Stoichiometric Interaction of the Epidermal Growth Factor Receptor with the Clathrin-associated Protein Complex AP-2

doi:10.1074/jbc.270.2.619

Stoichiometric Interaction of the Epidermal Growth Factor Receptor with the Clathrin-associated Protein Complex AP-2 (*)

From the (1)Department of Biochemistry and
(2)Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and the
(3)Department of Cell Biology, Harvard Medical School, and Center for Blood Research, Boston, Massachusetts 02115

↵^§ Present address: Dept. of Pharmacology, University of Colorado Health Science Center, 4200 East Ninth Ave., Denver, CO 80262.

Abstract

Plasma membrane clathrin-associated protein complexes (AP-2) have been shown to co-immunoprecipitate with the epidermal growth factor (EGF) receptor (Sorkin A., and Carpenter, G. (1993) Science 261, 612-615). Hence, we analyzed the stoichiometry of the EGF receptor interaction with AP-2 using a new antibody that efficiently immunoprecipitates native AP-2. EGF receptor•AP-2 complexes were isolated from S-labeled cells treated with EGF by EGF receptor affinity chromatography followed by precipitation with the antibody to AP-2. Quantitation of the relative molar concentrations of the proteins found in the complex revealed that 1 mol of AP-2 was associated with approximately 1.1 mol of EGF receptor. No other proteins were present in significant molar concentrations relative to AP-2, indicating that other proteins are not stoichiometrically involved in the interaction of EGF receptors and AP-2 in vivo. Co-immunoprecipitation experiments in cells expressing a mutant EGF receptor demonstrated that the cytoplasmic carboxyl-terminal 214 residues of the EGF receptor are essential for interaction with AP-2.

Footnotes

↵* This work was supported by National Institutes of Health Grants CA24071 (to G. C.), DK46817 (to A. S.), and GM36548 (to T. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

EGF

epidermal growth factor

DMEM

Dulbecco's modified Eagle's medium, AP-2, plasma membrane clathrin-associated protein complex

AP-1

Golgi clathrin-associated protein complex

CMF-PBS

Ca- and Mg-free phosphate-buffered saline

TGH

Triton X-100 solubilization buffer.
↵²W. Boll, K. Clairmont, and T. Kirchhausen, manuscript in preparation.
↵³A. Sorkin, and G. Carpenter, unpublished data.
- Received August 25, 1994.
- Revision received October 31, 1994.