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

项目摘要 大麻素受体（CB 1）是中枢神经系统中表达最丰富的GPCR， 滥用药物的目标，如大麻和合成大麻素中的精神活性成分。 虽然CB 1也是一个潜在的高价值治疗靶点，但利用这种关键受体实现治疗目标 由于一系列不希望有的副作用而变得复杂。对这些副作用的一种解释是 这暗示了CB 1被激活时的混杂性质，因为CB 1能够与多种 信号合作伙伴 通常，CB 1与抑制性G蛋白亚型（Gαi/o）偶联。然而，一些CB 1配体也引起了 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 CB 1可以表现出配体偏好性--一种配体与受体结合从而稳定独特受体的现象 构象选择性地促进（或抑制）与不同信号传导伴侣的相互作用。CB 1信号 也可以通过在正常（正构）配体结合口袋之外变构结合的配体来调节。 一种CB 1变构配体ORG 27569（ORG）显示出特别奇特的行为-它增加了与 结合CB 1的激动剂，但抑制G蛋白的受体活化。在调查 我们的实验室最近发现，ORG结合稳定了一种独特的 CB 1构象，其中G蛋白偶联所需的构象变化受到限制。 虽然这种独特的构象已被证明可以减少Gi介导的信号传导，但其完整的生理作用 仍然存在争议。此外，如何以及为什么ORG增加激动剂结合，以及它是否会导致 受体中的其他结构变化仍然未知。 本提案将通过三个具体目标探讨这些问题，这些目标旨在探讨和界定 通过偏向配体和变构调节剂操纵CB 1的分子机制。SA 1将 定义ORG如何影响构象变化，发生在正构配体结合口袋周围， 反应激动剂结合，使用新的荧光技术。这些实验将确定ORG 诱导该区域的交替结构，或者如果该关键区域的变化与构象无关， 信号转导胞质结构域的变化。SA 2将直接测试变构配体对CB 1的影响 需要更高级的受体多聚体，通过进行荧光和放射性配体结合研究， 在nanodsics中分离的单体CB 1。最后，SA 3将开发和使用新型生物传感器来量化和 直接比较配体偏倚和G蛋白亚型的生化和药理学参数 CB 1/G蛋白/配体信号传导复合物的选择性。这些实验不仅能解决 关于CB 1的问题，他们还将为学员提供经典和前沿的重要经验 用于研究GPCR结构和功能的药理学、生物化学和生物物理学方法。

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.