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

摘要

A lipid second messenger, phosphatidylinositol (3,4,5)-trisphosphate (PIP_3), is a signaling molecule that mediates central cellular events, such as growth, motility, and development by activating downstream proteins. Although functions of various PIP_3 binding partners have been unveiled, the various roles of PIP_3 have not been resolved thoroughly because of limitations of PIP_3 analysis. Herein, we describe a novel method for the analysis of relative PIP_3 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 PIP_3 binding units, pleckstrin homology domains (PHDs) of the general receptor for phosphoinositides 1 (GRP1), was expressed in cytosol (PP-LucC). In response to PIP_3 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 PIP_3 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 PIP_3 production on the plasma membrane by bioluminescence. This method enables further study of PIP_3 and supports versatile applications related to the PIP_3 amount.