Identification of a linear epitope in sortilin that partakes in pro-neurotrophin binding.
Sortilin acts as a cell surface receptor for pro-neurotrophins (pro-NT) that upon complex formation with the p75 neurotrophin receptor (p75(NTR)) is able to signal neuronal cell death. Here we screened a sortilin peptide library comprising 16-mer overlapping sequences for binding of the pro-domains of nerve growth factor and brain-derived neurotrophic factor. We find that a linear surface-exposed sequence, (163)RIFRSSDFAKNF(174), constitutes an important pro-NT binding epitope in sortilin. Systematic mutational analysis revealed residues Arg(163), Phe(165), Arg(166), and Phe(170) to be critical for the interaction. Expression of a sortilin mutant in which these four amino acids were substituted by alanines disrupted pro-NT binding without affecting receptor heterodimerization with p75(NTR) or binding of ligands that selectively engages the centrally located tunnel in the beta-propeller of sortilin. We furthermore demonstrate that a peptide comprising the ligand-binding epitope can prevent pro-NT-induced apoptosis in RN22 schwannoma cells.
Department of Medical Biochemistry, Membrane Receptors in Neuronal Disease (MIND) Center, University of Aarhus, OleWorms Allé, Aarhus C, Denmark.
Olga
O
Serup Andersen
Prisca
P
Boisguerin
Simon
S
Glerup
Sune
S
Skeldal
Rudolf
R
Volkmer
Thomas E
TE
Willnow
Anders
A
Nykjaer
Olav M
OM
Andersen
D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017209:Apoptosis; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009414:Nerve Growth Factors / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism; D017475:Receptors, Nerve Growth Factor / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014162:Transfection; D000099183:Sortilin
D033942:Adaptor Proteins, Vesicular Transport; D017931:DNA Primers; D000939:Epitopes; D008024:Ligands; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D011498:Protein Precursors; D017475:Receptors, Nerve Growth Factor; D011994:Recombinant Proteins; D000099183:Sortilin; C484065:NGFR protein, human