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，正在广泛研究，以了解其毒性和免疫潜力。它们激活CB 1和CB 2受体，并通过内源性大麻素系统发出信号。CB 2受体的表达在免疫系统的细胞中占主导地位，我们的新的初步研究结果表明，CB 2不仅是一种细胞外G蛋白偶联受体，而且在不同的位置和免疫细胞以不同的量表达。虽然人B细胞在细胞表面上表达CB 2，但它们也表达高细胞内浓度的CB 2。相反，T细胞不表达细胞外CB 2，但表达细胞内蛋白。此外，CB 2表达模式在恶性B细胞系中发生变化，并且在幼稚B细胞被激活时发生变化。这些新的发现使我们假设CB 2受体的表达，定位和贩运是将大麻素与特定信号传导和功能后果联系起来的关键特征。本论文的主要目的是：1）研究CB 2受体的内化和衔接蛋白对CB 2在免疫细胞中分布的影响。实验将确定CB 2在细胞内的位置，并评估位置是否因细胞类型而异，细胞内CB 2是否可与配体结合，大麻素是否在其进入细胞内CB 2的能力方面不同，以及达特受体内化，再循环和衔接蛋白在控制CB 2分布中的作用。目的2）研究大麻素在B细胞上表达CB 2之间的双向相互作用、B细胞活化对CB 2表达的影响以及大麻素调节B细胞分化和免疫球蛋白（IG）类别转换的能力。我们假设CB 2的表达是一个受调控的事件，其表达模式将使应答细胞偏向特定的分化途径。目的3）研究CB 2受体的定位和功能与信号传导的关系。将通过三种途径- ERK/MAPK、PI 3 K/AKT和细胞内Ca++评估具有不同CB 2表达模式的细胞和其中表达模式已被特异性操纵/激活的细胞的大麻素信号传导。通过这些研究的结论，我们希望更好地了解CB 2受体的位置，内化和贩运如何与大麻素对人类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.