Bioluminescent probes to analyze ligand-induced phosphatidylinositol 3,4,5-trisphosphate production with split luciferase complementation.
Anal Chem
; 85(23): 11352-9, 2013 Dec 03.
Article
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| MEDLINE
| ID: mdl-24195761
ABSTRACT
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.
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Fosfatos de Fosfatidilinositol
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Colorantes Fluorescentes
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Luciferasas
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Mediciones Luminiscentes
Límite:
Animals
Idioma:
En
Revista:
Anal Chem
Año:
2013
Tipo del documento:
Article
País de afiliación:
Japón