Bioluminescent Probes to Analyze Ligand-Induced Phosphatidylinositol 3,4,5-Trisphosphate Production with Split Luciferase Complementation

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• 作者：Lingzhi Yang ; Yusuke Nasu ; Mitsuru Hattori ; Hideaki Yoshimura ; Akira Kanno ; Takeaki Ozawa
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：December 3, 2013
• 年：2013
• 卷：85
• 期：23
• 页码：11352-11359
• 全文大小：389K
• 年卷期：v.85,no.23(December 3, 2013)
• ISSN：1520-6882

文摘

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 PIP₃ binding partners have been unveiled, the various roles of PIP₃ have not been resolved thoroughly because of limitations of PIP₃ analysis. Herein, we describe a novel method for the analysis of relative PIP₃ amount 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 PIP₃ binding units, pleckstrin homology domains (PHDs) of the general receptor for phosphoinositides 1 (GRP1), was expressed in cytosol (PP-LucC). In response to PIP₃ production, 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 PIP₃ amounts successfully upon stimulation with an agonist. We also demonstrated that the probes were applied for a high-throughput screening format and for monitoring of PIP₃ production on the plasma membrane by bioluminescence. This method enables further study of PIP₃ and supports versatile applications related to the PIP₃ amount.