Receptor recognition by histidine 16 of human epidermal growth factor via hydrogen-bond donor/acceptor interactions

Human epidermal growth factor (hEGF) and human transforming growth factor alpha (hTGFalpha) are prototypical of structurally related polypeptide mitogens which interact with the epidermal growth factor receptor (EGFR). Several determinants of receptor recognition that specify function have been proposed on the basis of structural criteria. This study evaluates the role of one such candidate, H16 of hEGF, by site-specific mutagenesis. When assayed for receptor tyrosine kinase stimulation using (Glu4,Tyr1)n as the exogenous substrate in vitro, the relative agonist activities of position 16 mutants range from 14-263% of wild-type hEGF. The rank order of potency was found to correlate with the relative receptor binding affinities of the mutants, which range from 7-272% of wild-type, as determined by radioreceptor competition assays. The mitogenic activity of the H16 mutants is similar to that of wild-type hEGF as determined by clonogenic assays using rat tracheal epithelial cells. While the colony forming efficiencies do not reflect significant differences in growth rate or survival characteristics in the presence of the hEGF variants, it is reduced to 1.6% in control cultures which lack EGF in the medium. The results show that H16 of hEGF, although not essential for mitogenic activity, optimizes receptor recognition by hydrogen-bond donor/acceptor interactions and may share this feature with H18 of hTGFalpha.