How Wnt ligands achieve specificity Wnt signaling is essential for development, tissue homeostasis, and disease. The 19 members of the Wnt family interact promiscuously with the 10 Frizzled receptors, raising the question of how ligand-specific discrimination is achieved in a biological context. Eubelen et al. used experiments in zebrafish to show that cells are equipped with decoding modules that bind Wnt with high specificity and trigger signal amplification via their recruitment into higher-order Frizzled signalosomes (see the Perspective by Kim and Goentoro). Thus, distinct Wnt ligand-receptor pairs can be targeted specifically for therapeutic purposes. Science, this issue p. eaat1178; see also p. 643 The Wnt decoding capacities of vertebrate cells and the functional diversification of Wnt family members are elucidated. INTRODUCTION Wnt signaling is an ancient signaling pathway that has accompanied the emergence of metazoans and is key to many developmental, physiological, and disease processes. Similar to other signaling pathways, gene families for both Wnt ligand and its corresponding Frizzled receptor have undergone extensive expansion during metazoan evolution. Vertebrate genomes harbor 19 closely related Wnt genes as well as 10 Frizzled genes. Gene duplication is typically considered a major driving force in the evolution of new biological functions through neo- or subfunctionalization of emerging paralogs. How this functional diversification of Wnt ligands is structurally and molecularly organized, however, remains poorly understood. The Wnt/Frizzled molecular interaction is mediated by residues conserved across both families. This promiscuous interaction is incompatible with monospecific recognition and, accordingly, when tested in pair-wise combinations, multiple Wnt ligands compete for binding to various Frizzled receptors. RATIONALE These observations raise the questions of how Wnt ligands achieve functional diversification and how cells interpret the intermingled expression patterns of simultaneous and sometimes conflicting Wnt signals. In some biological settings, cells may integrate all signaling inputs nondiscriminately and trigger appropriate responses by considering their total net balance. However, other biological processes exhibit strict Wnt ligand selectivity, despite complex Wnt/Frizzled expression landscapes. A prototypical example is provided by the exclusive control of mammalian forebrain and ventral spinal cord angiogenesis by Wnt7a and Wnt7b. Within this neurovascular unit, in order to respond to neural progenitor–derived Wnt7 by activating Wnt/β-catenin signaling, cerebral endothelial cells must express a membrane protein complex consisting of the adhesion G protein–coupled receptor (GPCR) Gpr124 and the glycosylphosphatidylinositol-anchored glycoprotein Reck. This Gpr124/Reck complex was recently reported to promote Wnt7-specific responses. RESULTS Using a combination of biophysical approaches and ligand-binding assays in genetically engineered cells, we demonstrate that ligand selectivity is conferred by Reck, which mediates Wnt7-specific binding in a Frizzled-independent manner. Reck orchestrates Wnt ligand discrimination by engaging the structurally disordered and highly divergent linker domain of Wnt7. The presence of Gpr124 is required to deliver Reck-bound Wnt7 to Frizzled by assembling higher-order Reck/Gpr124/Frizzled/Lrp5/6 complexes. This Gpr124 tethering function does not rely on its GPCR structure but instead on its combined capacity to interact with Reck extracellularly and recruit the Dishevelled scaffolding protein intracellularly. By bridging Gpr124 and Frizzled, Dishevelled recruits Wnt7, via its association with Reck, into dynamic Wnt/Frizzled signalosomes, resulting in increased local concentrations of ligand available for Frizzled signaling. CONCLUSION Our data reveal that cells are equipped with “Wnt-decoding modules” that distinguish between Wnt ligands that are otherwise very similar. They also reveal a critical role for the linker domain in Wnt ligand evolution and functional diversification. These mechanistic insights into the Wnt decoding capacities of vertebrate cells predict that additional Wnt decoding modules exist, enabling fine-tuning of cellular behaviors in response to other Wnt or Frizzled family members. These modules expand the diversity of proximal events in Wnt signaling, opening new therapeutic opportunities for conditions in which Wnt stimulation or inhibition are desirable at the membrane level. In particular, the mechanisms uncovered here provide an opportunity for the targeted treatment of human central nervous system neurovascular disorders. Task sharing for orchestrated Wnt7-specific cellular responses. (Top) Gpr124 and Reck cooperatively alter the cell’s perception of its Wnt microenvironment by selectively potentiating Wnt7 signals (cyan-tinted dots). (Bottom) Reck decodes Wnt ligands by establishing monospecific contacts with the highly divergent Wnt7 linker domain. Gpr124 links Reck-bound Wnt7 to Dishevelled. Dishevelled polymers by interacting simultaneously with Gpr124 and Fz assemble Wnt7-enriched signalosomes that trigger signaling through Fz receptors and Lrp5/6 co-receptors. Wnt signaling is key to many developmental, physiological, and disease processes in which cells seem able to discriminate between multiple Wnt ligands. This selective Wnt recognition or “decoding” capacity has remained enigmatic because Wnt/Frizzled interactions are largely incompatible with monospecific recognition. Gpr124 and Reck enable brain endothelial cells to selectively respond to Wnt7. We show that Reck binds with low micromolar affinity to the intrinsically disordered linker region of Wnt7. Availability of Reck-bound Wnt7 for Frizzled signaling relies on the interaction between Gpr124 and Dishevelled. Through polymerization, Dishevelled recruits Gpr124 and the associated Reck-bound Wnt7 into dynamic Wnt/Frizzled/Lrp5/6 signalosomes, resulting in increased local concentrations of Wnt7 available for Frizzled signaling. This work provides mechanistic insights into the Wnt decoding capacities of vertebrate cells and unravels structural determinants of the functional diversification of Wnt family members.

