Biochemical dissection of Anosmin‐1 interaction with FGFR1 and components of the extracellular matrix

Abstract

Anosmin-1, defective in Kallmann's syndrome, participates in the adhesion, migration and differentiation of different cell types in the CNS. Although not fully understood, the mechanisms of action of Anosmin-1 involve the interaction with different proteins, being the interaction with fibroblast growth factor receptor 1 (FGFR1) and the modulation of its signalling the best studied to date. Using glutathione-S-transferase pull-down assays we demonstrate that the FnIII.3 (Fibronectin-like type III) domain and the combination whey acidic protein-FnIII.1, but not each of them individually, interact with FGFR1. The interaction of the whey acidic protein-FnIII.1 domains is substantially reduced when the cysteine-rich region is present, suggesting a likely regulatory role for this domain. The introduction in FnIII.3 of any of the two missense mutations found in Kallmann's syndrome patients, E514K and F517L, abolished the interaction with FGFR1, what suggests an important role for these residues in the interaction. Interestingly, the chemoattraction of Anosmin-1 on rat neuronal precursors (NPs) via FGFR1 is retained by the N-terminal region of Anosmin-1 but not by FnIII.3 alone, and is lost in proteins carrying either one of the missense mutations, probably because of a highly reduced binding capacity to FGFR1. We also describe homophilic interaction Anosmin-1/Anosmin-1 via the FnIII repeats 1 and 4, and the interaction of FnIII.1 and FnIII.3 with Fibronectin and of FnIII.3 with Laminin.

FGF-2 and Anosmin-1 Are Selectively Expressed in Different Types of Multiple Sclerosis Lesions

“…The membranes were coated as described previously (Merchán et al, 2007;Murcia-Belmonte et al, 2010) and 4 ϫ 10 4 adult OPCs were seeded in the upper chamber in BS medium and different experimental conditions in the lower chamber as follows: (1) CHO-CT extract; (2) CHO-CT extract with FGF-2 (20 ng/ml; R&D Systems); (3) CHO-A1 extract; or (4) CHO-A1 extract with FGF-2 (20 ng/ml; R&D Systems). To block FGFRs, the adult OPCs were treated during the experiment with the FGFR blocker SU5402 (10 M; Calbiochem) as indicated, and the rest of the cultures were exposed to an equal volume of the vehicle dimethylsulfoxide (Sigma).…”

Invertebrate Models of Kallmann Syndrome: Molecular Pathogenesis and New Disease Genes

“…The evidence emerged from experiments performed in C. elegans , Drosophila and in cell culture and is also confirmed by clinical observations: cases of KS patients with mild sexual phenotypes carrying missense mutations that disrupt disulfide bonds in the WAP domain are well documented [55, 56]. The interaction between anosmin-1 and FGFR requires the WAP domain, and probably the CR region [57], which seem to have a regulatory role on the adhesiveness of the FnIII domains present in the same molecule Fig. ( 4a , black arrow).…”

The Adhesion Molecule Anosmin-1 in Neurology: Kallmann Syndrome and Beyond

“…Interaction between anosmin-1 and FGFR1 has been reported by co-immunoprecipitation (CoIP) (Ayari and SoussiYanicostas 2007 ;Bribián et al 2006 ;González-Martínez et al 2004 ), and it has been determined that the WAP and FnIII.1 domains together and the FnIII.3 domain by itself interact with FGFR1 (Hu et al 2009 ;Murcia-Belmonte et al 2010 ). Not surprisingly, some of the missense mutations in the KAL1 gene found in KS patients lie within the FnIII domains involved in the interaction with FGFR1 (N267K, E514K, F517L), impeding or greatly reducing the interaction with the receptor and rendering non-functional proteins (Cariboni et al 2004 ;Hu et al 2009 ;Murcia-Belmonte et al 2010 ). Mutations within the WAP domain have also been reported in KS patients, mainly missense mutations replacing some of the cysteine residues and presumably disrupting the formation of disulphide bonds and the correct folding or structure of the domain.…”

“…The best known mechanism of action of anosmin-1 is the interaction with the fi broblast growth factor receptor 1 (FGFR1) and the modulation of the activation of this receptor, linking two of the genes responsible for KS (Ayari and Soussi-Yanicostas 2007 ;Bribián et al 2006 ;Dode et al 2003 ;García-González et al 2010 ;González-Martínez et al 2004 ;Hu et al 2009 ;Murcia-Belmonte et al 2010 ). Interaction between anosmin-1 and FGFR1 has been reported by co-immunoprecipitation (CoIP) (Ayari and SoussiYanicostas 2007 ;Bribián et al 2006 ;González-Martínez et al 2004 ), and it has been determined that the WAP and FnIII.1 domains together and the FnIII.3 domain by itself interact with FGFR1 (Hu et al 2009 ;Murcia-Belmonte et al 2010 ). Not surprisingly, some of the missense mutations in the KAL1 gene found in KS patients lie within the FnIII domains involved in the interaction with FGFR1 (N267K, E514K, F517L), impeding or greatly reducing the interaction with the receptor and rendering non-functional proteins (Cariboni et al 2004 ;Hu et al 2009 ;Murcia-Belmonte et al 2010 ).…”

“…Approximately two-thirds of the mutations described in the KAL1 gene are deletions or frameshift or nonsense mutations that could affect the overall length or composition of the protein, suffi cient to account for the disease (Hu and Bouloux 2011 ), what confl icts with the fact that a truncated N-terminal protein comprising the CR-WAP-FnIII.1 domains, behaves as the full-length protein in some scenarios regarding some of the biological functions of anosmin-1 involving FGFR1 (Bülow et al 2002 ;González-Martínez et al 2004 ;Hu et al 2004 ;Murcia-Belmonte et al 2010 ). It has been suggested that the function of the different anosmin-1 domains could be determined or conditioned by the extracellular environment and that the protein could be involved in different phenotypic effects, depending on the cell type or the interaction with different receptors or binding proteins (Andrenacci et al 2006 ;Bülow et al 2002 ).…”