Chemical biology tools for investigating heteromeric complexes of cannabinoid receptors

项目摘要 Gi/o偶联的大麻素受体1和2(CB1和CB2)在中枢和外周组织中共同表达。 它们分别在膜可塑性、囊泡分泌、细胞迁移和 发炎。虽然CB1和CB2受体在结构上只有40%的同源性，但这两种受体都被2- 花生四烯基甘油(2-AG)，并与相同的G-蛋白结合。令人惊讶的是，相反的信号 在中枢和外周器官中选择性激活CB1或CB2后的结果已有报道。 这两种受体分别与已建立的蛋白质复合体相互作用。 异源三聚体G蛋白的作用，即通过与其他G蛋白的异构体复合体偶联 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，一个人的活动调节另一个人的活动。由于2-AG激活了这两个受体，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 该项目旨在将蛋白质工程和蛋白质组学相结合，以调查流行率、生物学和 活细胞中大麻素受体异构体的功能和组成。CB1、CB2及其 需要对各自的相互作用进行联合调查，以区分个体和协同作用对 细胞活动。最终研究CB1和CB2异构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体形成的新策略 需要采取临时解决的方式。异构化机制的速度和复杂性是 用传统技术捕捉具有挑战性的。我将开发新的分子工具来研究 大麻素受体异构体，包括成像工具以及用于表征和操纵CB1的工具 和CB2在分子水平上的相互作用。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，以及对 光作用下形成的异构体。这项K99/R00奖项将通过批判性培训扩大我的教育范围 蛋白质组学和前沿蛋白质工程技术。 目的1.研究大麻素异构体复合体的亚细胞位置和组成 感受器。 目的2.研究光诱导内源性受体异构体形成的生物学功能。

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.