Phosphoinositide and inositol phosphate analysis in lymphocyte activation.
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Phosphoinositide and inositol phosphate analysis in lymphocyte activation. / Sauer, Karsten; Huang, Yina Hsing; Lin, Hongying; Sandberg, Mark; Mayr, Georg W.
in: Curr Protoc Immunol, Jahrgang 11, 2009, S. 1.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - Phosphoinositide and inositol phosphate analysis in lymphocyte activation.
AU - Sauer, Karsten
AU - Huang, Yina Hsing
AU - Lin, Hongying
AU - Sandberg, Mark
AU - Mayr, Georg W.
PY - 2009
Y1 - 2009
N2 - Lymphocyte antigen receptor engagement profoundly changes the cellular content of phosphoinositide lipids and soluble inositol phosphates. Among these, the phosphoinositides phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) play key signaling roles by acting as pleckstrin homology (PH) domain ligands that recruit signaling proteins to the plasma membrane. Moreover, PIP2 acts as a precursor for the second messenger molecules diacylglycerol and soluble inositol 1,4,5-trisphosphate (IP3), essential mediators of PKC, Ras/Erk, and Ca2+ signaling in lymphocytes. IP3 phosphorylation by IP3 3-kinases generates inositol 1,3,4,5-tetrakisphosphate (IP4), an essential soluble regulator of PH domain binding to PIP3 in developing T cells. Besides PIP2, PIP3, IP3, and IP4, lymphocytes produce multiple other phosphoinositides and soluble inositol phosphates that could have important physiological functions. To aid their analysis, detailed protocols that allow one to simultaneously measure the levels of multiple different phosphoinositide or inositol phosphate isomers in lymphocytes are provided here. They are based on thin layer, conventional and high-performance liquid chromatographic separation methods followed by radiolabeling or non-radioactive metal-dye detection. Finally, less broadly applicable non-chromatographic methods for detection of specific phosphoinositide or inositol phosphate isomers are discussed. Support protocols describe how to obtain pure unstimulated CD4+CD8+ thymocyte populations for analyses of inositol phosphate turnover during positive and negative selection, key steps in T cell development.
AB - Lymphocyte antigen receptor engagement profoundly changes the cellular content of phosphoinositide lipids and soluble inositol phosphates. Among these, the phosphoinositides phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) play key signaling roles by acting as pleckstrin homology (PH) domain ligands that recruit signaling proteins to the plasma membrane. Moreover, PIP2 acts as a precursor for the second messenger molecules diacylglycerol and soluble inositol 1,4,5-trisphosphate (IP3), essential mediators of PKC, Ras/Erk, and Ca2+ signaling in lymphocytes. IP3 phosphorylation by IP3 3-kinases generates inositol 1,3,4,5-tetrakisphosphate (IP4), an essential soluble regulator of PH domain binding to PIP3 in developing T cells. Besides PIP2, PIP3, IP3, and IP4, lymphocytes produce multiple other phosphoinositides and soluble inositol phosphates that could have important physiological functions. To aid their analysis, detailed protocols that allow one to simultaneously measure the levels of multiple different phosphoinositide or inositol phosphate isomers in lymphocytes are provided here. They are based on thin layer, conventional and high-performance liquid chromatographic separation methods followed by radiolabeling or non-radioactive metal-dye detection. Finally, less broadly applicable non-chromatographic methods for detection of specific phosphoinositide or inositol phosphate isomers are discussed. Support protocols describe how to obtain pure unstimulated CD4+CD8+ thymocyte populations for analyses of inositol phosphate turnover during positive and negative selection, key steps in T cell development.
KW - Animals
KW - Humans
KW - Cell Separation methods
KW - Chromatography, High Pressure Liquid methods
KW - Chromatography, Ion Exchange methods
KW - Chromatography, Thin Layer methods
KW - Indicators and Reagents chemistry
KW - Inositol chemistry
KW - Inositol Phosphates analysis
KW - Lymphocyte Activation
KW - Lymphocytes chemistry
KW - Phosphatidylinositols analysis
KW - Radioactive Tracers
KW - Signal Transduction
KW - Staining and Labeling methods
KW - Animals
KW - Humans
KW - Cell Separation methods
KW - Chromatography, High Pressure Liquid methods
KW - Chromatography, Ion Exchange methods
KW - Chromatography, Thin Layer methods
KW - Indicators and Reagents chemistry
KW - Inositol chemistry
KW - Inositol Phosphates analysis
KW - Lymphocyte Activation
KW - Lymphocytes chemistry
KW - Phosphatidylinositols analysis
KW - Radioactive Tracers
KW - Signal Transduction
KW - Staining and Labeling methods
M3 - SCORING: Zeitschriftenaufsatz
VL - 11
SP - 1
JO - Curr Protoc Immunol
JF - Curr Protoc Immunol
SN - 1934-3671
ER -
