The EGF receptor binding of recombinant heregulin beta 1/EGF hybrids is blocked by heregulin residue glutamate 195

Defined sequences from the EGF-like domain of human heregulin-beta 1 (HRG beta 1) were recombined with a synthetic gene fro human epidermal growth factor (hEGF) in an attempt to locate receptor-specific determinants within the HRG beta 1 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-temimal HRG beta 1 residues. However, extending the N-terminal substitution to include to 20 HRG beta residues resulted in a 100-fold drop in relative EGFR binding. Both interruption of the major beta-sheet structure of hEGF by insertion of a three amino acid loop present in HRG beta 1 and replacement of nearly the entire C-terminal hEGF subdomain w;ith segments of HRG beta 1 sequence resulted in a 5-fold decreased EGFR affinity. The results presented here demonstrate that while a substantial protein of the hEGF and HRG beta 1 protein sequences were nearly interchangeable with regard to EGFR binding, the introduction of HRG beta 1 residue Glu195 effected a major decrease in EGFR binding. (C) 1996 Academic Press.