Dissection of Functional Sites on the Receptor for Epidermal Growth Factor

The epidermal growth factor receptor is a protein of 1186 amino acyl residues, which appears to be disposed as a 621-residue extracytoplasmic domain, a 23-residue transmembrane segment, and a 542-residue cytoplasmic domain. The binding of EGF to the extracytoplasmic domain of the receptor causes the dimerization of the receptor and the activation of a tyrosyl residue-specific protein kinase that forms part of the cytoplasmic domain of the receptor. We have applied a variety of techniques to identify individual residues within the receptor with specific receptor functions. Within the extracytoplasmic domain, residues adjacent to bound EGF are being identified by a strategy involving affinity cross-linking and protein microsequencing. Murine EGF (mEGF), which contains no Lys and therefore has the α-amino group as its only primary amine, and site-directed mutants of mEGF, which contain a single Lys and an Asn1 → Gln substitution in which the resulting Gln1 is chemically cyclized to pyroglutamate, are each modified on their single amino group with sulfo-N-succinimidyl 4-(fluorosulfonyl) benzoate. The resulting p-fluorosulfonylbenzoyl derivative of the EGF species is allowed to bind to the receptor, whereupon it reacts covalently with it. Limit proteolysis and sequencing are used to identify the modified residue of the receptor, e.g., mEGF modified on the α-amino terminus cross-links with Tyr101. Within the cytoplasmic domain, two residues essential for kinase activity have been identified. Lys721, identified by affinity labeling with 5′-(p-fluorosulfonylbenzoyl) adenosine and protein sequencing, appears to position the α- and β-phosphates of ATP within the active site and is essential for phosphoryl transfer. Asp813 appears to function as the catalytic base for phosphoryl transfer, as site-directed mutagenesis of Asp813 → Ala abolishes kinase activity.