CHARACTERIZATION OF HISTIDINE-RESIDUES ESSENTIAL FOR RECEPTOR-BINDINGAND ACTIVITY OF NERVE GROWTH-FACTOR

The role of the four histidine residues in receptor binding and activi ty of mouse nerve growth factor (NGF) was investigated using both site directed mutagenesis and chemical modification with diethyl pyrocarbo nate, Replacement of His-75 or His-84 with alanine resulted in decreas ed biological activity and decreased affinity for p140(trkA); however, with H75A only, a 5-fold increased affinity toward p75(LANR) was obse rved, The effect of simultaneous replacement of both His-75 and His-84 was neither additive nor synergistic. Slight perturbations in circula r dichroism spectra and weakened self-association of the mutants indic ated that His-75 and His-84 may be involved in stability, dimerization , and/or folding of NGF, Diethyl pyrocarbonate modification of His-4 a nd His-8 in the H75A/H84Q double mutant abolished neuritogenesis, bind ing to both receptors, and phosphorylation of p140(trkA) in PC12 cells , These chemi cal and mutational results confirm and clarify previous evidence for the involvement of His-75 and His-84 (Dunbar, J. C., Treg ear, G. W., and Bradshaw, R. A. (1984) J. Protein Chem, 3, 349-356) or His-4 and His-8 (Shih, A., Laramee, G. R., Schmelzer, C. H., Burton, L. E., and Winslow, J. W. (1994) J. Biol. Chem. 269, 27679-27686) in r eceptor binding of NGF. At least three and possibly all four histidine s, which are located in three spatially distinct regions, contribute t o maintenance of functional sites that are essential for receptor bind ing and activity of NGF.