Association of Epidermal Growth Factor Receptors with Coated Pit Adaptins via a Tyrosine Phosphorylation-regulated Mechanism

doi:10.1074/jbc.270.11.6320

Association of Epidermal Growth Factor Receptors with Coated Pit Adaptins via a Tyrosine Phosphorylation-regulated Mechanism (*)

From the Department of Medicine, University of California at San Diego, La Jolla, California 92093

^§ To whom correspondence should be addressed:
Dept. of Medicine 0650, University of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0650.
Tel.: 619-534-4310; Fax: 619-534-8193.

Abstract

We investigated the mechanism by which ligand-activated epidermal growth factor receptors (EGFR) associate with coated pit adaptor protein (AP) complexes. In vivo association, assayed by coimmunoprecipitation of AP with mutant EGFR, required tyrosine kinase activity, intact autophosphorylation sites, and the regulatory carboxyl terminus of EGFR. The role of autophosphorylation of EGFR in interaction with AP was examined in vitro using a BIAcore™ instrument. Purified active EGFR, immobilized on the biosensor surface, was reversibly autophosphorylated or dephosphorylated by treatment with ATP or phosphatase. Autophosphorylation of EGFR significantly increased AP binding. Once formed, EGFR•AP complexes were resistant to disassembly by dephosphorylation of EGFR or competition with phosphotyrosine, indicating that phosphorylated tyrosine residues do not directly participate in AP binding. Induction of conformational changes in EGFR by treatment with urea increased AP binding up to 10-fold in the absence of EGFR autophosphorylation. A recombinant EGFR carboxyl terminus specifically bound the AP complex and each of the isolated α- and β-subunits of AP2. We conclude that tyrosine autophosphorylation of EGFR exposes structural motif(s) in the carboxyl terminus of EGFR that interact specifically with AP2.

Footnotes

↵* This work was supported by National Institutes of Health Grant PO1 CA58689 and by the Markey Charitable Trust. 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:

EGFR

epidermal growth factor receptor(s)

EGF

epidermal growth factor

InsR

insulin receptor

AP

adaptor protein

IP

immunoprecipitation

Ab

antibody.
- Received November 2, 1994.
- Revision received January 9, 1995.