Reconstitution of a Frizzled8.Wnt3a.LRP6 Signaling Complex Reveals Multiple Wnt and Dkk1 Binding Sites on LRP6
Reconstitution of a Frizzled8.Wnt3a.LRP6 Signaling Complex Reveals Multiple Wnt and Dkk1 Binding Sites on LRP6
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DOI：
10.1074/jbc.m109.092130
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发表时间：
2010-03-19
影响因子：
4.8
通讯作者：
Hannoush, Rami N.
中科院分区：
文献类型：
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作者：
Bourhis, Eric;Tam, Christine;Hannoush, Rami N.
Wnt/β-连环蛋白信号传导在细胞表面通过分泌的Wnt与其受体Frizzled（Fz）和低密度脂蛋白受体相关蛋白5/6（LRP 5/6）的缔合而起始。这些分子相互作用的研究一直是一个重大的技术挑战，因为蛋白质一直无法获得足够的纯度和数量。在这份报告中，我们描述了昆虫细胞表达和纯化可溶性小鼠Fz 8富含半胱氨酸的结构域和人LRP 6胞外结构域，并表明它们抑制Wnt/β-连环蛋白信号在细胞测定。我们确定Wnt和Dickkopf 1（Dkk 1）与相关共受体的结合亲和力，并在体外重构Fz 8 CRD. Wnt 3a. LRP 6信号传导复合物。使用纯化的LRP 6片段，我们进一步表明，Wnt 3a结合到一个区域，仅包括第三和第四β-螺旋桨结构域的LRP 6（E3 E4）。令人惊讶的是，我们发现Wnt 9 b与LRP 6胞外结构域E1 E2的不同部分结合，并且我们证明Wnt 3a和Wnt 9 b可以同时与LRP 6结合。Dkk 1结合E1 E2和E3 E4片段，并与Wnt 3a和Wnt 9 b竞争结合LRP 6。LRP 6共受体上存在多个独立的Wnt结合位点，提示了Wnt信号复合物结构的新可能性，以及Dkk 1对Wnt/β-连环蛋白信号传导的广谱抑制模型。
Wnt/beta-catenin signaling is initiated at the cell surface by association of secreted Wnt with its receptors Frizzled (Fz) and low density lipoprotein receptor-related protein 5/6 (LRP5/6). The study of these molecular interactions has been a significant technical challenge because the proteins have been inaccessible in sufficient purity and quantity. In this report we describe insect cell expression and purification of soluble mouse Fz8 cysteine-rich domain and human LRP6 extracellular domain and show that they inhibit Wnt/beta-catenin signaling in cellular assays. We determine the binding affinities of Wnts and Dickkopf 1 (Dkk1) to the relevant co-receptors and reconstitute in vitro the Fz8 CRD.Wnt3a.LRP6 signaling complex. Using purified fragments of LRP6, we further show that Wnt3a binds to a region including only the third and fourth beta-propeller domains of LRP6 (E3E4). Surprisingly, we find that Wnt9b binds to a different part of the LRP6 extracellular domain, E1E2, and we demonstrate that Wnt3a and Wnt9b can bind to LRP6 simultaneously. Dkk1 binds to both E1E2 and E3E4 fragments and competes with both Wnt3a and Wnt9b for binding to LRP6. The existence of multiple, independent Wnt binding sites on the LRP6 co-receptor suggests new possibilities for the architecture of Wnt signaling complexes and a model for broad-spectrum inhibition of Wnt/beta-catenin signaling by Dkk1.