title: Hierarchy of binding sites for Grb2 and Shc on the epidermal growth factor receptor.
abstract: We analyzed the binding site(s) for Grb2 on the epidermal growth factor (EGF) receptor (EGFR), using cell lines overexpressing EGFRs containing various point and deletion mutations in the carboxy-terminal tail. Results of co-immunoprecipitation experiments suggest that phosphotyrosines Y-1068 and Y-1173 mediate the binding of Grb2 to the EGFR. Competition experiments with synthetic phosphopeptides corresponding to known autophosphorylation sites on the EGFR demonstrated that phosphopeptides containing Y-1068, and to a lesser extent Y-1086, were able to inhibit the binding of Grb2 to the EGFR, while a Y-1173 peptide did not. These findings were confirmed by using a dephosphorylation protection assay and by measuring the dissociation constants of Grb2's SH2 domain to tyrosine-phosphorylated peptides, using real-time biospecific interaction analysis (BIAcore). From these studies, we concluded that Grb2 binds directly to the EGFR at Y-1068, to a lesser extent at Y-1086, and indirectly at Y-1173. Since Grb2 also binds Shc after EGF stimulation, we investigated whether Y-1173 is a binding site for the SH2 domain of Shc on the EGFR. Both competition experiments with synthetic phosphopeptides and dephosphorylation protection analysis demonstrated that Y-1173 and Y-992 are major and minor binding sites, respectively, for Shc on the EGFR. However, other phosphorylation sites in the carboxy-terminal tail of the EGFR are able to compensate for the loss of the main binding sites for Shc. These analyses reveal a hierarchy of interactions between Grb2 and Shc with the EGFR and indicate that Grb2 can bind the tyrosine-phosphorylated EGFR directly, as well as indirectly via Shc.
authors.affiliation: Department of Pharmacology, New York University Medical Center, New York 10016.
authors.forename: A G
authors.initials: AG
authors.lastname: Batzer
authors.forename: D
authors.initials: D
authors.lastname: Rotin
authors.forename: J M
authors.initials: JM
authors.lastname: Ureña
authors.forename: E Y
authors.initials: EY
authors.lastname: Skolnik
authors.forename: J
authors.initials: J
authors.lastname: Schlessinger
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D001665:Binding Sites; D066246:ErbB Receptors / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D019000:Phosphotyrosine; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D010455:Peptides; D010750:Phosphoproteins; D011506:Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D066246:ErbB Receptors