Neurotrophin-3 acquires NGF-like activity after exchange to five 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 NGF-related neurotrophins, the members display different activities on different subset of neurons. Recent studies have shown that the various neurotrophins are ligands with high affinity to different receptors of the Trk family of tyrosine kinase receptors. We wanted to elucidate what specific parts of NGF replaced in neurotrophin-3 (NT-3) would result in NGF-like receptor binding and biological activity. By studying evolutionarily conserved amino acid sequences not shared by NT-3 and NGF and excluding parts which have been examined in earlier work with NGF and BDNF chimeras as well as taking advantage of the crystallographic data available for NGF, we decided to exchange three specific blocks of two or three amino acids in the human NT-3 backbone for the corresponding residues in NGF. The NGF residues Asn-Ile-Asn (43-45), Val-Phe (48,49) and Gln-Ala-Ala (96-98) were combined in pairs and are all shown 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-Ala. This mutant reached 90% NGF activity, based on survival of sympathetic neurons, stimulation of fibre outgrowth from sympathetic ganglia, the ability to block high affinity NGF binding to PC12 cells and phosphorylation of gp140trk. Thus, the three mutants with paired combinations 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 gained, although the mutated neurotrophin proteins do not lose the original NT-3 activity as ascertained by the stimulation of neurite outgrowth from the Remak ganglion. The three mutated sites are situated in two beta-loops at one end of the NGF molecule, forming a cleft that could specifically interact with high affinity to the signalling NGF receptor gp140trk.
Kullander
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K
Department of Developmental Biology, Uppsala University, Sweden.
Ebendal
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000166:cytology / Q000196:embryology; D002461:Cell Line, Transformed; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D005725:Ganglia, Autonomic / Q000166:cytology / Q000187:drug effects; D005728:Ganglia, Sympathetic / Q000166:cytology / Q000187:drug effects; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000494:pharmacology; D020933:Neurotrophin 3; D016716:PC12 Cells; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D020917:Receptor, trkA; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D009414:Nerve Growth Factors; D020933:Neurotrophin 3; D011518:Proto-Oncogene Proteins; D017475:Receptors, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; D020794:Receptor Protein-Tyrosine Kinases; D020917:Receptor, trkA