Use of the GRP1 PH domain as a tool to measure the relative levels of PtdIns(3,4,5)P3 through a protein-lipid overlay approach

Abstract

We describe a novel approach to the relative quantification of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] and its application to measure, in neutrophils, the activation of phosphoinositide 3-kinase (PI3K). This protein-lipid overlay-based assay allowed us to confirm and extend the observations, first, that N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation of primed human neutrophils leads to a transient and biphasic increase in PtdIns(3,4,5)P3 levels and, second, that the ability of fMLP to stimulate PtdIns(3,4,5)P3 accumulation in neutrophils isolated from mice carrying a Ras-insensitive (‘DASAA‘) knock-in of PI3Kγ (p110γDASAA/DASAA) is substantially dependent on the Ras binding domain of PI3Kγ.

Keywords

• phosphatidylinositol (3,4,5)-trisphosphate
• neomycin
• phosphoinositide 3-kinase
• general receptor for phosphoinositides-1
• protein-lipid overlay
• neutrophil