Novel Mechanisms of Fibroblast Growth Factor Receptor 1 Regulation by Extracellular Matrix Protein Anosmin-1

Abstract

Activation of fibroblast growth factor (FGF) signaling is initiated by a multiprotein complex formation between FGF, FGF receptor (FGFR), and heparan sulfate proteoglycan on the cell membrane. Cross-talk with other factors could affect this complex assembly and modulate the biological response of cells to FGF. We have previously demonstrated that anosmin-1, a glycosylated extracellular matrix protein, interacts with the FGFR1 signaling complex and enhances its activity in an IIIc isoformspecific and HS-dependent manner. The molecular mechanism of anosmin-1 action on FGFR1 signaling, however, remains unknown. Here, we show that anosmin-1 directly binds to FGFR1 with high affinity. This interaction involves domains in the N terminus of anosmin-1 (cysteine-rich region, whey acidic protein-like domain and the first fibronectin type III domain) and the D2-D3 extracellular domains of FGFR1. In contrast, anosmin-1 binds to FGFR2IIIc with much lower affinity and displays negligible binding to FGFR3IIIc. We also show that FGFR1-bound anosmin-1, although capable of binding to FGF2 alone, cannot bind to a FGF2⅐heparin complex, thus preventing FGFR1⅐FGF2⅐heparin complex formation. By contrast, heparinbound anosmin-1 binds to pre-formed FGF2⅐FGFR1 complex, generating an anosmin-1⅐FGFR1⅐FGF2⅐heparin complex. Furthermore, a functional interaction between anosmin-1 and the FGFR1 signaling complex is demonstrated by immunofluorescence co-localization and Transwell migration assays where anosmin-1 was shown to induce opposing effects during chemotaxis of human neuronal cells. Our study provides molecular and cellular evidence for a modulatory action of anosmin-1 on FGFR1 signaling, whereby binding of anosmin-1 to FGFR1 and heparin can play a dual role in assembly and activity of the ternary FGFR1⅐FGF2⅐heparin complex. FGF5 signaling plays an important role in a wide range of fundamental biological responses (1-3). Both FGF and FGFR bind to heparan sulfate (HS) and heparin, a highly sulfated type of HS produced in connective tissue mast cells. Heparan sulfate proteoglycans (HSPG) are the cell surface co-receptors essential for the formation of functional FGF⅐FGFR signaling complex (4, 5). There are four structurally related FGFRs (FGFR1-4), which consist of an extracellular ligand-binding region containing three immunoglobulin (Ig)-like domains (D1-D3), a single transmembrane domain, and a cytoplasmic domain with protein-tyrosine kinase catalytic activity. The 22 members of the FGF family bind to the interface formed by the D2/D3 domains and the linker between these domains (6, 7), whereas a conserved positively charged region in D2 serves as the HS binding site (8). An unusual stretch of seven to eight acidic residues designated as the "acid box" is present in the linker connecting D1 and D2. Alternative splicing events occur to generate various isoforms, including a truncated receptor lacking D1 and the D1-D2 linker or a full-length receptor that differs in the second half of D3, designated as IIIb and IIIc isoforms (5). Two cry...

Heparan sulfate 6-O-sulfotransferase 1 , a gene involved in extracellular sugar modifications, is mutated in patients with idiopathic hypogonadotrophic hypogonadism

“…Moreover, these in vivo findings are consistent with a model in which KAL1 may serve a modulatory role as a coligand with FGF in an HS-dependent manner as part of a ternary complex with FGFR, as previously suggested by cell culture studies (19). Oligogenic mutations in humans that directly or indirectly affect the genetic network comprising anosmin-1, the FGF-FGFR signaling module, and HS-modifying enzymes may thus synergize to cause the clinical phenotype of IHH (see below).…”

“…First, mutations in FGF/ FGFR signaling and in KAL1 are the most frequent among the known genetic lesions associated with nIHH/KS (found in 10% and 5% of patients, respectively) (24). Second, anosmin-1 can directly bind FGFR1 in vitro, and it colocalizes with FGFR1 in cell culture experiments (19). Third, pedigree I segregated mutations in both the FGFR1 and HS6ST1 genes in affected individuals, suggesting possible interactions between these two genes (see below).…”

“…Intriguingly, heparan 6-O-sulfation is also required for the function of FGF receptor 1 (FGFR1) and its ligand, FGF8 (16), and loss-of-function mutations in both genes are associated with human GnRH deficiency (17,18). Experiments in vitro have suggested that KAL1 modulates signaling via FGFR (19), but the genetic relationship between KAL1, FGFR, FGF, and HS remains elusive. In this paper, we investigated HS6ST1, a human homolog of C. elegans hst-6, as a candidate gene for GnRH deficiency and delineated the genetic interactions between these genes using C. elegans as a model.…”

Aspectos clínicos e moleculares do hipogonadismo hipogonadotrófico isolado congênito

“…(8) sugeriram que a anosmina-1 seria importante na sinalização do FGF e que os produtos dos genes KAL1 e FGFR1 poderiam interagir funcionalmente. De fato, foi observado que a anosmina-1 possui sítios de ligação para sulfato de heparina, moléculas importantes na formação do complexo FGF-FGFR1, e que essa proteína pode se ligar com alta afinidade diretamente ao FGFR1, atuando como um coligante modulador do complexo FGFR1-ligante (42) …”

“…Esses resultados sugerem que a HS6ST1 e a complexa rede de modificações das glicosaminoglicanas extracelulares são críticas para o desenvolvimento normal dos neurônios secretores de GnHR em humanos. Interessantemente, a HS6ST1 age em conjunto com outros genes associados ao hipogonadismo hipogonadotrófico, como o FGFR1 e o FGF8, consistente com o modelo de que a anosmina-1 atua como uma molécula coligante ao FGF na ativação do FGFR1 de maneira dependente de proteoglicanas sulfato de heparina (42,43,58).…”