Antibodies to the autophosphorylation sites of the epidermal growth factor receptor protein-tyrosine kinase as probes of structure and function.
Antisera were prepared against three synthetic peptides with amino acid sequences identical to those surrounding the three major autophosphorylation sites of the epidermal growth factor (EGF) receptor. The affinity-purified antibodies reacted strongly in an enzyme-linked immunosorbent assay against the immunizing peptide but showed little cross-reaction with the other two phosphorylation site peptides. EGF receptors labelled by autophosphorylation could be specifically precipitated by each of the phosphorylation site antibodies. The antibodies recognised EGF receptors labelled at each of the autophosphorylation sites, indicating that they could bind to the immunizing sequences irrespective of their states of phosphorylation. The antibodies were able to inhibit EGF receptor autophosphorylation without affecting EGF-stimulated tyrosine kinase activity towards exogenous peptide substrates, suggesting that the kinase and autophosphorylation sites were in distinct domains. Immunofluorescent staining of A431 cells showed that the autophosphorylation site sequences resided inside the cell. The autophosphorylation sites were shown to be within a domain of 20 000 mol. wt. which could be cleaved from the receptor through limited proteolysis by the calcium-dependent protease, calpain. The position of cleavage of the EGF receptor by the protease was mapped to lie between residues 996 and 1059. These results are discussed in the context of a model for the structure and function of the human EGF receptor.
W J
WJ
Gullick
J
J
Downward
M D
MD
Waterfield
D000595:Amino Acid Sequence; D000906:Antibodies; D002154:Calpain; D002294:Carcinoma, Squamous Cell; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000378:metabolism*; D000939:Epitopes / Q000032:analysis*; D066246:ErbB Receptors; D005455:Fluorescent Antibody Technique; D006801:Humans; D008715:Methionine / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000276:immunology / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000276:immunology / Q000378:metabolism*; D013462:Sulfur Radioisotopes
D000906:Antibodies; D000939:Epitopes; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D013462:Sulfur Radioisotopes; D004815:Epidermal Growth Factor; D008715:Methionine; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D002154:Calpain