Eps15 Is a Component of Clathrin-coated Pits and Vesicles and Is Located at the Rim of Coated Pits

doi:10.1074/jbc.271.46.28727

Eps15 Is a Component of Clathrin-coated Pits and Vesicles and Is Located at the Rim of Coated Pits*

From the ^‡ Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262, the
^∥ Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, and the
** Center for Blood Research, Boston, Massachusetts 02115

^¶To whom correspondence may be addressed:
Dept. of Pharmacology, University of Colorado Health Science Center, 4200 East Ninth Ave., Denver, CO 80262.
Tel.: 303-315-7252; Fax: 303-315-7097; E-mail: Alexander.Sorkin{at}USHSC.edu.
^‡‡To whom correspondence may be addressed:
Harvard Medical School, 200 Longwood Ave., Boston MA 02115.
Tel.: 617-278-3140; Fax: 617-278-3131; E-mail: kirchhausen{at}xtal0.harvard.edu.

Abstract

Eps15, a phosphorylation substrate of the epidermal growth factor (EGF) receptor kinase, has been shown to bind to the α-subunit of the clathrin-associated protein complex AP-2. Here we report that in cells, virtually all Eps15 interacts with the cytosol and membrane-bound forms of AP-2. This association is not affected by the treatment of cells with EGF. Immunofluorescence microscopy reveals nearly absolute co-localization of Eps15 with AP-2 and clathrin, and analysis by immunoelectron microscopy shows that the localization of membrane-associated Eps15 is restricted to the profiles corresponding to endocytic coated pits and vesicles. Unexpectedly, Eps15 was found at the edge of forming coated pits and at the rim of budding coated vesicles. This asymmetric distribution is in sharp contrast to the localization of AP-2 that shows an even distribution along the same types of clathrin-coated structures. These findings suggest several possible regulatory roles of Eps15 during the formation of coated pits.

Footnotes

↵^§ This work is the result of an equal contribution from the Sorkin and Kirchhausen laboratories.
↵* This work was supported by National Institutes of Health Grant DK46817 (to A. S.), a grant from Tobacco Council Research (to A. S.), a Fellowship from Ministerio de Educación y Cultura, Spain (to F. T.), and National Institutes of Health Grant GM36548 (to T. K.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

AP-2

plasma membrane clathrin-associated protein complex also referred to as adaptor

AP-1

Golgi clathrin-associated protein complex

CMF-PBS

Ca²⁺- and Mg²⁺-free phosphate-buffered saline

EGF

epidermal growth factor

GTPγS

guanosine 5′-O-(thiotriphosphate).
↵²K. Clairmont, W. Boll, and T. Kirchhausen, submitted for publication.
- Received August 30, 1996.
- Revision received September 19, 1996.