1 research outputs found
Bioluminescent Probes to Analyze Ligand-Induced Phosphatidylinositol 3,4,5-Trisphosphate Production with Split Luciferase Complementation
No full textA lipid second messenger, phosphatidylinositol
(3,4,5)-trisphosphate (PIP<sub>3</sub>), is a signaling molecule that
mediates central cellular events, such as growth, motility, and development
by activating downstream proteins. Although functions of various PIP<sub>3</sub> binding partners have been unveiled, the various roles of
PIP<sub>3</sub> have not been resolved thoroughly because of limitations
of PIP<sub>3</sub> analysis. Herein, we describe a novel method for
the analysis of relative PIP<sub>3</sub> 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<sub>3</sub> binding units,
pleckstrin homology domains (PHDs) of the general receptor for phosphoinositides
1 (GRP1), was expressed in cytosol (PP-LucC). In response to PIP<sub>3</sub> 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<sub>3</sub> 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<sub>3</sub> production
on the plasma membrane by bioluminescence. This method enables further
study of PIP<sub>3</sub> and supports versatile applications related
to the PIP<sub>3</sub> amount
