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

Abstract

SUMMARY 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)

Huntingtin-associated Protein 1 Interacts with Hepatocyte Growth Factor-regulated Tyrosine Kinase Substrate and Functions in Endosomal Trafficking

“…4, D-I). To further confirm the early endosomal localization of HAP1, HeLa cells were treated with Texas Red-conjugated EGF at 37°C for 10 min, under which conditions the internalized EGF proteins were almost exclusively localized to early endosomes (37,38). We observed that most of the HAP1-positive structures contained internalized EGF (Fig.…”

RNA helicase A is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus

“…However, the interaction of EGFR and RHA was inhibited by EGFR tyrosine kinase inhibitor in HeLa cells (Fig. S6D, Top), in which the level of EGFR expression is normal (1.4 × 10 5 /cell) (35). The impaired association of EGFR with RHA in HeLa cells upon treatment with tyrosine kinase inhibitor is caused by the absence of EGF/EGFR internalization and EGFR nuclear translocation in the presence of EGFR tyrosine kinase inhibitor (Fig.…”

Epidermal growth factor receptor mutation type iii transfected into a small cell lung cancer cell line is predominantly localized at the cell surface and enhances the malignant phenotype

“…In a recent study, EGFR was detected predominantly in clusters around the nonclathrin-coated plasma membrane. 50 EGFR has been shown to influence the level, phosphorylation state and localization of several proteins involved in the formation of or maintenance of tight junctions. These proteins includes ZO-1, ZO-2, ZO-3, cingulin, occludin, connexin43, nectin-2delta, cytoskeletal proteins and vinculin, actin and cadherins.…”