Characterization of site-specific antibodies to the erbB gene product and EGF receptor: inhibition of tyrosine kinase activity.
Site-specific antibodies were generated against the erbB protein and epidermal growth factor (EGF) receptor by immunizing rabbits with a synthetic peptide corresponding to amino acid residues 285-296 of the predicted AEV-H erbB protein sequence. This peptide region lies within the tyrosine kinase domain of erbB and EGF receptor. Antibodies directed against this region readily identified native and denatured forms of the erbB gene product and EGF receptor as demonstrated by immuneprecipitation and immunoblot analysis. The anti-peptide antibody immuneprecipitated a functional EGF binding receptor molecule. Scatchard analysis demonstrated a KD for 125I-labeled EGF binding of 40 nM, a value consistent with that of detergent solubilized EGF receptor. Immuneprecipitates, though able to bind EGF, were unable to transfer phosphate from gamma-labeled ATP in a standard phosphorylation reaction. In detergent solubilized extracts of crude A431 microsomes, the anti-peptide antibody inhibited in a dose dependent manner the autophosphorylation of EGF receptor as well as receptor mediated phosphorylation of exogenously added substrates. In addition, this anti-peptide antibody reduced the overall level of tyrosine kinase activity present in microsomes prepared from AEV-transformed erythroblasts. This site-specific antisera should be useful for understanding the role of EGF receptor and erbB tyrosine kinase activity and their link with cell proliferation.
D001355:Alpharetrovirus / Q000235:genetics*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000906:Antibodies; D001354:Avian Leukosis Virus / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D066246:ErbB Receptors; D007163:Immunosorbent Techniques; D009857:Oncogenes; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011817:Rabbits; D051381:Rats; D011956:Receptors, Cell Surface / Q000276:immunology*; D014764:Viral Proteins / Q000276:immunology*
D000596:Amino Acids; D000906:Antibodies; D011956:Receptors, Cell Surface; D014764:Viral Proteins; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases