Human epidermal growth factor receptor residue covalently cross-linked to epidermal growth factor.
An epidermal growth factor (EGF) receptor monoclonal antibody (mAb), mAb LA22, was used to analyze the covalent coupling of human EGF receptors to mouse EGF by the amine-reactive cross-linking agent disuccinimidyl suberate. A soluble Mr 105,000 truncated form of the receptor secreted by A-431 epidermoid carcinoma cells and consisting of the ligand-binding extracellular domain was cross-linked to 125I-labeled EGF. Digestion of this complex with an endoproteinase that specifically cleaves at the COOH side of glutamyl residue released a single radiolabeled glycosylated fragment of Mr 18,000 that reacted with mAb LA22. As the epitope for mAb LA22 resided between Ala-351 and Asp-364 of the mature receptor, this result localized the cross-linked receptor residue(s) to the 47-amino acid interval from Phe-321 to Glu-367. The receptor residue(s) involved in the covalent coupling of rat 125I-labeled transforming growth factor alpha was similarly localized to this region of the receptor. This receptor interval, which included two glycosylated asparaginyl residues at positions 328 and 337, contained but three amino acid residues that were potentially reactive with disuccinimidyl suberate: Lys-332, Lys-333, and Lys-336. Characterization of mAb LA22-reactive 125I-EGF-labeled receptor fragments generated by an endoproteinase specific for the COOH side of lysyl residue placed the NH2 termini of the two smallest fragments between the glycosylated residues Asn-328 and Asn-337. These results indicated that disuccinimidyl suberate cross-linked the NH2 group of EGF residue Asn-1 to the human EGF receptor residue Lys-336. Our results further suggest that EGF and transforming growth factor alpha, two members of the EGF family of peptide growth factors, interact with closely apposed or identical features of the receptor.
Wu
D G
DG
W. Alton Jones Cell Science Center, Inc., Lake Placid, NY 12946.
Wang
L H
LH
Chi
Y
Y
Sato
G H
GH
Sato
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004815:Epidermal Growth Factor / Q000302:isolation & purification / Q000378:metabolism*; D000939:Epitopes / Q000032:analysis; D066246:ErbB Receptors / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D051381:Rats; D013388:Succinimides / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D000939:Epitopes; D010446:Peptide Fragments; D013388:Succinimides; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; C019358:disuccinimidyl suberate