Krumins A M, Barber R
Department of Pharmacology, University of Texas-Houston Medical School, 77225, U.S.A.
Biochem Pharmacol. 1997 Jul 1;54(1):61-72. doi: 10.1016/s0006-2952(97)00147-0.
We have examined the effect of increased Gs protein levels on the abilities of three different beta2-agonists to induce GTP shifts and stimulate adenylyl cyclase response in an effort to investigate the kinetic association between the beta2-adrenergic receptor Gs and adenylyl cyclase. Agonist competition binding analysis and adenylyl cyclase concentration-response assays revealed that increases in Gs protein resulted in proportional increases in the areas of the GTP shift and adenylyl cyclase activity. Changes in the magnitude of the GTP shift were evaluated with a novel and straightforward approach for analyzing the GTP shift data that allowed us to determine the proportion of high agonist affinity binding receptor population and the apparent dissociation constant between the agonist bound receptor and Gs, regardless of the Gs protein level or the type of beta2-agonist. Using this method, we concluded that increased Gs results in the accumulation of the receptor population displaying high affinity towards agonist (HRGs) by increasing the number of receptor-Gs complexes (to a receptor:Gs protein ratio of about 0.7 at maximal Gs expression) without affecting the affinity between hormone bound receptor and Gs. Using the Gs protein levels determined with our novel analysis, we ran simulations using the theoretical shuttle model equation that relates the EC50 to available Gs. Fitting the simulations to experimental data required a receptor to catalytic unit ratio of 0.45 and revealed at least two distinct stages for beta2-agonist-stimulated adenylyl cyclase activity, namely, the activation of Gs by the beta2-adrenergic receptor (a step whose rate is dependent on the type of agonist used to stimulate activity), and the activation of adenylyl cyclase by active Gs (a step whose rate is independent of the type of agonist).
我们研究了Gs蛋白水平升高对三种不同β2-激动剂诱导GTP转移及刺激腺苷酸环化酶反应能力的影响，旨在探究β2-肾上腺素能受体、Gs与腺苷酸环化酶之间的动力学关联。激动剂竞争结合分析和腺苷酸环化酶浓度-反应测定表明，Gs蛋白增加导致GTP转移面积和腺苷酸环化酶活性成比例增加。我们采用一种新颖且直接的方法评估GTP转移幅度的变化，该方法用于分析GTP转移数据，使我们能够确定高激动剂亲和力结合受体群体的比例以及激动剂结合受体与Gs之间的表观解离常数，而不受Gs蛋白水平或β2-激动剂类型的影响。使用该方法，我们得出结论：Gs增加会通过增加受体-Gs复合物的数量（在最大Gs表达时受体与Gs蛋白的比例约为0.7），导致对激动剂具有高亲和力的受体群体（HRGs）积累，而不影响激素结合受体与Gs之间的亲和力。利用我们新颖分析方法测定的Gs蛋白水平，我们使用将EC50与可用Gs相关联的理论穿梭模型方程进行模拟。将模拟结果与实验数据拟合需要受体与催化单位的比例为0.45，并揭示了β2-激动剂刺激的腺苷酸环化酶活性至少有两个不同阶段，即β2-肾上腺素能受体激活Gs（该步骤的速率取决于用于刺激活性的激动剂类型），以及活性Gs激活腺苷酸环化酶（该步骤的速率与激动剂类型无关）。
