The EGF Receptor Binding of Recombinant Heregulinβ1/EGF Hybrids Is Blocked by Heregulin Residue Glutamate 195

Defined sequences from the EGF-like domain of human heregulin-β1 (HRGβ1) were recombined with a synthetic gene for human epidermal growth factor (hEGF) in an attempt to locate receptor-specific determinants within the HRGβ1 molecule that blocks its inappropriate association with the EGF receptor (EGFR). Receptor competition assays detected only minor changes in relative EGFR affinity for those hybrids containing up to 12 N-terminal HRGβ1 residues. However, extending the N-terminal substitution to include 20 HRGβ1 residues resulted in a 100-fold drop in relative EGFR binding. Both interruption of the major β-sheet structure of hEGF by insertion of a three amino acid loop present in HRGβ1 and replacement of nearly the entire C-terminal hEGF subdomain with segments of HRGβ1 sequence resulted in a 5-fold decreased EGFR affinity. The results presented here demonstrate that while a substantial portion of the hEGF and HRGβ1 protein sequences were nearly interchangeable with regard to EGFR binding, the introduction of HRGβ1 residue Glu195 effected a major decrease in EGFR binding.