An investigation of the mechanisms governing allosterism and G-protein selectivity of the human cannabinoid receptor, CB1. âââ

项目总结 大麻素受体(CB1)是中枢神经系统中表达最丰富的GPCR，并且 滥用药物的目标，如大麻和合成大麻中的精神活性成分。 虽然CB1也是一个潜在的高价值的治疗靶点，但利用这个关键的受体达到治疗目标 因一系列不良副作用而变得复杂。对这些副作用的一种解释是 暗示了CB1在激活时的混杂性质，因为CB1能够与各种 信令合作伙伴。 典型地，Cb1与抑制性G蛋白亚型(Gαi/o)偶联。然而，一些CB1配体也会引起 受体与其他G蛋白亚型，如GαS和GαQ偶联。因此，一些化合物靶向 CB1可以表现出配基偏向--一种与受体结合的配基稳定独特受体的现象 选择性地促进(或抑制)与不同信号伙伴的相互作用的构象。CB1信令 也可以受到变构结合的配体的调节，在正常(正构体)配体结合口袋之外。 CB1变构配体ORG27569(ORG)表现出特别的特殊行为-它增加了 激动剂与CB1结合，但抑制G蛋白的受体激活。在调查结构的同时 这个明显的悖论背后的机制，我们的实验室最近发现，ORG结合稳定了一个独特的 CB1构象，限制G蛋白偶联所需的构象变化。 虽然这种独特的构象已经被证明减少了GI介导的信号传递，但它的全部生理作用 仍然存在争议。此外，ORG如何和为什么增加激动剂的结合，以及它是否导致 该受体的其他结构变化尚不清楚。 本提案将通过三个特定目标(SA)来探讨这些问题，这些目标旨在探索和定义 通过偏置配体和变构调节剂操纵CB1的分子机制。SA1将 定义ORG如何影响发生在正位配体结合口袋周围的构象变化 对激动剂结合的反应，使用新的荧光技术。这些实验将确定ORG是否 在这个区域诱导替代结构，或者如果这个关键区域的变化与构象脱钩 信号胞质结构域的变化。SA2将直接测试变构配体对CB1的影响 通过开展荧光和放射性配基结合研究来要求更高阶的受体多聚体 纳米物理中分离的单体CB1。最后，SA3将开发和使用新型生物传感器来量化和 直接比较配体偏向和G蛋白亚型的生化和药理参数 CB1/G蛋白/配体信号复合体的选择性。这些实验不仅解决了关键问题 关于CB1的问题，他们还将为学员提供重要的经验，无论是经典的还是前沿的 方法在药理学、生物化学和生物物理学中用于研究GPCRs的结构和功能。

PROJECT SUMMARY The cannabinoid receptor (CB1) is the most abundantly expressed GPCR in the central nervous system, and the target of drugs of abuse like the psychoactive components in marijuana and synthetic cannabinoids. While CB1 is also a potentially high-value therapeutic target, exploiting this key receptor for therapeutic goals has been complicated by a wide array of undesired side-effects. One explanation for these side-effects has implicated the promiscuous nature of CB1 when activated, as CB1 is capable of coupling with a variety of signaling partners. Canonically, CB1 couples to inhibitory G-protein subtypes (Gαi/o). However, some CB1 ligands also cause the receptor to couple with other G-protein subtypes, such as Gαs and Gαq. Thus, some compounds targeting CB1 can exhibit ligand bias—a phenomenon whereby ligand binding to a receptor stabilizes a unique receptor conformation that selectively promotes (or inhibits) interactions with different signaling partners. CB1 signaling can also be modulated by ligands that bind allosterically, outside the normal (orthosteric) ligand binding pocket. One CB1 allosteric ligand, ORG27569 (ORG), shows especially peculiar behavior—it increases binding of agonists binding to CB1, yet inhibits receptor activation of G-proteins. While investigating the structural mechanisms underlying this apparent paradox, our lab recently found that ORG-binding stabilizes a unique CB1 conformation, one in which the conformational changes necessary for G-protein coupling are restricted. While this unique conformation has been shown to reduce Gi-mediated signaling, its full physiological role remains controversial. Moreover, how and why ORG increases agonist binding, and whether or not it causes other structural changes in the receptor is still not known. This proposal will explore these issues through three Specific Aims (SAs) designed to explore and define the molecular mechanisms involved in manipulation of CB1 by biased ligands and allosteric modulators. SA1 will define how ORG affects conformational changes that occur around the orthosteric ligand binding pocket in response to agonist binding, using novel fluorescence techniques. These experiments will determine if ORG induces alternate structures in this area, or if changes in this key region are decoupled from conformational changes in the signaling cytoplasmic domain. SA2 will directly test if the effect of allosteric ligands on CB1 require higher-order receptor multimers by carrying out fluorescent and radioligand binding studies of monomeric CB1 isolated in nanodsics. Finally, SA3 will develop and use novel biosensors to quantify and directly compare biochemical and pharmacological parameters underlying ligand bias and G-protein subtype selectivity at the CB1/G-protein/ligand signaling complex. Not only will these experiments address key questions about CB1, they will also provide vital experience for the trainee in both classical and cutting-edge methods in pharmacology, biochemistry, and biophysics used for the study of GPCR structure and function.