DIRECT IDENTIFICATION OF RESIDUES OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR IN CLOSE PROXIMITY TO THE AMINO TERMINUS OF BOUND EPIDERMAL GROWTH-FACTOR

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 (I-125-mEGF) by this technique and the I-125-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-treated I-125-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 modified I-125-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.