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Journal of Histochemistry and Cytochemistry, Vol. 48, 21-34, January 2000, Copyright © 2000, The Histochemical Society, Inc.

ARTICLE

Immunocytochemical Localization of Shc and Activated EGF Receptor in Early Endosomes After EGF Stimulation of HeLa Cells

Morten P. Oksvolda, Ellen Skarpena, Birgitte Lindemana, Norbert Roosb, and Henrik S. Huitfeldta
a Laboratory for Toxicopathology, Institute of Pathology, The National Hospital, University of Oslo, Oslo, Norway
b Electron Microscopical Unit for Biological Sciences, Department of Biology, University of Oslo, Oslo, Norway

Correspondence to: Henrik S. Huitfeldt, Lab for Toxicopathology, Inst. of Pathology, The National Hospital, U. of Oslo, N-0027 Oslo, Norway.

After binding of epidermal growth factor (EGF), the EGF receptor (EGFR) becomes autophosphorylated via tyrosine. The ligand-activated receptor is internalized by endocytosis and subsequently degraded in the lysosomal pathway. To follow EGFR activation after EGF stimulation, we generated antisera to the EGFR phosphotyrosine sites pY992 and pY1173. The SH2 region of Shc binds to both these sites. Both antisera identified EGFR after EGF binding and did not crossreact with the unactivated receptor. The intracellular distribution of phosphorylated EGFR after ligand binding was traced by two-color immunofluorescence confocal microscopy and immunoelectron microscopy. Before EGF stimulation EGFR was primarily located along the cell surface. When internalization of activated EGFR was inhibited by incubation with EGF on ice, Y992- and Y1173-phosphorylated EGFR were located along the plasma membrane. Ten minutes after internalization at 37C, Y992- and Y1173-phosphorylated EGFR were almost exclusively located in early endosomes, as shown by co-localization with EEA1. Immunoelectron microscopy confirmed that phosphorylated EGFR was located in intracellular vesicles resembling early endosomes. After EGF stimulation, the adaptor protein Shc redistributed to EGFR-containing early endosomes. Our results indicate that EGFR activation of Shc via tyrosine-phosphorylated Y992 and Y1173 occurred in early endocytic compartments, and support a role for membrane trafficking in intracellular signaling. (J Histochem Cytochem 48:21–33, 2000)

Key Words: HeLa cells, EGF receptor, Shc, signal transduction, endosomes, immunofluorescence, immunoelectron microscopy


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