Mutations in the cytoplasmic domain of EGF receptor affect EGF binding and receptor internalization.

Abstract

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.

The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase.

“…Additionally, a 10-amino-acid insertion just downstream of the ATP-binding domain of v-ErbB resulted in temperature-sensitive transforming and protein-tyrosine kinase activities (Ng and Privalsky 1986). And, reminiscient of our own results, a 4-amino-acid insertion at residue 708 of the human protein abolished tyrosine kinase activity of the immunoprecipitated receptor but not its ability to stimulate ligand-dependent DNA synthesis (Prywes et al 1986). Valine 743 is conserved in human, mouse and rat EGFRs, as well as in Her2/ErbB2/Neu, and it is conservatively replaced by an isoleucine in Her4/ErbB4.…”

“…For example, cytoplasmic sequences have been implicated previously in the appearance of high, but not low, affinity EGF-binding sites on the cell surface, and some mutations that abolish tyrosine kinase activity (e.g., a 4-amino-acid insertion at residue 888) also prevented expression of the high affinity state (Prywes et al 1986). However, it should be noted that we did not observe major differences in the apparent binding affinities of normal and wa-2 EGFRs.…”

“…Altematively, the wa-2 mutation could influence EGFR trafficking. Thus, certain tyrosine kinaseinactivating mutations, including the aforementioned residue 888 insertion (Prywes et al 1986), also prevented EGFR down-regulation (Chen et al 1987;Glenney et al 1988), and the specific mutation of lysine 721 in the ATP-binding pocket altered cellular routing of EGFR such that internalized receptors were recycled to the cell surface (Honegger et al 1987. Although mutations wa-2 encodes a defective EGF receptoi that effectively block EGFR down-regulation might generally be expected to enhance receptor signaling, the possibility exists that less drastic mutations (e.g., like that of wa-2) might influence EGFR routing in a manner that is detrimental to normal signaling, for example, by repressing a normal level of receptor recycling.…”

Differential requirement of EGF receptor and its tyrosine kinase for AP-1 transactivation induced by EGF and TPA

“…The intrinsic tyrosine kinase activity of the EGFR has been shown to be responsible for many pleiotropic intracellular effects resulting from ligand stimulation (Wells, 1999). In contrast, several reports have also suggested that specific aspects of EGFR function are independent of the intriasic tyrosine kinase activity (Prywes et al, 1986;Chen et al, 1987). In the present study, we determined if EGFR and its tyrosine kinase activation are necessary for EGF-or TPA-induced AP-1 activation.…”

“…The EGFR has intrinsic protein tyrosine kinase activity, which is responsible for many of the pleotropic intracellular effects (Schlessinger, 2000;Simon, 2000). Several reports have suggested that specific aspects of EGF receptor function are independent of the intrinsic tyrosine kinase activity (Prywes et al, 1986). Since the above results show that EGFR is necessary for TPA-and EGF-induced AP-1 activation, we ask whether the tyrosine kinase of EGFR plays a role in TPA-induced AP-1 activation.…”