GPR17-CysLT受体二聚化调节小胶质细胞激活的作用及机制

It has been confirmed that the G protein-coupled receptors, P2Y-like GPR17 and cysteinyl leukotriene receptors (CysLT1R and CysLT2R), relate to microglial activation after brain ischemia. In microglia and other cells, the inhibitory effects of GPR17 on CysLT1R responses have been revealed. G protein-coupled receptors are frequently interacted as dimerization, resulting in modulation of their intracellular trafficking (including internalization), signaling and functional regulation. However, such interactions in microglia remain unknown. In this project, in rat primary microglia and the mouse microglial line BV2 cells, the methods including co-immunoprecipitation, FRET, PLA, intracellular protein trafficking assay, and phagocytosis assay will be applied. We plan to answer the following questions: (1) whether dimers of GPR17/CysLT1R and/or GPR17/CysLT2R are formed, and thereby alter the roles of CysLT1R and/or CysLT2R in regulation of microglial activation; (2) whether the dimerization with GPR17 results in alteration of the internalization (such as nuclear translocation) and signaling of CysLT1R and CysLT2R. The purpose of this project is to highlight the mechanisms underlying regulatory roles of these receptors in microglial activation, and to provide a new consideration for the relevant pharmacological studies.

G蛋白偶联的P2Y样受体GPR17及半胱氨酰白三烯受体（CysLT1R和CysLT2R）与脑缺血后小胶质细胞激活的关系，已被证实；在小胶质细胞和其他细胞还发现GPR17对CysLT1R的抑制效应。G蛋白偶联受体常以二聚化形式相互作用，影响受体向细胞内移动、信号转导及功能调节，但尚不清楚小胶质细胞内有无这种相互作用。本项目在原代小胶质细胞和小胶质细胞株BV2细胞，采用免疫共沉淀、FRET、PLA、蛋白细胞内移动分析、吞噬功能分析等方法，拟阐明：（1）GPR17与CysLT1R和/或CysLT2R是否形成二聚体，而影响CysLT1R和/或CysLT2R对小胶质细胞激活的调节；（2）二聚化对CysLT1R和/或CysLT2R的影响，是否通过改变受体内移（如核移位）以及信号转导。本项目旨在深入认识这些受体对小胶质细胞激活的调节机制，并提供新的药理学研究思路。

本项目基本完成预定计划，积极探索了GPR17-CysLTR二聚化，并进一步深入研究了小胶质细胞激活的调节，取得以下主要成绩：（1）与其他研究者合作，建立了GFP小片段（split-GFP）标记GPR17和CysLTR，研究膜受体(GPCRs)的可视化方法，并首先研究了GPR17在活细胞的内移特点。（2）确定了CysLT2R作为调节脑缺血损伤后激活小胶质细胞、诱导炎症反应的主要受体亚型。（3）证实了CysLT2R拮抗剂（HAMI3379）具有抗大鼠脑缺血后神经损伤及炎症反应的保护作用。（4）证明CysLT2R激活后，经由Ca2+增高ERK1/2磷酸化Egr-1表达上调诱导炎症因子IL-8产生的信号途径。（5）在CysLT1R以及NAMPT调节小胶质细胞激活等方面有新的发现。本项目发表SCI论文7篇，为进一步研究GPR17-CysLT 受体二聚化建立了一种可视化新方法，并且，深入阐明了CysLT2R的在脑缺血损伤中的病理学及药理学意义。