Unit

UNIT 11.1 Phosphoinositide and Inositol Phosphate Analysis in Lymphocyte Activation

  1. Karsten Sauer1,
  2. Yina Hsing Huang2,
  3. Hongying Lin3,
  4. Mark Sandberg4,
  5. Georg W. Mayr3

Published Online: 1 NOV 2009

DOI: 10.1002/0471142735.im1101s87

Current Protocols in Immunology

Current Protocols in Immunology

How to Cite

Sauer, K., Huang, Y. H., Lin, H., Sandberg, M. and Mayr, G. W. 2009. Phosphoinositide and Inositol Phosphate Analysis in Lymphocyte Activation. Current Protocols in Immunology. 87:11.1:11.1.1–11.1.46.

Author Information

  1. 1

    The Scripps Research Institute, La Jolla, California

  2. 2

    Washington University School of Medicine, St. Louis, Missouri

  3. 3

    University Medical Center Hamburg-Eppendorf, Hamburg, Germany

  4. 4

    Genomics Institute of the Novartis Research Foundation (GNF), San Diego, California

Publication History

  1. Published Online: 1 NOV 2009
  2. Published Print: NOV 2009

Abstract

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. Curr. Protoc. Immunol. 87:11.1.1-11.1.46. © 2009 by John Wiley & Sons, Inc.

Keywords:

  • lymphocyte;
  • inositol;
  • phosphoinositide;
  • phospholipid;
  • second messenger;
  • T cell;
  • thymocyte;
  • signal transduction;
  • IP3;
  • IP4;
  • IP5;
  • IP6;
  • PIP2;
  • PIP3;
  • HPLC;
  • MDD-HPLC