Grb2/Ash binds directly to tyrosines 1068 and 1086 and indirectly to tyrosine 1148 of activated human epidermal growth factor receptors in intact cells.
The activation of receptor tyrosine kinases generates tyrosine-phosphorylated recognition motifs for the binding of signaling proteins containing Src homology 2 domains. We determined the binding sites of Grb2/Ash, an Src homology 2 domain-containing adaptor protein, within epidermal growth factor (EGF) receptors, using Chinese hamster ovary cells overexpressing human EGF receptor mutants in which one of the autophosphorylation sites was retained. In intact cells, the amount of Grb2/Ash coimmunoprecipitated with mutant receptors retaining tyrosines 992, 1068, 1086, 1148, or 1173 was approximately 10, 85, 55, 50, or 20% of wild-type levels, respectively. The association of Grb2/Ash with in vitro autophosphorylated EGF receptor mutants was detectable in those retaining either tyrosines 1068 or 1086 but not in other mutants including those retaining tyrosine 1148. In peptide inhibition assay, phosphorylated peptides representing tyrosines 1068 and 1086 inhibited the binding of Grb2/Ash to in vitro autophosphorylated wild-type EGF receptors, whereas the other peptides representing tyrosines 992, 1148, and 1173 failed to inhibit the binding. Given that tyrosine 1148 of the activated EGF receptor is a major binding site of Shc (Okabayashi, Y., Kido, Y., Okutani, T., Sugimoto, Y., Sakaguchi, K., and Kasuga, M. (1994) J. Biol. Chem. 269, 18674-18678), these results indicate that tyrosines 1068 and 1086 of activated human EGF receptors are direct high affinity binding sites of Grb2/Ash and that tyrosine 1148 is an indirect binding site through Shc in intact cells.
Okutani
T
T
Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
Okabayashi
Y
Y
Kido
Y
Y
Sugimoto
Y
Y
Sakaguchi
K
K
Matuoka
K
K
Takenawa
T
T
Kasuga
M
M
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D066246:ErbB Receptors / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D006801:Humans; D051379:Mice; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495949:Grb2 protein, mouse; D011506:Proteins; D014443:Tyrosine; D066246:ErbB Receptors; D016392:Proto-Oncogene Proteins pp60(c-src)