Structural instability of a constitutively active G protein-coupled receptor - Agonist-independent activation due to conformational flexibility
Structural instability of a constitutively active G protein-coupled receptor - Agonist-independent activation due to conformational flexibility
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DOI：
10.1074/jbc.272.5.2587
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发表时间：
1997-01-31
影响因子：
4.8
通讯作者：
Kobilka, BK
中科院分区：
文献类型：
--
作者：
Gether, U;Ballesteros, JA;Kobilka, BK
几个结构域中的突变可导致G蛋白偶联受体的激动剂非依赖性、组成性激活。然而，结构和分子的变化，组成性地打开一个G蛋白偶联受体的性质仍然未知。在这里，我们显示的证据表明，组成型激活突变体的β（2）肾上腺素能受体（CAM）的特点是结构不稳定性和夸张的构象反应配体结合。CAM的结构不稳定性可以通过与野生型受体相比，纯化的受体在37 ℃下的变性速率增加4倍来证明。用构象敏感和半胱氨酸反应性荧光团N，N ′-二甲基-N-（碘乙酰基）-N ′-（7-硝基苯-2-氧杂-1，3-二唑-4-基）乙二胺标记的纯化CAM的光谱分析进一步表明，激动剂和拮抗剂在CAM中引起比野生型蛋白更深刻的结构变化。我们认为，赋予β 2肾上腺素能受体组成性活性的突变消除了一些稳定的构象约束，使CAM更容易经历非活性和活性状态之间的转换，使受体更容易变性。
Mutations in several domains can lead to agonist-independent, constitutive activation of G protein-coupled receptors. However, the nature of the structural and molecular changes that constitutively turn on a G protein-coupled receptor remains unknown. Here we show evidence that a constitutively activated mutant of the beta(2) adrenergic receptor (CAM) is characterized by structural instability and an exaggerated conformational response to ligand binding. The structural instability of CAM could be demonstrated by a 4-fold increase in the rate of denaturation of purified receptor at 37 degrees C as compared with the wild type receptor. Spectroscopic analysis of purified CAM labeled with the conformationally sensitive and cysteine-reactive fluorophore, N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine, further indicated that both agonist and antagonist elicit more profound structural changes in CAM than in the wild type protein. We propose that the mutation that confers constitutive activity to the beta(2) adrenergic receptor removes some stabilizing conformational constraints, allowing CAM to more readily undergo transitions between the inactive and the active states and making the receptor more susceptible to denaturation.