Monitoring ligand-mediated internalization of G protein-coupled receptor as a novel pharmacological approach
Monitoring ligand-mediated internalization of G protein-coupled receptor as a novel pharmacological approach
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DOI：
10.1016/j.lfs.2006.08.022
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发表时间：
2006-12-03
期刊：
影响因子：
6.1
通讯作者：
Tsujimoto, Gozoh
中科院分区：
文献类型：
--
作者：
Fukunaga, Shin'ichi;Setoguchi, Shingo;Tsujimoto, Gozoh
G蛋白偶联受体（GPCR）的激动剂活化导致受体蛋白通过称为内化的内吞作用过程远离细胞表面重新分布到内部细胞区室中。通过使用荧光试剂如绿色荧光蛋白（GFP），受体内化的可视化在实验上已经变得可行。在这项研究中，我们研究了是否可以利用配体介导的内化的GPCR的药理学评价。我们获得了表达GFP标记的GPCR的细胞的荧光图像，并通过图像处理定量评估配体介导的内化。使用β 2-肾上腺素受体和加压素V-1a受体作为分别与Gs和Gq偶联的模型GPCR，我们首先检查这些GFP标记的GPCR是否表现出适当的药理学。针对每种受体的多种特异性激动剂和拮抗剂的受体内化效力的排序与之前在配体结合研究中观察到的一致。除了化学配体诱导的内化之外，这种基于细胞的荧光成像系统成功地监测了质子敏感GPCR TDAG 8和游离脂肪酸敏感GPCR GPR 120的内化。结果表明，监测受体内化可以是一个有用的方法，药理学特性的GPCR和孤儿GPCR的配体的捕捞。(c)2006年爱思唯尔公司All rights reserved.
Agonist activation of a G protein-coupled receptor (GPCR) results in the redistribution of the receptor protein away from the cell surface into internal cellular compartments through a process of endocytosis known as internalization. Visualization of receptor internalization has become experimentally practicable by using fluorescent reagents such as green fluorescent protein (GFP). In this study, we examined whether the ligand-mediated internalization of a GPCR can be exploited for pharmacological evaluations. We acquired fluorescent images of cells expressing GFP-labeled GPCRs and evaluated the ligand-mediated internalization quantitatively by image processing. Using beta(2)-adrenoceptor and vasopressin V-1a receptor as model GPCRs that couple to Gs and Gq, respectively, we first examined whether these GFP-tagged GPCRs exhibited appropriate pharmacology. The rank order of receptor internalization potency for a variety of agonists and antagonists specific to each receptor corresponded well with that previously observed in ligand binding studies. In addition to chemical ligand-induced internalization, this cell-based fluorescence imaging system successfully monitored the internalization of the proton-sensing GPCR TDAG8, and that of the free fatty acid-sensitive GPCR GPR120. The results show that monitoring receptor internalization can be a useful approach for pharmacological characterization of GPCRs and in fishing for ligands of orphan GPCRs. (c) 2006 Elsevier Inc. All rights reserved.