Introduction: Autosomal dominant Kallmanns syndrome (AKS) results from mutations within the fibroblast growth factor receptor 1 (FGFR1: KAL-2). The FGFR family of tyrosine kinases are involved in a multitude of biological processes from embryogenesis to adult homeostasis. The crystal structure of FGF-FGFR-heparan sulphate (HS) ternary complex has provided a basis for understanding the way in which FGF, FGFR and HS cooperate to assist FGFR dimerization. Anosmin-1, KAL-1 gene product is mutated in X-linked KS. We have previously demonstrated that anosmin-1 can modulate neuronal differentiation of human embryonic olfactory neuroblast through the interaction with FGFR1/FGF2/HS complex to stimulate p42/44 and p38 MAP kinase and cdc42/Rac, acting as a novel modulator for FGFR1 signalling.
Aim: To understand the role of anosmin-1 in FGFR1/FGF2/HS ternary complex assembly.
Methods: ELISA was performed using immobilised murine FGFR1IIIc and decasaccharide derived from bovine lung heparin. The 6His-tagged wild type anosmin-1 and its three KS-associated missense mutations were used as soluble ligands. Bound anosmin-1 was detected by using anti-His HRP conjugated antibody. The solution structure of anosmin-1 was studied by analytical ultracentrifugation, X-ray scattering and homology modelling.
Results: Anosmin-1 binds to FGFR1IIIc in a dose-dependent manner, while BSA and three missense mutants do not. The presence of FGF2 and heparin does not change the binding of anosmin-1 to FGFR1IIIc. Anosmin-1 binds to oligosaccharide as short as the 10mer. The six domains of anosmin-1 are in an extended arrangement with flexible inter-domain linker.
Conclusion: Anosmin-1 can directly bind to FGFR1 with the requirement of its first and third FnIII domains and to oligosaccharide with only ten saccharide residues. The extended domain arrangement of anosmin-1 may provide the binding platform for multi-molecules of FGFR1/FGF2/HS complex; this assembly presumably amplifies and synergises downstream FGFR1 signalling, thereby eliciting a receptor-specific cellular response within olfactory system ontogenesis.