Identification of epidermal growth factor receptor residues proximal to the C-terminus of bound EOF

To identify amino acyl residues of the EOF receptor (EGFR) adjacent to bound EGF, we have constructed a triple mutant of murine EGF, N1Q/H22Y/R45K, for use in affinity crosslinking studies. The triple mutant was constructed to introduce a primary amino group (R4SK) into the Cterminal region of mEGF. Cyclization of Ql to form pyroglutamate (pE) blocked the N-terminal a-amino group, the only other primary amino group in the EGF mutant, limiting the site of crosslinking to K45 and permitting identification of receptor residues proximal to the C-terminus of bound EGF. 125I-labelledNlpE/H22Y/R45Kwas reacted with the heterobifunctional crosslinking reagent sulfo-N-succinimidyl 4-(fluorosulfonyl)benzoate, and the resulting modified EGF was incubated with A431 vesicles bearing EGFR. Incubation resulted in specific crosslinking of labelled mutant to EGFR. The covalent complex was then partially purified and digested using endoproteinase Lys C. Lys C digestion yielded a unique radiolabelled peptide that was purified by immunoprecipitation and SDS/Tricine gel electrophoresis and subjected to microsequencing. The resulting sequence matched that of a Lys C fragment of EGFR situated near the interface of receptor subdomains 3 and 4.