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Journal of Histochemistry and Cytochemistry, Vol. 50, 697-708, May 2002, Copyright © 2002, The Histochemical Society, Inc.

ARTICLE

A Monoclonal Antibody to Visualize PtdIns(3,4,5)P3 in Cells

Riyan Chena, Veronica H. Kangc, Jian Chenb, Joseph C. Shoped, Javad Torabinejadd, Daryll B. DeWalda,d, and Glenn D. Prestwicha,b
a Center for Cell Signaling, Salt Lake City, Utah
b Department of Medicinal Chemistry, The University of Utah, Salt Lake City, Utah
c Echelon Research Laboratories, Salt Lake City, Utah
d Department of Biology, Utah State University, Logan, Utah

Correspondence to: Glenn D. Prestwich, Center for Cell Signaling, 421 Wakara Way, Suite 360, Salt Lake City, UT 84108. E-mail: gprestwich@pharm.utah.edu

Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] is a second messenger produced in response to agonist stimulation. Traditionally, visualization of phosphoinositide polyphosphates (PtdInsPn) in living cells is accomplished using chimeric green fluorescent protein (GFP)–pleckstrin homology (PH) domain proteins, while PtdInsPn quantitation is accomplished by extraction and separation of radiolabeled cellular PtdInsPns. Here we describe preparation of a covalent protein–PtdIns(3,4,5)P3 immunogen, characterization of binding selectivity of an anti-PtdIns(3,4,5)P3 IgM, and immunodetection of PtdIns(3,4,5)P3 in stimulated mammalian cells. This antibody has greater than three orders of magnitude selectivity for binding PtdIns(3,4,5)P3 relative to its precursor, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and is therefore optimal for studies of cell function. The immunodetection in platelet-derived growth factor (PDGF)-stimulated NIH 3T3 cells was benchmarked against HPLC analysis of [3H]-myo-inositol-labeled cellular PtdInsPns. In addition, the changes in subcellular amounts and localizations of both PtdIns(3,4,5)P3 and PtdIns(4,5)P2 in stimulated NIH 3T3 fibroblasts and human neutrophils were observed by immunofluorescence. In insulin- or PDGF-stimulated fibroblasts, PtdIns(3,4,5)P3 levels increased in the cytoplasm, peaking at 10 min. In contrast, increases in the PtdIns(4,5)P2 levels were detected in nuclei, corresponding to the production of new substrate following depletion by phosphoinositide (PI) 3-kinase. (J Histochem Cytochem 50:697–708, 2002)

Key Words: phosphoinositide, anti-lipid IgM, immunofluorescence, growth factor stimulation, phosphoinositide 3-kinase, photoaffinity labeling, cell signaling, second messenger, neutrophil, fibroblast, nuclear lipids


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