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Eps15 is a substrate for the tyrosine kinase of the epidermal growth factor receptor (EGFR) and is characterized by the presence of a novel protein:protein interaction domain, the EH domain. Eps15 also stably binds the clathrin adaptor protein complex AP-2. Previous work demonstrated an essential role for eps15 in receptor-mediated endocytosis. In this study we show that, upon activation of the EGFR kinase, eps15 undergoes dramatic relocalization consisting of 1) initial relocalization to the plasma membrane and 2) subsequent colocalization with the EGFR in various intracellular compartments of the endocytic pathway, with the notable exclusion of coated vesicles. Relocalization of eps15 is independent of its binding to the EGFR or of binding of the receptor to AP-2. Furthermore, eps15 appears to undergo tyrosine phosphorylation both at the plasma membrane and in a nocodazole-sensitive compartment, suggesting sustained phosphorylation in endocytic compartments. Our results are consistent with a model in which eps15 undergoes cycles of association:dissociation with membranes and suggest multiple roles for this protein in the endocytic pathway.

The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein.

We report the localization over the cell surface and the early steps of antibody-induced internalization of the product of the erbB-2 proto-oncogene, structurally related to the epidermal growth factor receptor (EGFR). We show that erbB-2/p 185 is mostly excluded from endocytic pits on the cell surface. Incubation at 37 degrees C with an anti-erbB-2/p185 monoclonal antibody induces the rapid entry of the protein into the cell. Similar internalization is shown by a chimeric molecule EGFR/erbB-2 in response to EGF. Both the timing and the pathway of internalization followed by the erbB-2/p185 appear totally similar to those described for the EGFR. At variance with the normal erbB-2/p185, two mutant activated erbB-2 proteins are frequently localized within endocytic pits of the cell surface, indicating that mutations in the transmembrane regions may determine constitutive internalization of the protein.

The fracture immunolabel technique, which permits assessment of the partition of transmembrane proteins with the inner or outer leaflets of the freeze-fractured membrane, was used to analyze the behavior on fracture of epidermal growth factor (EGF) receptors over the plasma membranes of A431 cells. The receptors partition mainly with the outer leaflet of the freeze-fractured plasma membranes, whereas they become associated with the inner leaflet when they are occupied by the ligand. This modified partition is even more evident after receptor clustering induced by incubation with EGF at 37 degrees C. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) decreases the number of receptors over both inner and outer leaflets. An effect similar to that induced by the ligand is obtained when receptor aggregation is achieved using anti-receptor monoclonal antibodies (MAb). The modified partition therefore indicates receptor activation and appears to be a consequence of receptor cross-linking rather than to reflect a conformational change of the receptor molecule. Parallel immunolabeling with anti-phosphotyrosine antibodies of freeze-fractured EGF-treated A431 cells reveals that the receptors, when activated, are associated only with the inner leaflet of the plasma membrane.

The localization of epidermal growth factor (EGF) receptors over the plasma membranes of human epidermoid carcinoma A431 cells was analyzed at the electron microscopic level using surface replica techniques and conventional thin sections, in combination with immunocytochemistry. Immunolabeling was performed using two distinct monoclonal antibodies directed against the extracellular portion of the receptor, followed by protein A-colloidal gold conjugates. Unexpectedly, with the first monoclonal antibody used, the distribution of the receptors in both unfixed and glutaraldehyde-fixed cells was clearly regionalized, showing a preferential localization of the immunolabeling at the cell periphery as well as over the areas rich in microvilli and in coated and uncoated pits. A similar pattern of distribution was observed also with the other monoclonal antibody, but only when the cells were fixed with glutaraldehyde before immunolabeling. Treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate modifies this distribution, inducing a more disperse pattern. Our observations suggest that a minor group of EGF receptors, which may represent the high-affinity receptors, presents a regional distribution, similar to that described for typical recycling receptors.