Hu Youli, Guimond Scott E, Travers Paul, Cadman Steven, Hohenester Erhard, Turnbull Jeremy E, Kim Soo-Hyun, Bouloux Pierre-Marc
Centre for Neuroendocrinology, University College London Medical School, Royal Free Campus, London NW32PF.
J Biol Chem. 2009 Oct 23;284(43):29905-20. doi: 10.1074/jbc.M109.049155. Epub 2009 Aug 20.
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 isoform-specific 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 FGF2.heparin complex, thus preventing FGFR1.FGF2.heparin complex formation. By contrast, heparin-bound anosmin-1 binds to pre-formed FGF2.FGFR1 complex, generating an anosmin-1.FGFR1.FGF2.heparin 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 FGFR1.FGF2.heparin complex.
成纤维细胞生长因子（FGF）信号的激活是由FGF、FGF受体（FGFR）和细胞膜上的硫酸乙酰肝素蛋白聚糖之间形成多蛋白复合物启动的。与其他因子的相互作用可能会影响这种复合物的组装，并调节细胞对FGF的生物学反应。我们之前已经证明，anosmin-1是一种糖基化的细胞外基质蛋白，它与FGFR1信号复合物相互作用，并以IIIc亚型特异性和HS依赖性方式增强其活性。然而，anosmin-1对FGFR1信号作用的分子机制仍然未知。在这里，我们表明anosmin-1以高亲和力直接结合FGFR1。这种相互作用涉及anosmin-1 N端的结构域（富含半胱氨酸区域、乳清酸性蛋白样结构域和第一个III型纤连蛋白结构域）以及FGFR1的D2-D3细胞外结构域。相比之下，anosmin-1与FGFR2IIIc的结合亲和力要低得多，与FGFR3IIIc的结合可忽略不计。我们还表明，与FGFR1结合的anosmin-1虽然能够单独结合FGF2，但不能结合FGF2-肝素复合物，从而阻止FGFR1-FGF2-肝素复合物的形成。相比之下，与肝素结合的anosmin-1与预先形成的FGF2-FGFR1复合物结合，形成anosmin-1-FGFR1-FGF2-肝素复合物。此外，免疫荧光共定位和Transwell迁移试验证明了anosmin-1与FGFR1信号复合物之间的功能相互作用，其中anosmin-1在人神经元细胞趋化过程中表现出相反的作用。我们的研究为anosmin-1对FGFR1信号的调节作用提供了分子和细胞证据，即anosmin-1与FGFR1和肝素的结合可以在三元FGFR1-FGF2-肝素复合物的组装和活性中发挥双重作用。
