通过重组APEX2-FGF1融合蛋白进行邻近标记揭示了FGF1与蛋白聚糖CD44和CSPG4的相互作用,Biochemistry

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通过重组APEX2-FGF1融合蛋白进行邻近标记揭示了FGF1与蛋白聚糖CD44和CSPG4的相互作用
Biochemistry ( IF 2.9 ) Pub Date : 2018-05-29 00:00:00 , DOI: 10.1021/acs.biochem.8b00120
Yan Zhen _{1,

2} , Ellen Margrethe Haugsten _{2,

3} , Sachin Kumar Singh _{2,

3} , Jørgen Wesche _{2,

3}

Affiliation

成纤维细胞生长因子1（FGF1）与靶细胞表面的特定FGF受体（FGFR）结合以启动细胞内信号传导。尽管硫酸乙酰肝素蛋白聚糖（HSPG）是公认的共受体，但尚不确定在细胞表面是否存在FGF1的额外结合位点。为了解决这个问题，我们设计并测试了一种在细胞表面鉴定FGF1新结合位点的方法，该结合位点也可能适用于其他蛋白质配体。我们构建了APEX2-FGF1融合蛋白，以在融合蛋白与细胞表面结合后对蛋白进行近端生物素标记。通过信号传导测定对融合蛋白进行功能验证后，我们使用此方法来鉴定活细胞细胞表面上FGF1的结合位点。我们通过检测FGFR4证实了我们方法的可行性，FGF1的众所周知的特异性受体。我们随后使用RPE1细胞筛选了新型相互作用子，并鉴定了蛋白聚糖CSPG4（NG2）和CD44。我们发现FGF1通过其肝素结合部分结合CD44。此外，我们发现FGF1与CSPG4和CD44都在细胞表面共定位，表明这些受体充当了存储FGF1的储存分子。重要的是，我们的数据表明重组配体– APEX2融合蛋白可用于鉴定细胞表面上的新型受体相互作用。我们发现FGF1与CSPG4和CD44共同定位在细胞表面，提示这些受体充当了存储FGF1的储存分子。重要的是，我们的数据表明重组配体– APEX2融合蛋白可用于鉴定细胞表面上的新型受体相互作用。我们发现FGF1与CSPG4和CD44共同定位在细胞表面，这表明这些受体充当了存储FGF1的储存分子。重要的是，我们的数据表明重组配体– APEX2融合蛋白可用于鉴定细胞表面上的新型受体相互作用。

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Proximity Labeling by a Recombinant APEX2–FGF1 Fusion Protein Reveals Interaction of FGF1 with the Proteoglycans CD44 and CSPG4

Fibroblast growth factor 1 (FGF1) binds to specific FGF receptors (FGFRs) at the surface of target cells to initiate intracellular signaling. While heparan sulfate proteoglycans (HSPGs) are well-described coreceptors, it is uncertain whether there are additional binding sites for FGF1 at the cell surface. To address this, we devised and tested a method to identify novel binding sites for FGF1 at the cell surface, which may also be applicable for other protein ligands. We constructed an APEX2–FGF1 fusion protein to perform proximal biotin labeling of proteins following binding of the fusion protein to the cell surface. After functional validation of the fusion protein by a signaling assay, we used this method to identify binding sites for FGF1 on cell surfaces of living cells. We confirmed the feasibility of our approach by detection of FGFR4, a well-known and specific receptor for FGF1. We subsequently screened for novel interactors using RPE1 cells and identified the proteoglycans CSPG4 (NG2) and CD44. We found that FGF1 binds CD44 through its heparin-binding moiety. Moreover, we found that FGF1 was colocalized with both CSPG4 and CD44 at the cell surface, suggesting that these receptors act as storage molecules that create a reservoir of FGF1. Importantly, our data demonstrate that recombinant ligand–APEX2 fusion proteins can be used to identify novel receptor interactions on the cell surface.

更新日期：2018-05-29

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