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A correction to this article has been published: J. Cell Biol. 137 (1) 259
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© The Rockefeller University Press, 0021-9525/1997//811 $5.00
The Journal of Cell Biology, Volume 136, Number 4, , 1997 811-821

Article

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



Sanne van Delft*, Christopher Schumacher{ddagger}, Willem Hage§, Arie J. Verkleij*, and Paul M.P. van Bergen en Henegouwen*

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

Abbreviations used in this paper: AP, adaptor protein; CSLM, confocal laser scanning microscope; G, vesicular stomatitis virus G protein; HC, heavy chain; LC, light chain; NH, hemagglutinin; TAPS, N-Tris[hydroxymethyl]methyl-3-amino-propanesulfonic acid buffer.

Please address all correspondence to Sanne van Delft, Department of Molecular Cell Biology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. Tel.: 31-30-253-3349; Fax: 31-30-251-3655; E-mail: sanne{at}emsaserv.biol.ruu.nl


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