THE ROLE OF ASPARAGINE-32 IN FORMING THE RECEPTOR-BINDING EPITOPE OF HUMAN EPIDERMAL GROWTH-FACTOR

The highly conserved asparagine residue at position 32 (Asn32) in the 'hinge' region of epidermal growth factor (EGF) separates the N- and C -terminal structural motifs of the EGF molecule and is therefore an ap propriate target for structure - function studies. Analogs of human EG F (hEGF) were generated in which Asn32 was substituted with aspartate, glycine, isoleucine, lysine, proline and tryptophan. The relative aff inity of the EGF receptor for mutant hEGF analogs was determined by ra dioreceptor competition assay. A wide range of receptor affinities was observed depending on the amino acid substitution. N32K and N32W hEGF analogs had relatively high receptor affinity, while the N32G and N32 D analogs showed decreased affinity, 35% and 25% respectively, relativ e to wild type hEGF. However, no binding of the N32P analog was detect ed by radioreceptor competition assay. The N32P mutant displayed an NM R spectrum significantly different from that of native wild type hEGF, indicating gross structural perturbation. In contrast, the N32K and N 32D analogs exhibited spectra similar to that of native wild type hEGF . Genetically combining the N32D hEGF with an hEGF species having eith er the mutation L26G in the N-terminal region or L47A in the C-termina l region, generated double-mutant hEGF species which had relative affi nities essentially equal to the product of the relative affinities of the parent hEGF mutants, indicating functionally independent changes i n ligand - receptor interaction. These studies indicate the requiremen t for H-bond donor functionality in the side chain of residue number 3 2 in forming a fully competent receptor-binding epitope.