The cysteine-rich region and the whey acidic protein domain are essential for anosmin-1 biological functions

Authors

  • Pedro F. Esteban,

    Corresponding author
    • Grupo de Neurobiología del Desarrollo-GNDe, Hospital Nacional de Parapléjicos, Toledo, Spain
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    • These two authors contributed equally to this manuscript.
  • Verónica Murcia-Belmonte,

    1. Grupo de Neurobiología del Desarrollo-GNDe, Hospital Nacional de Parapléjicos, Toledo, Spain
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    • These two authors contributed equally to this manuscript.
  • Diego García-González,

    1. Grupo de Neurobiología del Desarrollo-GNDe, Hospital Nacional de Parapléjicos, Toledo, Spain
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  • Fernando de Castro

    Corresponding author
    • Grupo de Neurobiología del Desarrollo-GNDe, Hospital Nacional de Parapléjicos, Toledo, Spain
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Address correspondence and reprint requests to Dr. Pedro F. Esteban or Dr. Fernando de Castro, Grupo de Neurobiología del Desarrollo-GNDe, Hospital Nacional de Parapléjicos, Finca “La Peraleda”, s/n, E-45071-Toledo, Spain. E-mails: pfesteban@externas.sescam.jccm.es; fdec@sescam.jccm.es

Abstract

The protein anosmin-1, coded by the KAL1 gene responsible for the X-linked form of Kallmann syndrome (KS), exerts its biological effects mainly through the interaction with and signal modulation of fibroblast growth factor receptor 1 (FGFR1). We have previously shown the interaction of the third fibronectin-like type 3 (FnIII) domain and the N-terminal region of anosmin-1 with FGFR1. Here, we demonstrate that missense mutations reported in patients with KS, C172R and N267K did not alter or substantially reduce, respectively, the binding to FGFR1. These substitutions annulled the chemoattraction of the full-length protein over subventricular zone (SVZ) neuronal precursors (NPs), but they did not annul it in the N-terminal-truncated protein (A1Nt). We also show that although not essential for binding to FGFR1, the cysteine-rich (CR) region is necessary for anosmin-1 function and that FnIII.3 cannot substitute for FnIII.1 function. Truncated proteins recapitulating nonsense mutations found in KS patients did not show the chemotropic effect on SVZ NPs, suggesting that the presence behind FnIII.1 of any part of anosmin-1 produces an unstable protein incapable of action. We also identify the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway as necessary for the chemotropic effect exerted by FGF2 and anosmin-1 on rat SVZ NPs.

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