NEUROTROPHIN-3 ACQUIRES NGF-LIKE ACTIVITY AFTER EXCHANGE TO 5 NGF AMINO-ACID-RESIDUES - MOLECULAR ANALYSIS OF THE SITES IN NGF MEDIATING THE SPECIFIC INTERACTION WITH THE NGF HIGH-AFFINITY RECEPTOR

Despite the large sequence similarity around 55-60% among the known NG F-related neurotrophins, the members display different activities on d ifferent subset of neurons. Recent studies have shown that the various neurotrophins are ligands with high affinity to different receptors o f the Trk family of tyrosine kinase receptors. We wanted to elucidate what specific parts of NGF replaced in neurotrophin-3 (NT-3) would res ult in NGF-like receptor binding and biological activity. By studying evolutionarily conserved amino acid sequences not shared by NT-3 and N GF and excluding parts which have been examined in earlier work with N GF and BDNF chimeras as well as taking advantage of the crystallograph ic data available for NGF, we decided to exchange three specific block s of two or three amino acids in the human NT-3 backbone for the corre sponding residues in NGF. The NGF residues Asn-Ile-Asn (43-45), Val-Ph e (48,49) and Gln-Ala-Ala (96-98) were combined in pairs and are all s hown to contribute NGF-like activity in the context of NT-3. The most efficient NGF-like transformation was obtained by the exchange of Pro- Val and Leu-Val-Gly in NT-3 to the NGF residues Val-Phe and Gln-Ala-Al a. This mutant reached 90% NGF activity, based on survival of sympathe tic neurons, stimulation of fibre outgrowth from sympathetic ganglia, the ability to block high affinity NGF binding to PC12 cells and phosp horylation of gp140(trk). Thus, the three mutants with paired combinat ions of the NGF residues as well as the NT-3 housing all three blocks of NGF residues were able to mimic NGF activity. This activity is gain ed, although the mutated neurotrophin proteins do not lose the origina l NT-3 activity as ascertained by the stimulation of neurite outgrowth from the Remak ganglion.