Selective Covalent Labeling of Tag-Fused GPCR Proteins on Live Cell Surface with a Synthetic Probe for Their Functional Analysis
Selective Covalent Labeling of Tag-Fused GPCR Proteins on Live Cell Surface with a Synthetic Probe for Their Functional Analysis
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DOI：
10.1021/ja910703v
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发表时间：
2010-07-14
影响因子：
15
通讯作者：
Hamachi, Itaru
中科院分区：
文献类型：
--
作者：
Nonaka, Hiroshi;Fujishima, Sho-hei;Hamachi, Itaru
用小分子探针进行选择性蛋白质标记是一种阐明活细胞中蛋白质功能的通用方法。在本文中，我们报道了一种利用小功能分子在细胞表面表达的标签融合g蛋白偶联受体（GPCR）蛋白的共价标记方法。该方法采用多肽标签和分子探针的选择性快速反应，分子探针包括含有半胱氨酸的短CA6D4x2标签（CAAAAAADDDDGDDDD）和含有活性α -氯乙酰基片段的四核Zn(II)-DpaTyr探针。在生理条件下，用不同功能基团的Zn(II)-DpaTyr探针在短时间（10-30分钟）内选择性地对缓动素受体（B2R）和乙酰胆碱受体（m1AchR）等标签融合的gpcr进行共价标记。用荧光团附加的Zn(II)-DpaTyr探针进行标记，可以通过荧光显示HEK293细胞表面的gpcr。用附加生物素的探针进行标记，允许将生物素单元引入gpcr。利用生物素-链霉亲和素的特异性相互作用，该生物素标签被用于荧光生物成像研究和标记后的gpcr印迹分析。这种标记方法的实用性在gpcr的几个功能分析中得到了证明，例如gpcr刺激响应内化的荧光可视化和含有内化gpcr的核内体的pH变化。
Selective protein labeling with a small molecular probe is a versatile method for elucidating protein functions in living cells. In this paper, we report a covalent labeling method of tag-fused G-protein coupled receptor (GPCR) proteins expressing on cell surfaces utilizing small functional molecules. This method employs the selective and rapid reaction of a peptide tag and a molecular probe, which comprises the cysteine-containing short CA6D4x2 tag (CAAAAAADDDDGDDDD) and a tetranuclear Zn(II)-DpaTyr probe containing a reactive alpha-chloroacetyl moiety. The covalent labeling of tag-fused GPCRs such as bradykinin receptor (B2R) and acetylcholine receptor (m1AchR) selectively proceeded under physiological conditions during short incubation (10-30 min) with Zn(II)-DpaTyr probes bearing various functional groups. Labeling with fluorophore-appended Zn(II)-DpaTyr probes enabled visualization of the GPCRs on the surface of HEK293 cells by fluorescence. Labeling with the biotin-appended probe allowed introduction of a biotin unit into the GPCRs. This biotin label was utilized for fluorescence bioimaging studies and postlabeling blotting analysis of the labeled GPCRs by use of the specific biotin-streptavidin interaction. The utility of this labeling method was demonstrated in several function analyses of GPCRs, such as fluorescence visualization of the stimuli-responsive internalization of GPCRs and pH change in endosomes containing the internalized GPCRs.