title: 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)P(3)] 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)P(3) levels and, second, that the ability of fMLP to stimulate PtdIns(3,4,5)P(3) accumulation in neutrophils isolated from mice carrying a Ras-insensitive ('DASAA') knock-in of PI3Kgamma (p110gamma(DASAA/DASAA)) is substantially dependent on the Ras binding domain of PI3Kgamma.
authors.affiliation: The Babraham Institute, Cambridge, UK.
authors.forename: Hervé
authors.initials: H
authors.lastname: Guillou
authors.forename: Charlotte
authors.initials: C
authors.lastname: Lécureuil
authors.forename: Karen E
authors.initials: KE
authors.lastname: Anderson
authors.forename: Sabine
authors.initials: S
authors.lastname: Suire
authors.forename: G John
authors.initials: GJ
authors.lastname: Ferguson
authors.forename: Chris D
authors.initials: CD
authors.lastname: Ellson
authors.forename: Alexander
authors.initials: A
authors.lastname: Gray
authors.forename: Nullin
authors.initials: N
authors.lastname: Divecha
authors.forename: Phillip T
authors.initials: PT
authors.lastname: Hawkins
authors.forename: Len R
authors.initials: LR
authors.lastname: Stephens
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002478:Cells, Cultured; D058544:Class Ib Phosphatidylinositol 3-Kinase; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
chemical_list: D007527:Isoenzymes; D018129:Phosphatidylinositol Phosphates; D018160:Receptors, Cytoplasmic and Nuclear; C060974:phosphatidylinositol 3,4,5-triphosphate; C040985:phosphatidylinositol receptors; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019869:Phosphatidylinositol 3-Kinases; D058544:Class Ib Phosphatidylinositol 3-Kinase; C546760:PIK3CG protein, human; C546764:Pik3cg protein, mouse