Antibodies against highly conserved sites in the epidermal growth factor receptor tyrosine kinase domain as probes for structure and function.
We generated anti-peptide antibodies against four highly conserved sequences in the kinase domain and against two nonconserved sequences surrounding autophosphorylation sites in the carboxyl-terminal domain of the epidermal growth factor receptor (EGFR). These antibodies were used to examine topology and function in catalysis of specific sequences. Two of the highly conserved sites, HRD (residues 811-818) and DFG (residues 827-838), appeared to participate in catalysis since alpha HRD and alpha DFG but not the other anti-peptide antibodies inhibited EGFR kinase activity. Examination of the topology of the six sites revealed that epitopes in all except the HRD site appeared to be exposed to antibody binding in the EGFR. The conditions that caused increased exposure of the HRD site to interaction with antibody included autophosphorylation, addition of the ionic detergent sodium dodecyl sulfate (SDS), and elevation in temperature from 4 to 34 degrees C.
Brown
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NA
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland 97201-3098.
Compton
L A
LA
Clinton
G M
GM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000906:Antibodies / Q000378:metabolism*; D001665:Binding Sites; D001666:Binding Sites, Antibody; D002384:Catalysis; D017124:Conserved Sequence; D004815:Epidermal Growth Factor / Q000494:pharmacology; D000939:Epitopes / Q000276:immunology; D066246:ErbB Receptors / Q000737:chemistry / Q000276:immunology* / Q000502:physiology; D007163:Immunosorbent Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000276:immunology* / Q000502:physiology
D000906:Antibodies; D000939:Epitopes; D010446:Peptide Fragments; D010710:Phosphates; D004815:Epidermal Growth Factor; D000255:Adenosine Triphosphate; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases