Antibodies against highly conserved sites in the epidermal growth factor receptor tyrosine kinase domain as probes for structure and function

Abstract

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.

A kinase-negative epidermal growth factor receptor that retains the capacity to stimulate DNA synthesis.

“…Mutation of the aspartyl residue homologous to Asp-166 of cAPK in the Tyr kinases v-fps (16) and c-kit (17,18) has also been shown to eliminate in vitro kinase activity. In the case of the EGFR, an antibody specific for a sequence of the EGFR including Asp-813, the residue homologous to Asp-166 of cAPK, has been shown to inhibit kinase activity (19). To test whether Asp-813 of the human EGFR is catalytically essential, we used site-directed mutagenesis to convert Asp-813 to Ala (D813A).…”

Generation of a polyclonal antibody that simultaneously detects multiple Ser/Thr protein kinases

“…Brown et al produced anti-peptide antibodies against four highly conserved sequences in the kinase domain of human epidermal growth factor receptor kinase [15]. These antibodies were used for examination of topology and catalytic functions of these sequences in this receptor kinase.…”

A kinase-negative epidermal growth factor receptor that retains the capacity of stimulate DNA synthesis

“…Mutation of the aspartyl residue homologous to Asp-166 of cAPK in the Tyr kinases v-fps (16) and c-kit (17,18) has also been shown to eliminate in vitro kinase activity. In the case of the EGFR, an antibody specific for a sequence of the EGFR including Asp-813, the residue homologous to Asp-166 of cAPK, has been shown to inhibit kinase activity (19). To test whether Asp-813 of the human EGFR is catalytically essential, we used site-directed mutagenesis to convert Asp-813 to Ala (D813A).…”