Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB1
Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB1
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DOI：
10.1073/pnas.1500895112
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发表时间：
2015-07-07
影响因子：
11.1
通讯作者：
Farrens, David L.
中科院分区：
文献类型：
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作者：
Fay, Jonathan F.;Farrens, David L.
G蛋白偶联受体(GPCRs)是令人惊讶的灵活分子，它可以做的远远不止是简单地打开G蛋白。有些甚至表现出偏向的信号，其中相同的受体优先激活不同的G蛋白或arrestin信号通路，这取决于配体结合的类型。这种行为发生的原因尚不清楚，但它既可以发生在传统的配体上，也可以发生在正构受体结合口袋外的变构结合的配体上。在这里，我们在大麻受体CB1中寻找这些现象背后的结构机制。我们的工作集中在变构配体Org 27569上，它对CB1有一个不寻常的作用-它同时增加激动剂结合，降低G蛋白激活，并诱导偏向信号。使用经典的药理结合研究，我们发现Org 27569与CB1上一个独特的变构位点结合，并表明它可以单独作用(不需要激动剂共同结合)。通过诱变研究，我们发现Org 27569的结合能力与受体处于多少能与G蛋白偶联的活性构象有关。利用这些数据，我们估计了非活化态和活化态之间的能量差。最后，定点荧光标记研究表明，Org 27569稳定的CB1结构与拮抗剂或激动剂稳定的结构不同且独特。具体地说，与G蛋白激活相关的跨膜螺旋6(TM6)运动被阻止，但同时，螺旋8/TM7运动被增强，这表明Org 27569诱导偏向信号的能力可能是一种机制。
G protein-coupled receptors (GPCRs) are surprisingly flexible molecules that can do much more than simply turn on G proteins. Some even exhibit biased signaling, wherein the same receptor preferentially activates different G-protein or arrestin signaling pathways depending on the type of ligand bound. Why this behavior occurs is still unclear, but it can happen with both traditional ligands and ligands that bind allosterically outside the orthosteric receptor binding pocket. Here, we looked for structural mechanisms underlying these phenomena in the marijuana receptor CB1. Our work focused on the allosteric ligand Org 27569, which has an unusual effect on CB1-it simultaneously increases agonist binding, decreases G-protein activation, and induces biased signaling. Using classical pharmacological binding studies, we find that Org 27569 binds to a unique allosteric site on CB1 and show that it can act alone (without need for agonist cobinding). Through mutagenesis studies, we find that the ability of Org 27569 to bind is related to how much receptor is in an active conformation that can couple with G protein. Using these data, we estimated the energy differences between the inactive and active states. Finally, site-directed fluorescence labeling studies show the CB1 structure stabilized by Org 27569 is different and unique from that stabilized by antagonist or agonist. Specifically, transmembrane helix 6 (TM6) movements associated with G-protein activation are blocked, but at the same time, helix 8/TM7 movements are enhanced, suggesting a possible mechanism for the ability of Org 27569 to induce biased signaling.