Phosphorylation of tyrosine 992, 1068, and 1086 is required for conformational change of the human epidermal growth factor receptor C-terminal tail

We reported previously that a conformation-specific antibody, Ab P2, to a 1 6-amino acid peptide (Glu-Gly-Tyr-Lys-Lys-Lys-Tyr-Gln-Gln-Val-Asp-Glu-Glu-P he-Leu-Arg) of the cytoplasmic domain of the P-type platelet-derived growth factor receptor also recognizes the epidermal growth factor (EGF) receptor . Although the antibody is not directed to phosphotyrosine, it recognizes i n immunoprecipitation the activated and hence phosphorylated form of both r eceptors. In P2 peptide, there are two tripeptide sequences, Asp-Glu-Glu an d Tyr-Gln-Gln, that are also present in the EGF receptor. Our present studi es using either EGF receptor C-terminal deletion mutants or point mutations (Tyr-->Phe) and our previous studies on antibody inhibition by P2-derived peptides suggest that Gin-Gin in combination with Asp-Glu-Glu forms a high- affinity complex with Ab P2 and that such complex formation is dependent on tyrosine phosphorylation. Of the five phosphate acceptor sites in the EGF receptor, clustered in the extreme C-terminal tail, phosphorylation of thre e tyrosine residues (992, 1068, and 1086) located between Asp-Glu-Glu and G in-Gin is necessary for Ab P2 binding. In contrast, the acceptor sites Tyr 1173 and 1148 play no role in the conformation change. Asp-Glu-Glu and Gin- Gin are located 169 amino acids apart, and it is highly likely that the int eractions among three negatively charged phosphotyrosine residues in the re ceptor C terminus may result in the bending of the peptide chain in such a way that these two peptides come close to each other to form an antibody-bi nding site. Such a possibility is also supported by our finding that recept or dephosphorylation results in complete loss of Ab P2-binding activity. In conclusion, we have identified a domain within the cytoplasmic part of the EGF receptor whose conformation is altered by receptor phosphorylation; fu rthermore, we have identified the tyrosine residues that positively regulat e this conformation.