Activation of the β2-adrenergic receptor involves disruption of an ionic lock between the cytoplasmic ends of transmembrane segments 3 and 6
Activation of the β2-adrenergic receptor involves disruption of an ionic lock between the cytoplasmic ends of transmembrane segments 3 and 6
复制标题
DOI：
10.1074/jbc.m103747200
复制
发表时间：
2001-08-03
影响因子：
4.8
通讯作者：
Javitch, JA
中科院分区：
文献类型：
--
作者：
Ballesteros, JA;Jensen, AD;Javitch, JA
跨膜区段（TMs）3和6在细胞膜细胞质侧的移动在G蛋白偶联受体的激活中起重要作用。在此我们提供证据表明，存在一种离子锁，它在β(2)-肾上腺素能受体的非活性状态下限制TM3和TM6细胞质末端的相对移动性。我们提出，TM3细胞质末端高度保守的Arg - 131(3.50)既与相邻的Asp - 130(3.49)相互作用，也与TM6细胞质末端的Glu268(6.30)相互作用。这种离子相互作用网络现已得到视紫红质非活性状态的高分辨率结构的直接支持。我们假设这种相互作用网络有助于将受体限制在非活性状态，并且这种离子锁的释放可能是受体激活的关键步骤。为了验证这一假设，我们对β(2)-肾上腺素能受体中的Glu - 268(6.30)和Asp - 130(3.49)进行了电荷中和突变。我们单独以及组合观察到，用突变受体瞬时转染的COS - 7细胞中基础的和吲哚洛尔刺激的环磷腺苷（cAMP）积累显著增加。此外，基于TM6中Cys - 285(6.47)可及性的增加，我们提供了TM6构象重排的证据，这种构象重排与不同突变体的组成型活性程度高度相关。目前的实验数据以及近期视紫红质的高分辨率结构表明，Asp/Glu(3.49)、Arg(3.50)和Glu(6.30)之间的离子相互作用可能构成一个控制许多视紫红质样G蛋白偶联受体激活的共同开关。
The movements of transmembrane segments (TMs) 3 and 6 at the cytoplasmic side of the membrane play an important role in the activation of G-protein-coupled receptors. Here we provide evidence for the, existence of an ionic lock that constrains the relative mobility of the cytoplasmic ends of TM3 and TM6 in the inactive state of the beta (2)-adrenergic receptor. We propose that the highly conserved Arg-131(3.50) at the cytoplasmic end of TM3 interacts both with the adjacent Asp-130(3.49) and with Glu268(6.30) at the cytoplasmic end of TM6. Such a network of ionic interactions has now been directly supported by the high-resolution structure of the inactive state of rhodopsin. We hypothesized that the network of interactions would serve to constrain the receptor in the inactive state, and the release of this ionic lock could be a key step in receptor activation. To test this hypothesis, we made charge-neutralizing mutations of Glu-268(6.30) and of Asp-130(3.49) in the beta (2)-adrenergic receptor. Alone and in combination, we observed a significant increase in basal and pindolol-stimulated cAMP accumulation in COS-7 cells transiently transfected with the mutant receptors. Moreover, based on the increased accessibility of Cys-285(6.47) in TM6, we provide evidence for a conformational rearrangement of TM6 that is highly correlated with the extent of constitutive activity of the different mutants. The present experimental data together with the recent high-resolution structure of rhodopsin suggest that ionic interactions between Asp/Glu(3.49), Arg(3.50), and Glu(6.30) may constitute a common switch governing the activation of many rhodopsin-like G-protein-coupled receptors.