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A more recent version of this article appeared on March 1, 2007

Papers In Press, published online ahead of print November 27, 2006
J. Lipid Res., doi:10.1194/jlr.D600038-JLR200
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Submitted on September 20, 2006
Revised on November 9, 2006
Accepted on November 27, 2006

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

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

Inositide, The Babraham Institute, Cambridge CB223AT

Corresponding Author: len.stephens{at}bbsrc.ac.uk

We describe a novel approach to 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, firstly, that fMLP stimulation of primed human neutrophils leads to a transient and biphasic increase in PtdIns(3,4,5)P3 levels and, secondly, 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 PI3Kgamma (p110gammaDASAA/DASAA) is substantially dependent on the Ras Binding Domain (RBD) of PI3Kgamma.


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