LINKING CANNABINOID RECEPTOR TYPE 2 (CB2) BIOLOGY TO FUNCTION

描述（由申请人提供）：大麻素，大麻的主要生物活性成分，正在被广泛研究，以了解其毒性和免疫治疗潜力。它们激活CB1和CB2受体，并通过内源性大麻素系统发出信号。CB2受体的表达在免疫系统细胞中占主导地位，我们新的初步发现表明CB2不仅是细胞外g蛋白偶联受体，而且在免疫细胞中以不同的位置和不同的量表达。人B细胞在细胞表面表达CB2的同时，也在细胞内表达高浓度的CB2。相比之下，T细胞不表达细胞外CB2，而表达细胞内蛋白。此外，CB2表达模式在恶性B细胞系中发生变化，当naïve B细胞被激活时也会发生变化。这些新发现使我们假设CB2受体的表达、位置和贩运是将大麻素与特定信号和功能后果联系起来的关键特征。目的：1)研究CB2受体内化及其受体蛋白在免疫细胞内CB2分布中的作用。实验将确定CB2在细胞内的位置，并评估其位置是否因细胞类型而异，细胞内CB2是否可与配体结合，大麻素是否在其访问细胞内CB2的能力上存在差异，以及受体内化、再循环和接头蛋白在控制CB2分布中所起的作用。目的2)研究CB2在B细胞上表达的双向相互作用，B细胞活化对CB2表达的影响，以及大麻素调节B细胞分化和免疫球蛋白（Ig）类转换的能力。我们假设CB2的表达是一个受调控的事件，其表达模式会使应答细胞偏向于特定的分化途径。目的3)将信号传导与CB2受体的定位和功能作用联系起来。具有不同CB2表达模式的细胞和表达模式被特异性操纵/激活的细胞将通过三种途径- ERK/MAPK， PI3K/AKT和细胞内ca++来评估大麻素信号。通过这些研究的结论，我们希望更好地了解CB2受体的定位、内化和贩运如何与大麻素对人体免疫细胞的生物学效应联系起来。

DESCRIPTION (provided by applicant): Cannabinoids, the primary bioactive constituents of marijuana, are being extensively studied in order to understand their toxic and immunotherapeutic potential. They activate CB1 and CB2 receptors and signal through an endogenous cannabinoid system. Expression of the CB2 receptor predominates in cells from the immune system, and our novel preliminary findings suggest that CB2 is not only an extracellular G-protein- coupled receptor but is expressed at different locations and in different amounts by immune cells. While human B cells express CB2 on the cell surface, they also express a high intracellular concentration of CB2. In contrast, T cells do not express extracellular CB2 but express intracellular protein. In addition, CB2 expression patterns change in malignant B cell lines and change when naïve B cells are activated. These novel findings lead us to hypothesize that CB2 receptor expression, location, and trafficking are critical features tha link cannabinoids to specific signaling and functional consequences. Three specific aims are proposed: Aim 1) To investigate the role of CB2 receptor internalization and the use of adaptor proteins on the cellular distribution of CB2 in immune cells. Experiments will define CB2 location within the cell and assess if location differs by cell type, if intracellular CB2 is accessible to ligand binding, if cannabinoids differ in their ability to access intracellular CB2, and the role tat receptor internalization, recycling and adaptor proteins play in controlling CB2 distribution. Aim 2) To investigate the two-way interaction between CB2 expression on B cells, the impact of B cell activation on CB2 expression, and the capacity for cannabinoids to regulate B cell differentiation and immunoglobulin (Ig) class switching. We hypothesize that CB2 expression is a regulated event, and its expression pattern will bias responder cells toward specific differentiation pathways. Aim 3) To link signaling to location and the functional role of CB2 receptors. Cells with different CB2 expression patterns and cells in which the expression patterns have been specifically manipulated / activated will be assessed for cannabinoid signaling via three pathways - ERK/MAPK, PI3K/AKT, and intracellular Ca++. By the conclusion of these studies, we hope to better understand how CB2 receptor location, internalization, and trafficking link to the biologic effects of cannabinoids on human immune cells.