Immunocytochemical localization of Shc and activated EGF receptor in early endosomes after EGF stimulation of HeLa cells

J Histochem Cytochem. 2000 Jan;48(1):21-33. doi: 10.1177/002215540004800103.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing*
  • Adaptor Proteins, Vesicular Transport*
  • Binding, Competitive
  • Biological Transport
  • Cell Compartmentation
  • Cell Membrane / metabolism
  • Endosomes / ultrastructure*
  • Enzyme-Linked Immunosorbent Assay
  • Epidermal Growth Factor / pharmacology*
  • ErbB Receptors / isolation & purification*
  • Fluorescent Antibody Technique
  • HeLa Cells
  • Humans
  • Intracellular Membranes / metabolism
  • Microscopy, Immunoelectron
  • Phosphoproteins / isolation & purification*
  • Phosphorylation
  • Proteins / isolation & purification*
  • Shc Signaling Adaptor Proteins
  • Src Homology 2 Domain-Containing, Transforming Protein 1

Substances

  • Adaptor Proteins, Signal Transducing
  • Adaptor Proteins, Vesicular Transport
  • Phosphoproteins
  • Proteins
  • SHC1 protein, human
  • Shc Signaling Adaptor Proteins
  • Src Homology 2 Domain-Containing, Transforming Protein 1
  • Epidermal Growth Factor
  • ErbB Receptors