Direct Identification of Residues of the Epidermal Growth Factor Receptor in Close Proximity to the Amino Terminus of Bound Epidermal Growth Factor

• Published in: Proceedings of the National Academy of Sciences - PNAS 1992-08, Vol.89 (16), p.7801-7805
• Main Authors: Woltjer, Randall L., Lukas, Thomas J., Staros, James V.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Animals; Antibodies; binding; Binding Sites; Biochemistry; Biological and medical sciences; Cell Line; Cell receptors; Cell structures and functions; Centrifugation; Chromatography, Affinity; cross-linking; Cross-Linking Reagents; Cysteine - analysis; Electrophoresis; epidermal growth factor; Epidermal Growth Factor - isolation & purification; Epidermal Growth Factor - metabolism; ErbB Receptors - isolation & purification; ErbB Receptors - metabolism; Fundamental and applied biological sciences. Psychology; Gels; Hormone receptors. Growth factor receptors. Cytokine receptors. Prostaglandin receptors; Humans; identification; Iodine Radioisotopes; man; Mice; Molecular and cellular biology; Molecular Sequence Data; Phosphotyrosine; Protein Conformation; Proteins; Receptors; residues; Resins; Sequencing; sites; Tyrosine - analogs & derivatives
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.89.16.7801

We have recently developed a kinetically controlled, step-wise affinity cross-linking technique for specific, high-yield, covalent linkage of murine epidermal growth factor (mEGF) via its N terminus to the EGF receptor. EGF receptor from A431 cells was cross-linked to radiolabeled mEGF (125I-mEGF) by this technique and the125I-mEGF-receptor complex was purified and denatured. Tryptic digestion of this preparation gave rise to a unique radiolabeled peptide that did not comigrate with trypsin-treated125I-mEGF in SDS/Tricine gels but that could be immunoprecipitated with antibodies to mEGF. The immunoprecipitated peptide was isolated by electrophoresis in SDS/Tricine gels, eluted, and sequenced. The sequence was found to correspond to that of a tryptic peptide of the EGF receptor beginning with Gly-85, which is in domain I, a region N terminal to the first cysteine-rich region of the receptor. Selective loss of signal in the 17th sequencing cycle suggests that the point of attachment of N-terminally modified125I-mEGF to the receptor is Tyr-101. The data presented here provide identification by direct protein microsequencing of a site of interaction of EGF and the EGF receptor.