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

对人类大麻素受体 CB1 的变构和 G 蛋白选择性的机制的研究。

• 批准号: 10229399
• 负责人: Anthony D Shumate
• 金额: $ 4.6万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10229399
• 关键词: Address; Affect; Affinity; Agonist; Anxiety; Architecture; Area; Behavior; Binding; Biochemical; Biochemistry; Biological Assay; Biophysics; Biosensor; Chimera organism; Chimeric Proteins; Complex; Consumption; Couples; Coupling; Crystallization; Cytoplasmic Receptors; Cytoplasmic Tail; Drug Targeting; Endocannabinoids; Exhibits; Face; Fluorescence; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Go Alpha Subunit; Goals; Human; Individual; Investigation; Ligand Binding; Ligands; Marijuana; Mediating; Mental Depression; Methods; Molecular; Molecular Conformation; Monitor; Nature; Neuraxis; Pathway interactions; Pharmacology; Physiological; Property; Receptor Activation; Receptor Signaling; Reporting; Role; Signal Transduction; Signaling Protein; Specificity; Structure; Techniques; Testing; Therapeutic; Tryptophan; Work; cannabinoid receptor; design; drug development; drug of abuse; experience; experimental study; extracellular; insight; interest; monomer; novel; radioligand; receptor; receptor-mediated signaling; response; side effect; success; suicidal risk; synthetic cannabinoid; therapeutic target; tool

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.

项目摘要 大麻素受体（CB1）是中枢神经系统中最绝对表达的GPCR，并且 滥用药物的靶标，例如大麻和合成大麻素中的精神活性成分。 而CB1也是一个潜在的高价值治疗靶标，将此关键受体用于治疗目标 多种不希望的副作用使其复杂化。这些副作用的一种解释 激活时实现了CB1的混杂性，因为CB1能够与多种 信号伙伴。 从规范上讲，CB1夫妇抑制G蛋白亚型（GαI/O）。但是，有些CB1配体也导致 受体与其他G蛋白亚型（例如Gαs和GαQ）夫妇。那，有些化合物的靶向 CB1可以表现出配体偏置 - 一种现象，通过该现象，配体与接收器结合可以稳定唯一的接收器 选择性促进（或抑制）与不同信号伴侣的相互作用的构象。 CB1信号传导 也可以通过在正常（正常）配体结合袋之外进行变构的配体调节。 一个CB1变构配体，org27569（org），表现出特别奇特的行为 - 它增加了结合的结合 激动剂与CB1结合，但抑制了G蛋白的受体激活。在研究结构的同时 这种明显悖论的机制，我们的实验室最近发现，org结合稳定了独特的 CB1构象，其中限制了G蛋白耦合所需的构象变化。 尽管已显示这种独特的构象可减少胃肠道介导的信号传导，但其完整的生理作用 仍然有争议。此外，组织如何以及为什么增加激动剂的约束力，以及是否造成 接收器的其他结构变化仍然不知道。 该建议将通过旨在探索和定义的三个特定目标（SAS）探索这些问题 通过偏置配体和变构调节剂操纵CB1的分子机制。 SA1会 定义ORG如何影响构象变化发生的构象变化 使用新型荧光技术对激动剂结合的响应。这些实验将确定Org是否 诱导该领域的替代结构，或者如果该关键区域的变化与构象相反 信号传导细胞质结构域的变化。 SA2将直接测试变构配体对CB1的影响是否 通过进行荧光和放射性结合研究，需要高阶受体多聚体 纳米学中分离的单体CB1。最后，SA3将开发和使用新颖的生物传感器来量化和 直接比较配体偏置和G蛋白亚型的生化和药物参数 CB1/G蛋白/配体信号复合物的选择性。这些实验不仅会解决密钥 关于CB1的问题，他们还将为学员在古典和尖端提供重要体验 用于研究GPCR结构和功能的药理学，生物化学和生物物理学的方法。