The Role of Transmembrane Domain 3 in the Actions of Orthosteric, Allosteric, and Atypical Agonists of the M4 Muscarinic Acetylcholine Receptor
The Role of Transmembrane Domain 3 in the Actions of Orthosteric, Allosteric, and Atypical Agonists of the M4 Muscarinic Acetylcholine Receptor
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DOI：
10.1124/mol.111.070938
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发表时间：
2011-05-01
影响因子：
3.6
通讯作者：
Christopoulos, Arthur
中科院分区：
文献类型：
--
作者：
Leach, Katie;Davey, Anna E.;Christopoulos, Arthur
尽管发现了各种各样的新型M-4毒蕈碱乙酰胆碱（ACh）受体（mAChR）激动剂和变构调节剂，但对这些配体如何激活受体知之甚少。我们使用跨膜3（TMIII），一个关键区域参与G蛋白偶联受体激活的保守残基的定点突变，以探测原型正构mAChR激动剂，变构调节剂和“非典型”激动剂的结合和功能。我们发现，除了W108（3.28）A和L109（3.29）A（可能直接影响变构和正构位点之间的界面）和突变D112（3.32）N（可能导致氢键网络的整体破坏）之外，大多数突变不影响变构调节剂的结合。虽然许多突变影响信号传导，但我们没有鉴定出对任何一类激动剂（正构、变构或非典型）的功能活性重要的氨基酸，从而排除任何其他激动剂，这表明TMIII是刺激传递的关键，而与激动剂无关。我们还确定了两个关键残基，Trp 108（3.28）和Asp 112（3.32），这是必要的3-氨基-5-氯-6-甲氧基-4-甲基-噻吩并[2，3-B]吡啶-2-羧酸环丙基酰胺（LY 2033298）和乙酰胆碱之间的结合协同性的传输。最后，我们发现LY 2033298能够以与ACh信号传导功效负相关的方式在大多数测试的突变体中挽救功能受损的ACh信号传导，这表明LY 2033298对内源性激动剂介导的正协同性机制的关键部分涉及受体朝向活性构象的全局驱动。
Despite the discovery of a diverse range of novel agonists and allosteric modulators of the M-4 muscarinic acetylcholine (ACh) receptor (mAChR), little is known about how such ligands activate the receptor. We used site-directed mutagenesis of conserved residues in transmembrane 3 (TMIII), a key region involved in G protein-coupled receptor activation, to probe the binding and function of prototypical orthosteric mAChR agonists, allosteric modulators, and "atypical" agonists. We found that most mutations did not affect the binding of the allosteric modulators, with the exception of W108(3.28)A and L109(3.29)A (which may contribute directly to the interface between allosteric and orthosteric sites) and mutation D112(3.32)N (which may cause a global disruption of a hydrogen bond network). Although numerous mutations affected signaling, we did not identify amino acids that were important for the functional activity of any one class of agonist (orthosteric, allosteric, or atypical) to the exclusion of any others, suggesting that TMIII is key for the transmission of stimulus irrespective of the agonist. We also identified two key residues, Trp108(3.28) and Asp112(3.32), that are essential for the transmission of binding cooperativity between 3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298) and ACh. Finally, we found that LY2033298 was able to rescue functionally impaired signaling of ACh at the majority of mutants tested in a manner that was inversely correlated with the ACh signaling efficacy, indicating that a key part of the mechanism of the positive cooperativity mediated by LY2033298 on the endogenous agonist involves a global drive of the receptor toward an active conformation.