Role of the N-terminus of epidermal growth factor in ErbB-2/ErbB-3 binding studied by phage display.
Epidermal growth factor (EGF) binds with high affinity to the EGF receptor, also known as ErbB-1, but upon replacement of the N-terminal linear region by neuregulin (NRG) 1 or transforming growth factor (TGF) alpha sequences it gains in addition high affinity for ErbB-2/ErbB-3 heterodimers. However, these chimeras weakly bind to ErbB-3 alone. To further dissect the ligand binding selectivity of the ErbB network, we have applied the phage display technique to examine the role of the linear N-terminal region in EGF for interaction with ErbB-2/ErbB-3 heterodimers. A library of EGF variants was constructed in which residues 2, 3, and 4 were randomly mutated, followed by selection for binding to intact MDA-MB-453 cells that overexpress ErbB-2 and ErbB-3 but lack ErbB-1. Analysis of the selected phage EGF variants revealed clones with high binding affinity to ErbB-2/ErbB-3 while maintaining high affinity to ErbB-1. In these variants, Trp (or alternatively His) was almost exclusively present at position 2, while specific combinations of hydrophobic, basic, and small residues were found at positions 3 and 4. The mitogenic activity of the phage EGF variants corresponded with their relative binding affinity. Two of the selected EGF variants, EGF/WVS and EGF/WRS, were further characterized as recombinant proteins. In contrast to previously characterized chimeras of EGF with NRG-1 or TGF-alpha, these variants did not only show high binding affinity for ErbB-2/ErbB-3 heterodimers but also for ErbB-3 alone. These data show that the linear N-terminal region of EGF-like growth factors is directly involved in binding to ErbB-3.
Stortelers
Catelijne
C
Department of Cell Biology, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands.
Souriau
Christelle
C
van Liempt
Ellis
E
van de Poll
Monique L M
ML
van Zoelen
Everardus J J
EJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D001943:Breast Neoplasms; D017931:DNA Primers; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000737:chemistry*; D066246:ErbB Receptors / Q000378:metabolism; D005260:Female; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry*; D020893:Receptor, ErbB-3 / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D008024:Ligands; D019151:Peptide Library; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3; D011994:Recombinant Proteins