Scheer A, Fanelli F, Costa T, De Benedetti P G, Cotecchia S
Institut de Pharmacologie et Toxicologie, Université de Lausanne, Switzerland.
EMBO J. 1996 Jul 15;15(14):3566-78.
Site-directed mutagenesis and molecular dynamics simulations of the alpha 1B-adrenergic receptor (AR) were combined to explore the potential molecular changes correlated with the transition from R (inactive state) to R (active state). Using molecular dynamics analysis we compared the structural/dynamic features of constitutively active mutants with those of the wild type and of an inactive alpha 1B-AR to build a theoretical model which defines the essential features of R and R. The results of site-directed mutagenesis were in striking agreement with the predictions of the model supporting the following hypothesis. (i) The equilibrium between R and R depends on the equilibrium between the deprotonated and protonated forms, respectively, of D142 of the DRY motif. In fact, replacement of D142 with alanine confers high constitutive activity to the alpha 1B-AR. (ii) The shift of R143 of the DRY sequence out of a conserved 'polar pocket' formed by N63, D91, N344 and Y348 is a feature common to all the active structures, suggesting that the role of R143 is fundamental for mediating receptor activation. Disruption of these intramolecular interactions by replacing N63 with alanine constitutively activates the alpha 1B-AR. Our findings might provide interesting generalities about the activation process of G protein-coupled receptors.
将α1B - 肾上腺素能受体（AR）的定点诱变和分子动力学模拟相结合，以探索与从R（非活性状态）转变为R*（活性状态）相关的潜在分子变化。通过分子动力学分析，我们比较了组成型活性突变体与野生型以及非活性α1B - AR的结构/动力学特征，构建了一个定义R和R基本特征的理论模型。定点诱变的结果与该模型的预测惊人地一致，支持了以下假设。（i）R和R之间的平衡分别取决于DRY基序中D142的去质子化形式和质子化形式之间的平衡。事实上，用丙氨酸取代D142会赋予α1B - AR高组成型活性。（ii）DRY序列中的R143从由N63、D91、N344和Y348形成的保守“极性口袋”中移出是所有活性结构共有的特征，这表明R143的作用对于介导受体激活至关重要。用丙氨酸取代N63破坏这些分子内相互作用会使α1B - AR组成型激活。我们的发现可能为G蛋白偶联受体的激活过程提供有趣的一般性规律。
