Impact of membrane composition on cholecystokinin receptor structure and function

项目摘要/摘要 CCK1R作用于迷走神经传入神经元上的1型受体进行调节 饱腹感，对预防和治疗肥胖症很重要。虽然CCK1R激动剂可以显著减少摄食， 这类药效高、作用时间长的药物往往与副作用和潜在的副作用有关。 对健康人进行慢性治疗时不能容忍的毒性。现在也很清楚，人口的一个子集是 由于膜胆固醇对受体构象的影响而对CCK激动剂的作用不佳 功能失调的刺激-活动耦合，可能对先前的临床试验产生负面影响。我们的长期计划 目的是以一种更安全、更有效的方式靶向这种受体。潜在的假设是，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 评价GPCR药物靶向的新模式是实现其治疗潜力的关键障碍 CCK1R类药物。努力将重点放在获得膜环境的分子理解上 影响CCK1R的结构和功能，并利用这些见解寻找变构的机会 调节以纠正负面影响，并用于配体导向的偏向，以塑造信号和调节反应 这一治疗靶点的激活剂。我们将利用强大的、成熟的合作，反映 米勒和塞克斯顿博士的技能和经验具有很强的互补性。目标1提供了一个连贯的评估 原构体激动剂和变构刺激CCK1R启动的信号和调节事件 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、控制G蛋白效力的药理作用， 以及受体的隔离和运输，以及分子位点之间的变构协同作用 交互可以修改这些事件。这一目标还包括量化激动剂配体结合的动力学。 以及G蛋白周期中相互依赖的事件，产生异常的刺激活动与 胆固醇升高时CCK1R的高结合亲和力和信号减少。《目标2》探索了 这一过程的基础，侧重于受体螺旋束外脂质相互作用的部位及其影响 关于天然多肽配体对接在该受体胞外区域的模式。这些研究将利用 聚焦嵌合CCK1R：CCK2R构建，以及一系列定点突变体和光亲和 标记和荧光探针法。AIM 3为理解异常CCK提供了一个结构框架 CCK1R的刺激-活动耦合，利用我们最近在确定活性- 用冷冻电子显微镜研究激动剂占据的CCK1R与异源三聚体G蛋白复合体的状态结构。这些 研究为调查受影响的潜在药物靶点提供了独特的范式 它们所在的质膜的组成。

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.