Secreted frizzled-related protein-1 binds directly to wingless and is a biphasic modulator of Wnt signaling
Secreted frizzled-related protein-1 binds directly to wingless and is a biphasic modulator of Wnt signaling
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DOI：
10.1074/jbc.275.6.4374
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发表时间：
2000-02-11
影响因子：
4.8
通讯作者：
Rubin, JS
中科院分区：
文献类型：
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作者：
Üren, A;Reichsman, F;Rubin, JS
分泌型卷曲相关蛋白-1（sFRP-1）含有一个富含半胱氨酸的结构域，与卷曲蛋白的Wnt结合位点同源。为了便于sFRP-1的生化和生物学分析，我们开发了一种哺乳动物重组表达系统，其产量类似于3 mg纯化蛋白/升条件培养基。使用这种重组蛋白，我们证明了sFRP-1和Wg（无翅）在酶联免疫吸附和共沉淀试验中相互作用。令人惊讶的是，缺乏富含半胱氨酸的结构域的衍生物保留了结合Wg的能力。用放射性碘标记的sFRP-1进行的交联实验提供了sFRP-1和Wg直接彼此结合的明确证据。除了检测到尺寸与sFRP-1和Wg的1：1化学计量一致的交联复合物之外，我们还观察到尺寸表明存在第二个sFRP-1分子的较大复合物。这两种复合物的形成显着增强了最佳浓度的外源性肝素，强调硫酸乙酰肝素蛋白聚糖在Wnt结合和信号传导的潜在重要性。sFRP-1在犰狳稳定试验中对Wg活性产生双相作用，在低浓度下增加犰狳水平，但在较高浓度下降低。这些结果提供了关于sFRPs的Wnt结合和生物活性的新见解。
Secreted Frizzled-related protein-1 (sFRP-1) contains a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzleds. To facilitate the biochemical and biological analysis of sFRP-1, we developed a mammalian recombinant expression system that yields similar to 3 mg of purified protein/liter of conditioned medium. Using this recombinant protein, we demonstrated that sFRP-1 and Wg (wingless) interact in enzyme-linked immunosorbent and co-precipitation assays. Surprisingly, a derivative lacking the cysteine-rich domain retained the ability to bind Wg, Cross-linking experiments performed with radioiodinated sFRP-1 provided definitive evidence that sFRP-1 and Wg bind directly to each other. Besides detecting a cross-linked complex consistent in size with 1:1 stoichiometry of sFRP-1 and Wg, we also observed a larger complex whose size suggested the presence of a second sFRP-1 molecule. The formation of both complexes was markedly enhanced by an optimal concentration of exogenous heparin, emphasizing the potential importance of heparan-sulfate proteoglycan in Wnt binding and signaling. sFRP-1 exerted a biphasic effect on Wg activity in an armadillo stabilization assay, increasing armadillo level at low concentrations but reducing it at higher concentrations. These results provide new insights about the Wnt binding and biological activity of sFRPs.