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.

项目摘要/摘要 胆囊动蛋白作用于迷走神经神经元上存在的1型受体（CCK1R）以调节 饱腹感，对于预防和治疗肥胖症很重要。虽然CCK1R激动剂可以急性减少喂养 这种具有较高效力和较长作用持续时间的药物往往与副作用和潜力有关 对健康人的长期治疗不容忍毒性。现在也很明显，一部分人口是 由于膜胆固醇对受体构象的影响，对CCK激动剂的影响难治性 功能失调的刺激 - 活性耦合，可能会对先前的临床试验产生负面影响。我们的长期 目的是以更安全，更有效的方式靶向该受体。基本的假设是缺乏 对疾病状态（肥胖）和受体功能之间相互作用的机械理解，以及 对GPCR药物靶向的新型模式的欣赏是实现治疗潜力的关键障碍 CCK1R药物。努力将着重于获取分子对膜环境的理解 影响CCK1R的结构和功能，并利用这些见解来寻求变构的机会 调节以纠正负面影响，并使配体指导的偏差雕刻信号和调节反应 致此治疗靶点的激活剂。我们将利用强大，公认的合作，反映 高度互补的技能和DR的经验。米勒和塞克斯顿。 AIM 1提供连贯的评估 通过用直角激动剂和变构刺激CCK1R引发的信号传导和调节事件 药物和膜脂质对这些事件的影响。这提供了连接不同配体的机会 影响效应子参与，调节蛋白募集，控制G蛋白功效的药理， 以及受体隔离和运输，以及分子位点之间的变构合作 互动可以修改这些事件。该目标还包括量化激动剂配体结合的动力学 G蛋白周期相互依存的事件，产生异常的刺激活性耦合 高胆固醇中CCK1R的高结合亲和力和降低的信号传导。 AIM 2探索物理 此过程的基础，专注于受体螺旋束外脂质相互作用的位点及其影响 在自然肽配体的模式下，该受体的外生域的对接。这些研究将利用 聚焦的嵌合CCK1R：CCK2R构建体以及一系列定向突变体和光附属机构 标记和荧光探测。 AIM 3提供了理解异常CCK的结构框架 在CCK1R处进行刺激活动耦合，利用我们最近的结构突破来确定主动 使用冷冻EM，激动剂占CCK1R的状态结构与异三聚体G蛋白复合在一起。这些 研究为研究受到的潜在药物靶标的研究提供了独特的范式 它们居住的质膜的组成。