Bioluminescent Probes to Analyze Ligand-lnduced Phosphatidylinositol3,4,5-Trisphosphate Production with Split Luciferase Complementation
Bioluminescent Probes to Analyze Ligand-lnduced Phosphatidylinositol3,4,5-Trisphosphate Production with Split Luciferase Complementation
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用于分析配体诱导的磷脂酰肌醇3,4,5-三磷酸生产与分裂荧光素酶互补的生物发光探针
DOI：
10.1021/ac402278f
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发表时间：
2013
影响因子：
7.4
通讯作者：
A Kanno and T Ozawa
中科院分区：
文献类型：
--
作者：
L Yang;Y Nasu;M Hattori;H Yoshimura;A Kanno and T Ozawa
脂质第二信使磷脂酰肌醇(3,4,5)-三磷酸（PIP3）是一种信号分子，通过激活下游蛋白介导中心细胞事件，如生长、运动和发育。虽然各种pip3结合伙伴的功能已经被揭示，但由于pip3分析的局限性，pip3的各种作用并没有得到彻底的解决。在此，我们描述了一种基于荧光素酶片段自发互补的新方法来分析相对pip3量。荧光素酶的n端片段位于质膜上（LucN-pm）。荧光素酶的c端片段与pip3结合单元融合，即磷酸肌苷1 （GRP1）一般受体的pleckstrin同源结构域（phd）在细胞质（PP-LucC）中表达。为了响应pip3的产生，PP-LucC被带到质膜上并与LucN-pm共定位。LucN-pm和PP-LucC自发重组形成活性荧光素酶，产生生物发光恢复。我们在激动剂的刺激下成功地获得了与相对pip3量相对应的生物发光信号。我们还证明了该探针可用于高通量筛选格式，并通过生物发光监测质膜上pip3的产生。该方法可以进一步研究pip3，并支持与pip3量相关的多种应用程序。
A lipid second messenger, phosphatidylinositol (3,4,5)-trisphosphate (PIP3), is a signaling molecule that mediates central cellular events, such as growth, motility, and development by activating downstream proteins. Although functions of various PIP3binding partners have been unveiled, the various roles of PIP3have not been resolved thoroughly because of limitations of PIP3analysis. Herein, we describe a novel method for the analysis of relative PIP3amount based on spontaneous complementation of split luciferase fragments. An N-terminal fragment of a luciferase was located on the plasma membrane (LucN-pm). A C-terminal fragment of a luciferase fused with PIP3binding units, pleckstrin homology domains (PHDs) of the general receptor for phosphoinositides 1 (GRP1), was expressed in cytosol (PP-LucC). In response to PIP3production, PP-LucC was brought to the plasma membrane and colocalized with LucN-pm. The LucN-pm and PP-LucC reconstituted spontaneously to form an active luciferase, producing bioluminescence recovery. We obtained bioluminescence signals corresponding to relative PIP3amounts successfully upon stimulation with an agonist. We also demonstrated that the probes were applied for a high-throughput screening format and for monitoring of PIP3production on the plasma membrane by bioluminescence. This method enables further study of PIP3and supports versatile applications related to the PIP3amount.