Mutations in the cytoplasmic domain of EGF receptor affect EGF binding and receptor internalization.
Binding of epidermal growth factor (EGF) to its receptor results in a cascade of events that culminate in cell division. The receptor is present on the cell surface in two forms of high and low affinity binding for EGF. EGF binding activates the receptor's intracellular tyrosine kinase activity and subsequently causes the receptor to be rapidly internalized into the cell via clathrin-coated pits. We have cloned the EGF receptor cDNA into a retroviral expression vector and made mutations in vitro to investigate the function of different receptor domains. Deletion of cytoplasmic sequences abolishes high but not low affinity sites as well as impairing the ability of the protein to internalize into cells. Thus, cytoplasmic sequences must be involved in the regulation of high affinity sites and are required for EGF-induced receptor internalization. A four amino acid insertion mutation at residue 708 abolishes the protein-tyrosine kinase activity of the immunoprecipitated receptor. However, this receptor mutant exhibits both the high and low affinity states, internalizes efficiently and is able to cause cells to undergo DNA synthesis in response to EGF. Another four amino acid insertion mutation (residue 888) abolishes protein-tyrosine kinase activity, high affinity binding, internalization and mitogenic responsiveness. Finally, a chimaeric receptor composed of the extracellular EGF binding domain and the cytoplasmic v-abl kinase region transforms Rat-I cells. This chimaeric receptor possesses intrinsic protein tyrosine kinase activity which cannot be regulated by EGF. Moreover, EGF fails to induce the internalization of the chimaeric receptor.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D066246:ErbB Receptors / Q000235:genetics* / Q000378:metabolism; D007106:Immune Sera; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D009154:Mutation; D010957:Plasmids
D007106:Immune Sera; D004815:Epidermal Growth Factor; D004247:DNA; D066246:ErbB Receptors