Systematic Mapping of WNT-FZD Protein Interactions Reveals Functional Selectivity by Distinct WNT-FZD Pairs
Systematic Mapping of WNT-FZD Protein Interactions Reveals Functional Selectivity by Distinct WNT-FZD Pairs
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DOI：
10.1074/jbc.m114.612648
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发表时间：
2015-03-13
影响因子：
4.8
通讯作者：
Schulte, Gunnar
中科院分区：
文献类型：
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作者：
Dijksterhuis, Jacomijn P.;Baljinnyam, Bolormaa;Schulte, Gunnar
FZD1-10 类七次跨膜受体被脂糖蛋白 WNT 家族结合并激活，从而诱导复杂的信号通路网络。然而，迄今为止，哺乳动物 WNT 和 FZD 蛋白之间相互作用的特异性以及不同 WNT-FZD 对下游的信号级联下游尚未系统地得到解决。在这项研究中，我们通过使用生物层干涉测量法确定了各种WNT与FZD家族不同成员的结合亲和力，并表征了它们在细胞系统中的功能选择性。使用纯化的 WNT，我们发现不同的 FZD 富含半胱氨酸结构域更喜欢以快结合速率和慢解离速率结合不同的 WNT。在经过工程设计以过表达 FZD(2)、FZD(4) 或 FZD(5) 的基于 32D 细胞的系统中，我们发现 WNT-3A（但不是 WNT-4、-5A 或 -9B）通过 FZD(2/4/5) 激活 WNT-β-连环蛋白途径（通过 LRP6 磷酸化和 β-连环蛋白稳定化来测量）。令人惊讶的是，不同的 WNT-FZD 对对 DVL2 和 DVL3 的磷酸化表现出不同的影响，揭示了 32D 细胞中不同 WNT-FZD 对的 DVL 同工型选择性，这是以前未被认识到的。总之，我们提出了 WNT-FZD 富含半胱氨酸结构域相互作用的广泛图谱，并通过在表达单个 FZD 亚型的独特细胞系统中分析 WNT-FZD 对功能进行补充。差异性 WNT-FZD 结合和选择性功能读数表明，内源性 WNT 配体的进化具有对不同下游信号通路的内在自然偏好，这种现象在 FZD 靶向药物的设计中可能非常重要。
The seven-transmembrane-spanning receptors of the FZD1-10 class are bound and activated by the WNT family of lipoglycoproteins, thereby inducing a complex network of signaling pathways. However, the specificity of the interaction between mammalian WNT and FZD proteins and the subsequent signaling cascade downstream of the different WNT-FZD pairs have not been systematically addressed to date. In this study, we determined the binding affinities of various WNTs for different members of the FZD family by using bio-layer interferometry and characterized their functional selectivity in a cell system. Using purified WNTs, we show that different FZD cysteine-rich domains prefer to bind to distinct WNTs with fast on-rates and slow off-rates. In a 32D cell-based system engineered to overexpress FZD(2), FZD(4), or FZD(5), we found that WNT-3A (but not WNT-4, -5A, or -9B) activated the WNT-beta-catenin pathway through FZD(2/4/5) as measured by phosphorylation of LRP6 and beta-catenin stabilization. Surprisingly, different WNT-FZD pairs showed differential effects on phosphorylation of DVL2 and DVL3, revealing a previously unappreciated DVL isoform selectivity by different WNT-FZD pairs in 32D cells. In summary, we present extensive mapping of WNT-FZD cysteine-rich domain interactions complemented by analysis of WNT-FZD pair functionality in a unique cell system expressing individual FZD isoforms. Differential WNT-FZD binding and selective functional readouts suggest that endogenous WNT ligands evolved with an intrinsic natural bias toward different downstream signaling pathways, a phenomenon that could be of great importance in the design of FZD-targeting drugs.