The human epidermal growth factor (EGF) receptor (HER) family members cooperate 
in malignancy. Of this family, HER2 does not bind growth factors and HER3 does 
not encode an active tyrosine kinase. This diversity creates difficulty in 
creating pan-specific therapeutic HER family inhibitors. We have identified 
single amino acid changes in epidermal growth factor receptor (EGFR) and HER3 
which create high affinity sequestration of the cognate ligands, and may be used 
as receptor decoys to downregulate aberrant HER family activity. In silico 
modeling and high throughput mutagenesis were utilized to identify receptor 
mutants with very high ligand binding activity. A single mutation (T15S; EGFR 
subdomain I) enhanced affinity for EGF (two-fold), TGF-alpha (twenty-six-fold), 
and heparin-binding (HB)-EGF (six-fold). This indicates that T15 is an 
important, previously undescribed, negative regulatory amino acid for EGFR 
ligand binding. Another mutation (Y246A; HER 3 subdomain II) enhanced neuregulin 
(NRG)1-beta binding eight-fold, probably by interfering with subdomain II-IV 
interactions. Further work revealed that the HER3 subunit of an EGFR:HER3 
heterodimer suppresses EGFR ligand binding. Optimization required reversing this 
suppression by mutation of the EGFR tether domain (G564A; subdomain IV). This 
mutation resulted in enhanced ligand binding (EGF, ten-fold; TGF-alpha, 
thirty-four-fold; HB-EGF, seventeen-fold; NRG1-beta, thirty-one-fold). This 
increased ligand binding was reflected in improved inhibition of in vitro tumor 
cell proliferation and tumor suppression in a human non-small cell lung cancer 
xenograft model. In conclusion, amino acid substitutions were identified in the 
EGFR and HER3 ECDs that enhance ligand affinity, potentially enabling a 
pan-specific therapeutic approach for downregulating the HER family in cancer.