THE SIGNIFICANCE OF VALINE-33 AS A LIGAND-SPECIFIC EPITOPE OF TRANSFORMING-GROWTH-FACTOR-ALPHA

Although binding of epidermal growth factor (EGF) and transforming gro wth factor alpha (TGF alpha) to the EGF receptor (EGFR) is mutually co mpetitive, their binding is not identical, and their biological activi ties are not always equivalent, To probe for ligand-specific interacti ons, me have synthesized analogues of TGF alpha with modifications to the residue lying between the fourth and fifth cysteines (the ''hinge' '). Although this residue Lies in a structurally conserved region of t he protein, it is not conserved within the EGFR ligand family. Our res ults show that in TGF alpha there is a preference for a bulky hydropho bic hinge residue; this contrasts with EGF, for which a hydrogen bond donor functionality is preferred. Sequence analysis of the human EGFR Ligands revealed that the nature of the hinge residue correlated with the sequence in the B-loop beta-sheet. As this region is an important determinant in recognition of TGF alpha by the chicken EGFR, we assess ed the mitogenicity of the TGF alpha hinge mutants, as well as the oth er EGFR ligands, using chicken embryo fibroblasts. The preference of t he chicken EGFR for TGF alpha hinge mutants with hydrophobic side chai ns paralleled that of the human EGFR. Betacellulin and heparin-binding EGF-like growth factor also possess an hydrophobic hinge; both were a t least as potent as TGF alpha for chicken embryo fibroblasts. EGF and amphiregulin, both with hydrogen bond donor functionalities at their hinge, displayed markedly decreased in potency by comparison with TGF alpha. We propose that EGFR ligands can be subclassified into TGF alph a-like and EGF-like and that this is of functional significance, ident ifying a potential mechanism whereby EGFR can discriminate between its ligands.