Bioluminescent probes to analyze ligand-induced phosphatidylinositol 3,4,5-trisphosphate production with split luciferase complementation.

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 biolumin...