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* Department of Molecular Cell Biology, Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands; 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- Laboratory of Molecular Oncology, Rockefeller University, New York 10021; and § Hubrecht Laboratory, Netherlands Institute
for Developmental Biology, 3584 CT Utrecht, The Netherlands
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
-adaptin and clathrin. Upon EGF stimulation, Eps15
and -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.
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