Mutagenesis reveals a role for epidermal growth factor receptor extracellular subdomain TV in ligand binding

The extracellular domain of the epidermal growth factor (EGF) receptor (EGF R) comprises four subdomains (I-IV) and mediates binding of several differe nt poly-peptide ligands, including EGE, transforming growth factor-alpha, a nd heparin-binding EGF. Previous studies have predominantly implicated subd omain III in ligand binding. To investigate a possible role for sequences i n subdomain IV, we constructed several mutant EGFRs in which clusters of ch arged or aromatic amino acids mere replaced with alanine. Analysis of stabl y transfected Chinese hamster ovary cells expressing mutant EGFRs confirmed that they were present on the cell surface at levels approaching that of t he wild-type receptor. Although tyrosine phosphorylation of most mutants wa s markedly induced by EGF, a cluster mutation (mt25) containing four alanin e substitutions in the span of residues 521-527 failed to respond. EGF-indu ced tyrosine phosphorylation of an alternative mutant (Delta EN) with amino acids 518-589 deleted was also greatly diminished. Larger doses of EGF or heparin-binding EGF induced only weak tyrosine phosphorylation of mt25, whe reas the response to transforming growth factor-a was undetectable. These r esults suggest that mt25 might be defective with respect to either ligand b inding or receptor dimerization, Quantitative analyses showed that binding of I-125-EGF to mt25 and Delta EN was reduced to near background levels, wh ereas binding of EGF to other cluster mutants was reduced 60-70% compared w ith wild-type levels. Among the mutants, only mt25 and Delta EN failed to f orm homodimers or to transphosphorylate HER2/Neu in response to EGF treatme nt. Collectively, our results are the first to provide direct evidence that discrete subdomain IV residues are required for normal binding of EGF fami ly ligands, Significantly, they were obtained with the full-length receptor in vivo, rather than a soluble truncated receptor, which has been frequent ly used for structure/function studies of the EG;FR extracellular region.