THE FUNCTIONAL IMPORTANCE OF LEU15 OF HUMAN EPIDERMAL GROWTH-FACTOR IN RECEPTOR-BINDING AND ACTIVATION

The biological importance of Leu15 of epidermal growth factor (EGF) is suggested by its conservation through evolution, its critical locatio n in the domain-domain interface of EGF and its close proximity to Arg 41, a residue that is crucial for receptor binding and activation. Mut agenesis of Leu15 of human EGF (hEGF) was employed to examine the role of this residue in the ligand-receptor interaction. The relative rece ptor affinities of the hEGF variants, as determined by radioreceptor c ompetition assays, varied depending on the amino acid substitution. Th e L15F, L15W and L15V hEGF analogues had receptor affinities 45, 26 an d 18% respectively of wild type hEGF. The L15A and L15R analogues disp layed receptor affinities of only 2.4 and 1.6% relative to wild type h EGF. No binding of the L15E analogue was detected. The relative agonis t activities, as measured by receptor tyrosine kinase stimulation assa ys, generally followed a similar trend. The L15F, L15W and L15V analog ues stimulated the receptor kinase to a level (V-max) similar to that for wild type hEGF. A striking difference was observed between the L15 A and L15R variants; although having similar binding affinities, the L 15A mutant activated the receptor to only similar to 5% of the wild ty pe V-max in contrast to 53% for the L15R mutant. H-1-NMR analysis of t he L15R and L15A mutants showed only minor structural alterations that were not sufficient to account for the dramatic losses in binding and agonist activities. The results indicate that both the size and hydro phobicity of the gamma-branched aliphatic side chain of Leu15 of hEGF are important in the formation of a catalytically active ligand-recept or complex.