45 and 52 kDa forms of Gs alpha, respectively. [3H]Forskolin binding assays were used to assess the formation of high-affinity forskolin binding sites representing Gs alpha-adenylate cyclase complexes occurring in response to Gs alpha activation. Quantification of the adenylate cyclase complexes was facilitated by the permeabilization of cells with saponin. The addition of isoprenaline (isoproterenol) and guanosine 5′-[gamma-thio]trisphosphate to saponin-permeabilized myocytes results in the formation of 6 x 10(5) Gs alpha-adenylate cyclase complexes. Taken together, the data presented here demonstrate that, in a physiologically relevant setting, G-protein is present in large stoichiometric excess relative to both receptor and effector. In addition, we show that, overall, only modest signal amplification occurs between receptor and adenylate cyclase. Thus adenylate cyclase (rather than Gs) is the component distal to receptor that limits agonist-mediated increases in cyclic AMP production. Although limited data are as yet available for other G-protein-regulated effectors, we hypothesize that the stoichiometry of signalling components and the extent of signal amplification described for the beta-adrenergic response pathway will be applicable to other G-protein-coupled hormone receptor systems.