MUTATIONAL STUDIES OF CONSERVED RESIDUES IN THE DIMER INTERFACE OF NERVE GROWTH-FACTOR

An understanding of the structure-function relationship of nerve growt h factor (NGF) requires precise knowledge of all the residues and regi ons that participate in NGF receptor binding, receptor activation, and biological activity. Seven recombinant human NGF mutants having alani ne substituted for residues located either in the NGF dimer interface or beta-strand region were studied to determine the role of each amino acid residue in NGF biological activity. F86A, T91A, R100A, and R103A remained nearly Fully active with 61, 120, 91, and 73% of wildtype ac tivity, respectively, in the PC12 cell bioassay. Hydrophobic core and dimer interface residues Y52, F53, and F54 were studied in more detail . Y52A and F54A were expressed in very low levels, suggesting that the se two residues may be important for protein stability. Y52A retained full biological activity (91%). F53A had a 20- and 70-fold reduction i n biological activity and TrkA phosphorylation, respectively, with onl y a 5- to 10-fold effect on TrkA binding and no effect on low-affinity receptor binding. F54A had significantly decreased TrkA phosphorylati on and biological activity (40-fold). The results suggest that F53 and F54 may play a structural role in TrkA receptor activation subsequent to binding.