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

Hervé Guillou, Charlotte Lécureuil, Karen E. Anderson, Sabine Suire, G. John Ferguson, Chris D. Ellson, Alexander Gray, Nullin Divecha, Phillip T. Hawkins, Len R. Stephens

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    22 Citations (Scopus)

    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-methionylleucyl-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.

    Original languageEnglish
    Pages (from-to)726-732
    Number of pages7
    JournalJournal of Lipid Research
    Volume48
    Issue number3
    Early online date27 Nov 2006
    DOIs
    Publication statusPublished - Mar 2007

    Keywords

    • General receptor for phosphoinositides-1
    • Neomycin
    • Neutrophil
    • p110γ
    • Phosphatidylinositol (3,4,5)-trisphosphate
    • Phosphoinositide 3-kinase
    • Protein-lipid overlay
    • Ras

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