Association of epidermal growth factor receptors with coated pit adaptins via a tyrosine phosphorylation-regulated mechanism.
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 alpha- and beta-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.
Nesterov
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A
Department of Medicine, University of California at San Diego, La Jolla 92093.
Kurten
R C
RC
Gill
G N
GN
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D066246:ErbB Receptors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010649:Phenylalanine; D010766:Phosphorylation; D019000:Phosphotyrosine; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D010649:Phenylalanine; D066246:ErbB Receptors