Graeser D, Neubig R R
Department of Pharmacology, University of Michigan, Ann Arbor 48109-0626.
Mol Pharmacol. 1993 Mar;43(3):434-43.
Many different types of receptors couple to the inhibitory guanine nucleotide-binding protein (G protein) Gi. In NG108-15 neuroblastoma-glioma cells, alpha 2b-adrenergic, m4 muscarinic, and delta-opiate receptors all use Gi as a transducer. According to the ternary complex model of receptor-G protein interactions, agonists bind to these receptors with high affinity only in their G protein-associated form. Conversely, G protein affinity for the receptor is increased by agonist binding. We have developed an extended ternary complex model in which multiple receptors couple to a single G protein and we have examined two consequences of the model theoretically and experimentally. First, the simple ternary complex model can account for the observed high and low affinity agonist binding only when G protein is limiting; however, measurements show a significant excess of G protein over receptor. Could this paradox be explained by other receptors competing for the same G protein and limiting the amount of free G protein so that high and low affinity agonist binding would be seen? Our theoretical simulations show that this does not occur unless the receptors and G protein are present in a precise stoichiometric ratio and have an extremely high affinity, such as when agonists for both receptors are present. The second prediction of this model is that binding of an agonist at one receptor should produce competition for G protein used by another receptor. If the G protein pool were limiting and freely mobile, this would result in an unlabeled agonist at one receptor decreasing binding of a radiolabeled agonist to another receptor. Experimentally, the G protein was made limiting by a partial pertussis toxin treatment. Radioligand binding to alpha 2b-adrenergic and m4 muscarinic receptors in these pertussis toxin-treated NG108-15 membranes showed no cross-talk with the delta-opiate or muscarinic receptors, which are present in excess. This could occur because the different receptors interact with structurally different G proteins (e.g., distinct beta or gamma subunits). More likely it is because of limitations of the mobility of G proteins in the membrane due to 1) attachment to structural elements, such as the cytoskeleton, 2) sequestration in lipid pools, or 3) organization into slowly exchanging supramolecular complexes. These results show that we must reexamine the assumptions of the collision coupling and ternary complex models.
许多不同类型的受体与抑制性鸟嘌呤核苷酸结合蛋白（G蛋白）Gi偶联。在NG108 - 15神经母细胞瘤 - 胶质瘤细胞中，α2b - 肾上腺素能受体、m4毒蕈碱受体和δ - 阿片受体均将Gi用作转导分子。根据受体 - G蛋白相互作用的三元复合物模型，激动剂仅在其与G蛋白相关的形式下以高亲和力结合这些受体。相反，激动剂结合会增加G蛋白对受体的亲和力。我们开发了一种扩展的三元复合物模型，其中多个受体与单个G蛋白偶联，并且我们已经从理论和实验上研究了该模型的两个结果。首先，简单的三元复合物模型仅在G蛋白有限时才能解释观察到的高亲和力和低亲和力激动剂结合；然而，测量结果显示G蛋白明显过量于受体。这个悖论能否通过其他受体竞争相同的G蛋白并限制游离G蛋白的量来解释，从而观察到高亲和力和低亲和力激动剂结合呢？我们的理论模拟表明，除非受体和G蛋白以精确的化学计量比存在并且具有极高的亲和力，例如当两种受体的激动剂都存在时，否则不会发生这种情况。该模型的第二个预测是，一种受体上激动剂的结合应该会对另一种受体使用的G蛋白产生竞争。如果G蛋白池有限且可自由移动，这将导致一种受体上的未标记激动剂降低放射性标记激动剂与另一种受体的结合。在实验中，通过部分百日咳毒素处理使G蛋白变得有限。在这些经百日咳毒素处理的NG108 - 15膜中，放射性配体与α2b - 肾上腺素能受体和m4毒蕈碱受体的结合未显示与过量存在的δ - 阿片受体或毒蕈碱受体有串扰。这可能是因为不同的受体与结构不同的G蛋白相互作用（例如，不同的β或γ亚基）。更有可能的是，这是由于G蛋白在膜中的移动性受限，原因如下：1）附着于结构元件，如细胞骨架；2）隔离在脂质池中；3）组织成缓慢交换的超分子复合物。这些结果表明，我们必须重新审视碰撞偶联和三元复合物模型的假设。
