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

doi:10.1194/jlr.D600038-JLR200

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

Research output: Contribution to journal › Article

20 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 (p110γ^DASAA/DASAA) is substantially dependent on the Ras binding domain of PI3Kγ.

Original language	English
Pages (from-to)	726-732
Number of pages	7
Journal	Journal of Lipid Research
Volume	48
Issue number	3
Early online date	27 Nov 2006
DOIs	https://doi.org/10.1194/jlr.D600038-JLR200
Publication status	Published - 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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Guillou, H., Lécureuil, C., Anderson, K. E., Suire, S., Ferguson, G. J., Ellson, C. D., Gray, A., Divecha, N., Hawkins, P. T., & Stephens, L. R. (2007). 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. Journal of Lipid Research, 48(3), 726-732. https://doi.org/10.1194/jlr.D600038-JLR200

Guillou, Hervé ; Lécureuil, Charlotte ; Anderson, Karen E. ; Suire, Sabine ; Ferguson, G. John ; Ellson, Chris D. ; Gray, Alexander ; Divecha, Nullin ; Hawkins, Phillip T. ; Stephens, Len R. / 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. In: Journal of Lipid Research. 2007 ; Vol. 48, No. 3. pp. 726-732.

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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.",

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

author = "Herv{\'e} Guillou and Charlotte L{\'e}cureuil and Anderson, {Karen E.} and Sabine Suire and Ferguson, {G. John} and Ellson, {Chris D.} and Alexander Gray and Nullin Divecha and Hawkins, {Phillip T.} and Stephens, {Len R.}",

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

In: Journal of Lipid Research, Vol. 48, No. 3, 03.2007, p. 726-732.

Research output: Contribution to journal › Article

TY - JOUR

T1 - 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

AU - Guillou, Hervé

AU - Lécureuil, Charlotte

AU - Anderson, Karen E.

AU - Suire, Sabine

AU - Ferguson, G. John

AU - Ellson, Chris D.

AU - Gray, Alexander

AU - Divecha, Nullin

AU - Hawkins, Phillip T.

AU - Stephens, Len R.

PY - 2007/3

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AB - 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.

KW - General receptor for phosphoinositides-1

KW - Neomycin

KW - Neutrophil

KW - p110γ

KW - Phosphatidylinositol (3,4,5)-trisphosphate

KW - Phosphoinositide 3-kinase

KW - Protein-lipid overlay

KW - Ras

U2 - 10.1194/jlr.D600038-JLR200

DO - 10.1194/jlr.D600038-JLR200

M3 - Article

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JO - Journal of Lipid Research

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SN - 0022-2275

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