Antibodies to the ATP-binding site of the human epidermal growth factor (EGF) receptor as specific inhibitors of EGF-stimulated protein-tyrosine kinase activity.
A region of the primary amino acid sequence of the epidermal growth factor receptor (EGF) protein-tyrosine kinase, which is involved in ATP binding, was identified using chemical modification and immunological techniques. EGF receptor was 14C-labelled with the ATP analogue 5'-p-fluorosulphonylbenzoyladenosine and from a tryptic digest a single radiolabelled peptide was isolated. The amino acid sequence was determined to be residues 716-724 and hence lysine residue 721 is located within the ATP-binding site. Antisera were elicited in rabbits to a synthetic peptide identical to residues 716-727 of the EGF receptor and the homologous sequence in v-erb B transforming protein from avian erythroblastosis virus. The affinity-purified antibodies precipitated human ECF receptor from A431 cells and placenta, and the v-erb B protein from erythroblasts. The antibodies inhibited EGF-stimulated receptor protein-tyrosine kinase autophosphorylation and phosphorylation of an exogenous peptide substrate containing tyrosine. The antibodies did not immunoprecipitate the transforming proteins pp60v-src or P120gag-abl or cAMP-dependent protein kinase, proteins which have homologous but not identical sequences surrounding the lysine residue within the ATP-binding site, nor did they react with the platelet-derived growth factor receptor. The antibodies had no effect on the kinase activity of purified v-abl protein in solution. The antibodies may therefore be a specific inhibitor of the tyrosine kinase of the EGF receptor.
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000906:Antibodies / Q000276:immunology*; D001665:Binding Sites; D002250:Carbon Radioisotopes; D003429:Cross Reactions; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000032:analysis; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011956:Receptors, Cell Surface / Q000032:analysis / Q000276:immunology*
D000906:Antibodies; D002250:Carbon Radioisotopes; D009856:Oncogene Proteins, Viral; D011956:Receptors, Cell Surface; D004815:Epidermal Growth Factor; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D000241:Adenosine