ASSOCIATION AND COLOCALIZATION OF EPS15 WITH ADAPTER PROTEIN-2 AND CLATHRIN

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-a results in phosphorylation of Eps15, Stimulation of cells with PDGF or insulin did not lead to Eps15 phosphorylation, s uggesting that phosphorylation of Eps15 is a receptor-specific process . We demonstrate that Eps15 is constitutively associated with both alp ha-adaptin and clathrin. Upon EGF stimulation, Eps15 and alpha-adaptin are recruited to the EGF receptor. Using a truncated EGF receptor mut ant, we demonstrate that the regulatory domain of the cytoplasmic tail of the EGF receptor is essential for the binding of Eps15. Fractionat ion studies reveal that Eps15 is present in cell fractions enriched fo r plasma membrane and endosomal membranes. Immunofluorescence studies show that Eps15 colocalizes with adaptor protein-2 (AP-2) and partiall y with clathrin, No colocalization of Eps15 was observed with the earl y endosomal markers rab4 and rab5. These observations indicate that Ep s15 is present in coated pits and coated vesicles of the clathrin-medi ated endocytic pathway, but not in early endosomes, Neither AP-2 nor c lathrin are required for the binding of Eps15 to coated pits or coated vesicles, since in membranes lacking AP-2 and clathrin, Eps15 still s hows the same staining pattern, These findings suggest that Eps15 may play a critical role in the recruitment of active EGF receptors into c oated pit regions before endocytosis of ligand-occupied EGF receptors.