DISSECTION OF FUNCTIONAL SITES ON THE RECEPTOR FOR EPIDERMAL GROWTH-FACTOR

The epidermal growth factor receptor is a protein of 1186 amino acyl r esidues, which appears to be disposed as a 621-residue extracytoplasmi c domain, a 23-residue transmembrane segment, and a 542-residue cytopl asmic domain. The binding of EGF to the extracytoplasmic domain of the receptor causes the dimerization of the receptor and the activation o f a tyrosyl residue-specific protein kinase that forms part of the cyt oplasmic domain of the receptor. We have applied a variety of techniqu es to identify individual residues within the receptor with specific r eceptor functions. Within the extracytoplasmic domain, residues adjace nt to bound EGF are being identified by a strategy involving affinity cross-linking and protein microsequencing. Murine EGF (mEGF), which co ntains no Lys and therefore has the alpha-amino group as its only prim ary amine, and site-directed mutants of mEGF, which contain a single L ys and an Asn1 --> Gln substitution in which the resulting Gln1 is che mically cyclized to pyroglutamate, are each modified on their single a mino group with sulfo-N-succinimidyl 4-(fluorosulfonyl) benzoate. The resulting p-fluorosulfonylbenzoyl derivative of the EGF species is all owed to bind to the receptor, whereupon it reacts covalently with it. Limit proteolysis and sequencing are used to identify the modified res idue of the receptor, e.g., mEGF modified on the alpha-amino terminus cross-links with Tyr101. Within the cytoplasmic domain, two residues e ssential for kinase activity have been identified. Lys721, identified by affinity labeling with 5'-(p-fluorosulfonylbenzoyl) adenosine and p rotein sequencing, appears to position the alpha- and beta-phosphates of ATP within the active site and is essential for phosphoryl transfer . Asp813 appears to function as the catalytic base for phosphoryl tran sfer, as site-directed mutagenesis of Asp813 --> Ala abolishes kinase activity. (C) 1995 Academic Press, Inc.