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

We describe a novel approach to the relative quantification of phosphatidylinositol (3,4,5)-trisphosphate [Ptdlns(3,4,5)P-3] and its application to measure, in neutrophils, the activation of phosphoinositicle 3-kinase (PI3K). This protein-lipid overlay-based assay allowed us to confirm and extend the observations, first, that N-formyl-methionyl-leucyl-phenylaianine (fMLP) stimulation of primed human neutrophils leads to a transient and biphasic increase in Ptdlns(3,4,5)P-3 levels and, second, that the ability of fMLP to stimulate Ptdlns(3,4,5)P-3 accumulation in neutrophils isolated from mice carrying a Ras-insensitive ('DASAA') knock-in of PI3K gamma (p110 gamma(DASAA/DASAA)) is substantially dependent on the Ras binding domain of PI3K gamma.