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（CB 1和CB 2）在中枢和外周组织中共表达。 它们分别在膜可塑性、囊泡分泌、细胞迁移和细胞增殖中发挥重要作用。 炎症虽然CB 1和CB 2受体仅具有40%的结构同源性，但两者都被2-氨基-2-甲基-N-苯并[d]芘完全激活。 花生四烯酰甘油（2-AG），并与相同的G-蛋白。令人惊讶的是，相反的信号 已经报道了在中枢和外周器官中选择性激活CB 1或CB 2后的结果。 已知这两种受体分别与几种蛋白质复合物相互作用， 异三聚体G蛋白的作用，即通过与其他G蛋白偶联的异三聚体复合物 受体或受体酪氨酸激酶。共表达的CB 1和CB 2受体被认为是并列的 一个人的活动会调节另一个人的活动由于2-AG激活两种受体，我推测， 内源性大麻素协调CB 1和CB 2与其它蛋白质的异聚化机制。这 该项目旨在结合蛋白质工程和蛋白质组学来研究 活细胞中大麻素受体异聚体的功能和组成。CB 1、CB 2及其 需要联合研究各自的相互作用组，以区分个体与协同作用， 细胞活动最终研究CB 1和CB 2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蛋白。 此外，一种新的策略，以操纵这些蛋白质复合物的形成在空间和 需要时间分辨的方式。异聚化机制的速度和复杂性是 用传统的技术来捕捉是一个挑战。我将开发新的分子工具， 大麻素受体异聚体，包括成像工具和表征和操纵CB 1的工具 和CB 2相互作用体的研究。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 通过光形成异聚体。这个K99/R 00奖项将通过以下方面的关键培训来扩大我的教育范围： 蛋白质组学和尖端蛋白质工程技术。 目标1.研究大麻素异聚体复合物的亚细胞定位和组成 受体。 目标二。探讨光诱导内源性受体异聚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.