Impact of membrane composition on cholecystokinin receptor structure and function

膜成分对胆囊收缩素受体结构和功能的影响

• 批准号: 10541873
• 负责人: LAURENCE J MILLER
• 金额: $ 46.16万
• 依托单位: MAYO CLINIC ARIZONA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10541873
• 关键词: Acute; Address; Affect; Afferent Neurons; Affinity; Agonist; Binding; Biology; Cell membrane; Cholecystokinin; Cholecystokinin Receptor; Cholesterol; Chronic; Clinical Medicine; Clinical Trials; Collaborations; Complex; Coupling; Cryoelectron Microscopy; Data; Development; Disease; Docking; Drug Receptors; Drug Targeting; Environment; Event; Exhibits; Face; Fluorescence; G-Protein-Coupled Receptors; GTP-Binding Proteins; Heterotrimeric GTP-Binding Proteins; Hormones; Investigation; Kinetics; Knowledge; Lateral; Ligand Binding; Ligands; Link; Lipids; Mediating; Membrane; Membrane Lipids; Methodology; Molecular; Molecular Conformation; Obesity; Occupations; Peptides; Persons; Pharmaceutical Preparations; Pharmacology; Photoaffinity Labels; Physiological; Population; Prevention; Process; Public Health; Refractory; Regulation; Research Personnel; Resistance; Resolution; Satiation; Scaffolding Protein; Series; Signal Transduction; Sincalide; Site; Stimulus; Structure; System; Texture; Therapeutic; Therapeutic Effect; Toxic effect; Work; antagonist; dieting; drug development; drug discovery; experience; extracellular; feeding; genetic regulatory protein; insight; molecular site; mutant; negative affect; novel; obese person; obesity treatment; patient subsets; receptor; receptor function; recruit; response; screening; side effect; skills; structural biology; therapeutic target; trafficking

PROJECT SUMMARY/ABSTRACT Cholecystokinin acts on type 1 receptors (CCK1Rs) present on vagal afferent neurons to regulate satiety, important in prevention and treatment of obesity. While CCK1R agonists can acutely reduce feeding, such agents with high potency and long duration of action tend to be associated with side effects and potential toxicity not tolerated for chronic therapy of healthy people. It is also now clear that a subset of the population is refractory to effects of CCK agonists, due to impact of membrane cholesterol on receptor conformation and dysfunctional stimulus-activity coupling, likely negatively affecting previous clinical trials. Our long term objective is to target this receptor in a safer and more effective way. The underlying hypothesis is that lack of mechanistic understanding of interplay between disease states (obesity) and receptor function, and under- appreciation of novel modes of GPCR drug targeting are key barriers to realizing the therapeutic potential of CCK1R drugs. Efforts will focus on acquiring molecular understanding of how the membrane environment affects CCK1R structure and function, and utilizing these insights to pursue opportunities for allosteric modulation to correct negative impact, and for ligand-directed bias to sculpt signaling and regulatory responses to activators of this therapeutic target. We will utilize a strong, well-established collaboration, reflecting the highly complementary skills and experience of Drs. Miller and Sexton. Aim 1 provides a coherent assessment of signaling and regulatory events initiated by stimulation of CCK1R with orthosteric agonists and allosteric drugs, and impact of membrane lipids on these events. This provides the opportunity to link distinct ligand pharmacologies to impact effector engagement, regulatory protein recruitment, control of G protein efficacy, and receptor sequestration and trafficking, as well as how allosteric cooperativity between sites of molecular interaction can modify these events. This aim also includes quantification of kinetics of agonist ligand binding and the events of the G protein cycle that are interdependent, yielding aberrant stimulus-activity coupling with high binding affinity and reduced signaling at CCK1R in elevated cholesterol. Aim 2 explores the physical basis for this process, focusing on the site of lipid interaction outside the receptor helical bundle, and its impact on the mode of natural peptide ligand docking at the ectodomain of this receptor. These studies will utilize focused chimeric CCK1R:CCK2R constructs, as well as a series of site-directed mutants, and photoaffinity labeling and fluorescence probing. Aim 3 provides a structural framework for understanding aberrant CCK stimulus-activity coupling at CCK1R, utilizing our recent structural breakthroughs in determination of active- state structures of agonist-occupied CCK1R in complex with heterotrimeric G proteins using cryo-EM. These studies provide a unique paradigm for the investigation of potential drug targets that are affected by the composition of the plasma membrane in which they reside.

项目概要/摘要 胆囊收缩素作用于迷走神经传入神经元上的 1 型受体 (CCK1R) 来调节 饱腹感对于预防和治疗肥胖很重要。虽然 CCK1R 激动剂可以急剧减少进食， 此类高效且作用持续时间长的药物往往会产生副作用和潜在的风险。 健康人的长期治疗无法耐受毒性。现在也清楚的是，人口的一个子集是 由于膜胆固醇对受体构象的影响，对 CCK 激动剂的作用无效 功能失调的刺激-活动耦合，可能会对之前的临床试验产生负面影响。我们的长期 目标是以更安全、更有效的方式靶向该受体。基本假设是缺乏 对疾病状态（肥胖）和受体功能之间相互作用的机制理解，以及 对 GPCR 药物靶向新模式的认识是实现 GPCR 治疗潜力的关键障碍 CCK1R药物。努力的重点是获得对膜环境如何进行分子理解。 影响 CCK1R 结构和功能，并利用这些见解来寻求变构机会 调节以纠正负面影响，并通过配体定向偏差来塑造信号传导和监管反应 该治疗靶点的激活剂。我们将利用强大、完善的合作，反映 博士的技能和经验高度互补。米勒和塞克斯顿。目标 1 提供连贯的评估 由正构激动剂和变构刺激 CCK1R 引发的信号传导和调节事件 药物，以及膜脂对这些事件的影响。这提供了连接不同配体的机会 影响效应器参与、调节蛋白募集、G蛋白功效控制的药理学， 和受体隔离和运输，以及分子位点之间的变构协同作用如何 交互可以修改这些事件。该目标还包括激动剂配体结合动力学的量化 和 G 蛋白循环的事件是相互依赖的，产生与 在胆固醇升高时，CCK1R 具有高结合亲和力并减少信号传导。目标 2 探索物理 该过程的基础，重点关注受体螺旋束外部脂质相互作用的位点及其影响 关于天然肽配体对接在该受体的胞外域的模式。这些研究将利用 聚焦嵌合 CCK1R:CCK2R 构建体，以及一系列定点突变体和光亲和力 标记和荧光探测。目标 3 提供了理解异常 CCK 的结构框架 CCK1R 的刺激-活动耦合，利用我们最近在确定活性- 使用冷冻电镜观察激动剂占据的 CCK1R 与异三聚体 G 蛋白复合物的状态结构。这些 研究为研究受药物影响的潜在药物靶标提供了独特的范例 它们所在的质膜的组成。