Association and Colocalization of Eps15 with Adaptor Protein-2 and Clathrin

• Published in: The Journal of cell biology Vol. 136; no. 4; pp. 811 - 821
• Main Authors: van Delft, Sanne, Schumacher, Christopher, Hage, Willem, Verkleij, Arie J., Paul M. P. van Bergen en Henegouwen
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 24.02.1997; The Rockefeller University Press
• Subjects: 3T3 Cells; Adaptor Protein Complex alpha Subunits; Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Animals; Antibodies; Calcium-Binding Proteins - drug effects; Calcium-Binding Proteins - metabolism; Calcium-Binding Proteins - physiology; Cell lines; Cell membranes; Cells; Cellular biology; Clathrin - metabolism; Clathrin - physiology; Coated Pits, Cell-Membrane - chemistry; Coated vesicles; Endocytosis; Epidermal Growth Factor - metabolism; Epidermal Growth Factor - pharmacology; ErbB Receptors - metabolism; ErbB Receptors - physiology; Insulin; Insulin - pharmacology; Intracellular Signaling Peptides and Proteins; Membrane Proteins - metabolism; Membrane Proteins - physiology; Mice; Phosphoproteins - drug effects; Phosphoproteins - metabolism; Phosphoproteins - physiology; Phosphorylation; Platelet-Derived Growth Factor - pharmacology; Protein Structure, Tertiary; Proteins; Receptors; Subcellular Fractions - metabolism; Transforming Growth Factor alpha - pharmacology
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.136.4.811

Eps15 has been identified as a substrate of the EGF receptor tyrosine kinase. In this report, we show that activation of the EGF receptor by either EGF or TGF-α results in phosphorylation of Eps15. Stimulation of cells with PDGF or insulin did not lead to Eps15 phosphorylation, suggesting that phosphorylation of Eps15 is a receptor-specific process. We demonstrate that Eps15 is constitutively associated with both α-adaptin and clathrin. Upon EGF stimulation, Eps15 and α-adaptin are recruited to the EGF receptor. Using a truncated EGF receptor mutant, we demonstrate that the regulatory domain of the cytoplasmic tail of the EGF receptor is essential for the binding of Eps15. Fractionation studies reveal that Eps15 is present in cell fractions enriched for plasma membrane and endosomal membranes. Immunofluorescence studies show that Eps15 colocalizes with adaptor protein-2 (AP-2) and partially with clathrin. No colocalization of Eps15 was observed with the early endosomal markers rab4 and rab5. These observations indicate that Eps15 is present in coated pits and coated vesicles of the clathrin-mediated endocytic pathway, but not in early endosomes. Neither AP-2 nor clathrin are required for the binding of Eps15 to coated pits or coated vesicles, since in membranes lacking AP-2 and clathrin, Eps15 still shows the same staining pattern. These findings suggest that Eps15 may play a critical role in the recruitment of active EGF receptors into coated pit regions before endocytosis of ligand-occupied EGF receptors.