Impact of membrane composition on cholecystokinin receptor structure and function

项目总结/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（CCK 1 Rs）， 饱腹感，对预防和治疗肥胖有重要意义。虽然CCK 1 R激动剂可以急性减少进食， 这些具有高效力和长作用持续时间的药剂往往与副作用和潜在的 健康人的慢性治疗不能耐受毒性。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， 刺激-活动耦合功能失调，可能对之前的临床试验产生负面影响。我们的长期 目的是以更安全和更有效的方式靶向该受体。潜在的假设是， 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药物靶向新模式的认识是实现GPCR治疗潜力的关键障碍 CCK 1 R药物努力将集中在获取分子理解如何膜环境 影响CCK 1 R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，并为配体导向的偏见，造型信号和监管反应 这个治疗靶点的激活剂。我们将利用一个强大的，建立良好的合作，反映了 米勒博士和塞克斯顿博士的技能和经验高度互补。目标1提供了连贯的评估 通过用正构激动剂和变构激动剂刺激CCK 1 R引发的信号传导和调节事件 药物和膜脂对这些事件的影响。这提供了将不同的配体 影响效应子接合、调节蛋白募集、G蛋白功效控制的药理学， 受体螯合和运输，以及如何变构协同作用的网站之间的分子 交互可以修改这些事件。该目的还包括激动剂配体结合动力学的定量 以及相互依赖的G蛋白周期事件，产生异常的刺激活性偶联， 高结合亲和力和降低胆固醇升高时CCK 1 R的信号传导。目标2探索物理 这一过程的基础，重点是受体螺旋束外的脂质相互作用的位点，及其影响 天然肽配体在该受体的胞外域对接的模式。这些研究将利用 聚焦嵌合CCK 1 R：CCK 2 R构建体，以及一系列定点突变体， 标记和荧光探测。目的3为理解CCK异常提供了一个结构框架 在CCK 1 R的刺激活性耦合，利用我们最近在活性测定方面的结构突破， 使用cryo-EM的激动剂占据的CCK 1 R与异源三聚体G蛋白复合物的状态结构。这些 这些研究为研究受药物影响的潜在药物靶点提供了独特的范例。 它们所处的质膜的组成。

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.