N-glycosylation of fibroblast growth factor receptor 1 regulates ligand and heparan sulfate co-receptor binding
N-glycosylation of fibroblast growth factor receptor 1 regulates ligand and heparan sulfate co-receptor binding
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DOI：
10.1074/jbc.m601248200
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发表时间：
2006-09-15
影响因子：
4.8
通讯作者：
Fernig, David G.
中科院分区：
文献类型：
--
作者：
Duchesne, Laurence;Tissot, Berangere;Fernig, David G.
成纤维细胞生长因子（FGF）对细胞功能的调节是通过由酪氨酸激酶、FGFR和硫酸肝素（HS）受体组成的双受体系统进行的。人fgfr中一些潜在n -糖基化位点的突变导致受体过度激活的表型特征。为了确定n -糖基化如何影响FGFR功能，我们在中国仓鼠卵巢细胞中产生了与FGFR1-IIIc细胞外配体结合域相对应的可溶性和膜结合重组受体。两种形式的FGFR1-IIIc都被观察到严重的n -糖基化，并在SDS-PAGE上作为一系列50至75 kDa的多条带迁移，而去糖基化受体在32 kDa迁移，与多肽的预期分子量相对应。光学生物传感器和石英晶体微平衡耗散结合实验表明，从FGFR1-IIIc中去除n-聚糖导致受体与FGF-2和肝素衍生低聚糖（细胞HS的代表）的结合增加。这种作用是通过n -糖基化介导的，它降低了FGF-2和肝素寡糖受体的结合速率常数。通过质谱分析，n -聚糖显示了双天线和三天线核心聚焦的复合体型结构的优势，携带一个，两个和/或三个唾液酸。对受体蛋白上这种聚糖结构的建模表明，至少有一些可能被策略性地定位，以干扰受体与FGF配体和/或HS共受体的相互作用。因此，受体的n -聚糖代表了调控fgf活性的另一途径。
The regulation of cell function by fibroblast growth factors (FGF) occurs through a dual receptor system consisting of a receptor-tyrosine kinase, FGFR and the glycosaminoglycan heparan sulfate (HS). Mutations of some potential N-glycosylation sites in human fgfr lead to phenotypes characteristic of receptor overactivation. To establish how N-glycosylation may affect FGFR function, soluble- and membrane-bound recombinant receptors corresponding to the extracellular ligand binding domain of FGFR1-IIIc were produced in Chinese Hamster Ovary cells. Both forms of FGFR1-IIIc were observed to be heavily N-glycosylated and migrated on SDS-PAGE as a series of multiple bands between 50 and 75 kDa, whereas the deglycosylated receptors migrated at 32 kDa, corresponding to the expected molecular weight of the polypeptides. Optical biosensor and quartz crystal microbalance-dissipation binding assays show that the removal of the N-glycans from FGFR1-IIIc caused an increase in the binding of the receptor to FGF-2 and to heparin-derived oligosaccharides, a proxy for cellular HS. This effect is mediated by N-glycosylation reducing the association rate constant of the receptor for FGF-2 and heparin oligosaccharides. N-Glycans were analyzed by mass spectrometry, which demonstrates a predominance of bi- and tri-antennary core-fucosylated complex type structures carrying one, two, and/or three sialic acids. Modeling of such glycan structures on the receptor protein suggests that at least some may be strategically positioned to interfere with interactions of the receptor with FGF ligand and/or the HS co-receptor. Thus, the N-glycans of the receptor represent an additional pathway for the regulation of the activity of FGFs.