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.

项目总结/文摘