Chemical biology tools for investigating heteromeric complexes of cannabinoid receptors

用于研究大麻素受体异聚复合物的化学生物学工具

• 批准号: 10447093
• 负责人: Aurelien Laguerre
• 金额: $ 0.35万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10447093
• 关键词: 2-arachidonylglycerol; Affect; Agonist; Amber; Amino Acids; Antibodies; Award; Beta Cell; Binding; Binding Proteins; Biological Assay; Biological Process; Biology; CNR1 gene; CNR2 gene; Calcium; Cell model; Cells; Chemicals; Codon Nucleotides; Complex; Coupled; Development; Diabetes Mellitus; Dissociation; Dyes; Education; Endocannabinoids; Enzymes; Epitopes; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genetic; Genetic Code; Heterotrimeric GTP-Binding Proteins; Imaging Device; Individual; Inflammation; Label; Ligands; Ligation; Light; Lighting; Location; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Membrane; Microscopy; Modification; Molecular; Monitor; Monoacylglycerol Lipases; Mutation; Nature; Obesity; Organ; Outcome; Peptides; Peripheral; Pharmacology; Play; Point Mutation; Postdoctoral Fellow; Prevalence; Protein Engineering; Proteins; Proteomics; Receptor Protein-Tyrosine Kinases; Reporting; Resolution; Role; Signal Pathway; Signal Transduction; Specific qualifier value; Speed; System; Techniques; Terminator Codon; Tissues; Training; Vesicle; Visualization; Work; antagonist; base; cannabinoid receptor; cell motility; crosslink; endogenous cannabinoid system; extracellular; human disease; insulin secretion; new therapeutic target; novel; novel strategies; novel therapeutic intervention; optogenetics; peptide I; protein complex; protein crosslink; receptor; receptor binding; recruit; sensor; spatiotemporal; tool

Project Summary The Gi/o coupled cannabinoid receptors 1 and 2 (CB1 and CB2) co-express in central and peripheral tissues. They individually play fundamental roles in membrane plasticity, vesicle secretion, cell migration, and inflammation. While CB1 and CB2 receptors share only 40% structural homology, both are fully activated by 2- arachidonoylglycerol (2-AG) and associate with the same G-proteins. Surprisingly, opposite signaling outcomes have been reported after selective activation of CB1 or CB2 in both central and peripheral organs. The two receptors are individually known to interact with several protein complexes beyond the established role of heterotrimeric G-proteins, namely through heteromeric complexes with other G protein-coupled receptors or receptor tyrosine kinases. Co-expressed CB1 and CB2 receptors are suggested to be juxtaposed in a way that one's activation regulates the other's activity. As 2-AG activates both receptors, I hypothesize that the endocannabinoid coordinates heteromerization mechanisms of CB1 and CB2 with other proteins. This project aims at combining protein engineering and proteomics to investigate the prevalence, the biological functions and the composition of cannabinoid receptor heteromers in living cells. CB1, CB2 and their respective interactomes need to be jointly investigated to distinguish individual from synergistic effects on cellular activity. Investigating the biological function of CB1 and CB2 heteromers in living cells ultimately requires novel tools to activate, label and purify cannabinoid receptors and their interacting proteins. Additionally, a new strategy to manipulate the formation of these protein complexes in a spatially and temporally resolved manner is needed. The speed and complexity of heteromerization mechanisms are challenging to capture with traditional techniques. I will develop new molecular tools for the study of cannabinoid receptor heteromers including imaging tools and those to characterize and manipulate the CB1 and CB2 interactomes at the molecular level. The techniques involved in this project include cannabinoid receptor visualization and photo-crosslinking, reversible manipulation of 2-AG levels, and manipulation of heteromer formation by light. This K99/R00 award will broaden my education through critical training in proteomics and cutting-edge protein engineering techniques. Aim 1. To investigate subcellular locations and composition of heteromeric complexes of cannabinoid receptors. Aim 2. To investigate the biological function of endogenous receptor heteromer formation by light.

项目摘要 GI/O耦合大麻素受体1和2（CB1和CB2）在中央和外围组织中共表达。 他们在膜可塑性，囊泡分泌，细胞迁移和 炎。尽管CB1和CB2受体仅具有40％的结构同源性，但两者都被2-完全激活 蛛网膜（2-AG）并与相同的G蛋白相关。令人惊讶的是，相反的信号传导 在中央和周围器官中选择性激活CB1或CB2后，结果已有报道。 已知两个受体与已建立的几种蛋白质复合物相互作用 异三聚体G蛋白的作用，即通过其他G蛋白偶联的异质体复合物 受体或受体酪氨酸激酶。建议将共表达的CB1和CB2受体并列 在某种程度上，一个人的激活调节对方的活动。当2-Ag激活两个受体，我假设 内源性大麻素协调CB1和CB2与其他蛋白质的异构化机制。这 项目旨在将蛋白质工程和蛋白质组学结合起来研究患病率，即生物学 活细胞中大麻素受体异构体的功能和组成。 CB1，CB2及其 需要共同研究各自的相互作用体，以区分个人与对 细胞活性。研究活细胞中CB1和CB2异构体的生物学功能最终 需要新颖的工具来激活，标记和纯化大麻素受体及其相互作用的蛋白质。 此外，一种在空间和 需要时间解决方式。异构化机制的速度和复杂性是 用传统技术捕获的挑战。我将开发用于研究的新分子工具 大麻素受体异构体，包括成像工具以及来表征和操纵CB1 和CB2在分子水平上相互作用。该项目所涉及的技术包括大麻素 受体可视化和照相链接，可逆操纵2级水平以及对 光线形成异构体。这项K99/R00奖将通过关键的培训来扩大我的教育 蛋白质组学和尖端的蛋白质工程技术。 目的1。研究大麻素的杂体配合物的亚细胞位置和组成 受体。 目的2。研究内源性受体异构体形成的生物学功能。