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

基本信息

批准号：
10190434
负责人：
Aurelien Laguerre
金额：
$ 9.9万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10190434
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 Structure System Techniques Terminator Codon Tissues Training Vesicle Visualization Work 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

项目摘要

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.

项目概要 Gi/o 偶联大麻素受体 1 和 2（CB1 和 CB2）在中枢和外周组织中共同表达。它们各自在膜可塑性、囊泡分泌、细胞迁移和炎。虽然 CB1 和 CB2 受体仅具有 40% 的结构同源性，但两者均被 2- 花生四烯酰甘油 (2-AG) 并与相同的 G 蛋白结合。令人惊讶的是，相反的信号中枢和外周器官选择性激活 CB1 或 CB2 后的结果已有报道。已知这两种受体分别与几种蛋白质复合物相互作用，超出了已建立的范围。异源三聚体 G 蛋白的作用，即通过与其他 G 蛋白偶联的异源复合物受体或受体酪氨酸激酶。建议并列共表达 CB1 和 CB2 受体在某种程度上，一个人的激活调节另一个人的活动。由于 2-AG 激活两种受体，我假设内源性大麻素协调 CB1 和 CB2 与其他蛋白质的异聚机制。这项目旨在结合蛋白质工程和蛋白质组学来研究患病率、生物学活细胞中大麻素受体异聚体的功能和组成。 CB1、CB2 及其需要联合研究各自的相互作用组，以区分个体与协同效应细胞活动。最终研究 CB1 和 CB2 异聚体在活细胞中的生物学功能需要新的工具来激活、标记和纯化大麻素受体及其相互作用的蛋白质。此外，一种在空间和空间上操纵这些蛋白质复合物形成的新策略需要暂时解决的方式。异聚化机制的速度和复杂性是用传统技术捕捉具有挑战性。我将开发新的分子工具来研究大麻素受体异聚体，包括成像工具以及表征和操纵 CB1 的工具和分子水平上的 CB2 相互作用组。该项目涉及的技术包括大麻素受体可视化和光交联、2-AG 水平的可逆操作以及光形成异聚体。这个 K99/R00 奖项将通过关键培训拓宽我的教育范围蛋白质组学和尖端蛋白质工程技术。目标 1. 研究大麻素异聚复合物的亚细胞位置和组成受体。目的2.研究光形成内源性受体异聚体的生物学功能。