The binding epitopes of neurotrophin-3 to its receptors trkC and gp75 and the design of a multifunctional human neurotrophin.
Survival and maintenance of vertebrate neurons are influenced by neurotrophic factors which mediate their signal by binding to specific cell surface receptors. We determined the binding sites of human neurotrophin-3 (NT-3) to its receptors trkC and gp75 by mutational analysis and compared them to the analogous interactions of nerve growth factor (NGF) with trkA and gp75. The trkC binding site extends around the central beta-strand bundle and in contrast to NGF does not make use of non-conserved loops and the six N-terminal residues. The gp75 epitope is dominated by loop residues and the C-terminus of NT-3. A novel rapid biological screening procedure allowed the identification of NT-3 mutants that are able to signal efficiently through the non-preferred receptors trkA and trkB, which are specific for NGF and BDNF respectively. Mutation of only seven residues in NT-3 resulted in a human neurotrophin variant which bound to all receptors of the trk family with high affinity and efficiently supported the survival of NGF-, BDNF- and NT-3-dependent neurons. Our results suggest that the specificity among neurotrophic factors is not solely encoded in sequence diversity, but rather in the way each neurotrophin interacts with its preferred receptor.
Urfer
R
R
Department of Protein Engineering, Genentech Inc., South San Francisco, CA 94080.
Tsoulfas
P
P
Soppet
D
D
Escandón
E
E
Parada
L F
LF
Presta
L G
LG
D000595:Amino Acid Sequence; D000818:Animals; D002454:Cell Differentiation; D003198:Computer Simulation; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D000939:Epitopes; D005818:Genetic Engineering; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009414:Nerve Growth Factors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009417:Nerve Tissue / Q000254:growth & development*; D020933:Neurotrophin 3; D016716:PC12 Cells; D011485:Protein Binding; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D020800:Receptor, Nerve Growth Factor; D020812:Receptor, trkC; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D000939:Epitopes; D008562:Membrane Glycoproteins; D009414:Nerve Growth Factors; D020933:Neurotrophin 3; D020800:Receptor, Nerve Growth Factor; D017475:Receptors, Nerve Growth Factor; D011994:Recombinant Proteins; D020794:Receptor Protein-Tyrosine Kinases; D020812:Receptor, trkC