Lipid G Protein-coupled Receptor Ligand Identification Using β-Arrestin PathHunter™ Assay
Lipid G Protein-coupled Receptor Ligand Identification Using β-Arrestin PathHunter™ Assay
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DOI：
10.1074/jbc.m806516200
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发表时间：
2009-05-01
影响因子：
4.8
通讯作者：
Chen, Yu Alice
中科院分区：
文献类型：
--
作者：
Yin, Hong;Chu, Alan;Chen, Yu Alice
据报道，越来越多的孤儿G蛋白偶联受体（GPCR）被脂质配体激活，如溶血磷脂酸、1-磷酸鞘氨醇（S1 P）和大麻素，已经有很好的受体。这些新的配体声明是有争议的，由于缺乏独立的确认或相互矛盾的报告。我们使用β-抑制蛋白PathHunter（TM）测定系统，一种新开发的通用GPCR测定形式，其测量β-抑制蛋白与GPCR的结合，以评估脂质受体和配体配对。该测定消除了亲本细胞上内源性受体的干扰，因为它测量了由标记受体特异性产生的信号，并且该信号紧邻受体活化的下游。我们筛选了大量新的“脱孤儿”受体（GPR 23、GPR 92、GPR 55、G2 A、GPR 18、GPR 3、GPR 6、GPR 12和GPR 63）和对照受体，以对抗类似于400种脂质分子的集合，试图以无偏的方式鉴定受体配体。GPR 92被证实是一种溶血磷脂酸受体，对焦磷酸法呢酯和香叶基香叶基二磷酸的反应较弱。假定的大麻素受体GPR 55对AM 251、利莫那班和溶血磷脂酰肌醇反应强烈，但对内源性大麻素反应非常弱。G2 A受体被证实是一种氧化的游离脂肪酸受体。此外，我们发现，3，3 '-二吲哚基甲烷，一种来自十字花科蔬菜的膳食分子，具有已知的抗癌特性，是CB 2受体部分激动剂，结合亲和力约为1 μ M。3，3-二吲哚甲烷在RAW264.7细胞中的抗炎作用显示部分由CB 2介导。
A growing number of orphan G-protein-coupled receptors (GPCRs) have been reported to be activated by lipid ligands, such as lysophosphatidic acid, sphingosine 1-phosphate (S1P), and cannabinoids, for which there are already well established receptors. These new ligand claims are controversial due to either lack of independent confirmations or conflicting reports. We used the beta-arrestin PathHunter (TM) assay system, a newly developed, generic GPCR assay format that measures beta-arrestin binding to GPCRs, to evaluate lipid receptor and ligand pairing. This assay eliminates interference from endogenous receptors on the parental cells because it measures a signal that is specifically generated by the tagged receptor and is immediately downstream of receptor activation. We screened a large number of newly "deorphaned" receptors (GPR23, GPR92, GPR55, G2A, GPR18, GPR3, GPR6, GPR12, and GPR63) and control receptors against a collection of similar to 400 lipid molecules to try to identify the receptor ligand in an unbiased fashion. GPR92 was confirmed to be a lysophosphatidic acid receptor with weaker responses to farnesyl pyrophosphate and geranylgeranyl diphosphate. The putative cannabinoid receptor GPR55 responded strongly to AM251, rimonabant, and lysophosphatidylinositol but only very weakly to endocannabinoids. G2A receptor was confirmed to be an oxidized free fatty acid receptor. In addition, we discovered that 3,3'-diindolylmethane, a dietary molecule from cruciferous vegetables, which has known anti-cancer properties, to be a CB2 receptor partial agonist, with binding affinity around 1 mu M. The anti-inflammatory effect of 3,3-diindolylmethane in RAW264.7 cells was shown to be partially mediated by CB2.