中文翻译：

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Wnt 配体特异性信号传导的分子机制

Wnt 配体如何实现特异性 Wnt 信号传导对于发育、组织稳态和疾病至关重要。Wnt 家族的 19 个成员与 10 个卷曲受体混杂地相互作用，提出了如何在生物学背景下实现配体特异性区分的问题。尤贝伦等人。在斑马鱼中使用的实验表明，细胞配备了解码模块，这些模块可以高度特异性地结合 Wnt，并通过将它们募集到更高阶的卷曲信号小体中来触发信号放大（参见 Kim 和 Goentoro 的观点）。因此，不同的 Wnt 配体-受体对可以专门针对治疗目的。科学，这个问题 p。eaat1178; 另见第。643 阐明了脊椎动物细胞的 Wnt 解码能力和 Wnt 家族成员的功能多样化。引言 Wnt 信号是一种古老的信号通路，伴随着后生动物的出现，是许多发育、生理和疾病过程的关键。与其他信号通路类似，Wnt 配体及其相应卷曲受体的基因家族在后生动物进化过程中经历了广泛的扩展。脊椎动物基因组包含 19 个密切相关的 Wnt 基因以及 10 个 Frizzled 基因。基因复制通常被认为是通过新兴旁系同源物的新功能或亚功能化来进化新生物功能的主要驱动力。然而，Wnt 配体的这种功能多样化是如何在结构和分子上组织的，仍然知之甚少。Wnt/Frizzled 分子相互作用由两个家族中保守的残基介导。这种混杂的相互作用与单特异性识别不相容，因此，当以成对组合进行测试时，多个 Wnt 配体竞争结合各种卷曲受体。基本原理这些观察提出了以下问题：Wnt 配体如何实现功能多样化以及细胞如何解释同时且有时相互冲突的 Wnt 信号的混合表达模式。在某些生物环境中，细胞可以无差别地整合所有信号输入，并通过考虑它们的总净平衡来触发适当的反应。然而，其他生物过程表现出严格的 Wnt 配体选择性，尽管 Wnt/Frizzled 表达环境复杂。Wnt7a 和 Wnt7b 对哺乳动物前脑和腹侧脊髓血管生成的独家控制提供了一个典型的例子。在这个神经血管单元中，为了通过激活 Wnt/β-catenin 信号来响应神经祖细胞衍生的 Wnt7，脑内皮细胞必须表达一种膜蛋白复合物，该复合物由粘附 G 蛋白偶联受体 (GPCR) Gpr124 和糖基磷脂酰肌醇-锚定糖蛋白 Reck。最近有报道称这种 Gpr124/Reck 复合物可促进 Wnt7 特异性反应。结果 在基因工程细胞中结合使用生物物理方法和配体结合测定，我们证明了配体选择性是由 Reck 赋予的，它以一种独立于卷曲蛋白的方式介导 Wnt7 特异性结合。Reck 通过结合 Wnt7 的结构无序和高度发散的接头域来协调 Wnt 配体区分。通过组装更高阶的 Reck/Gpr124/Frizzled/Lrp5/6 复合物，需要 Gpr124 的存在才能将 Reck-bound Wnt7 传递给 Frizzled。这种 Gpr124 束缚功能不依赖于其 GPCR 结构，而是依赖于其在细胞外与 Reck 相互作用并在细胞内募集蓬乱的支架蛋白的综合能力。通过桥接 Gpr124 和 Frizzled，Disheveled 通过其与 Reck 的关联将 Wnt7 招募到动态 Wnt/Frizzled 信号体中，从而导致可用于 Frizzled 信号传导的配体的局部浓度增加。结论 我们的数据显示，细胞配备了“Wnt 解码模块”，可以区分非常相似的 Wnt 配体。它们还揭示了接头域在 Wnt 配体进化和功能多样化中的关键作用。这些对脊椎动物细胞 Wnt 解码能力的机械见解预测存在额外的 Wnt 解码模块，从而能够微调细胞行为以响应其他 Wnt 或 Frizzled 家族成员。这些模块扩展了 Wnt 信号传导中近端事件的多样性，为需要在膜水平上进行 Wnt 刺激或抑制的病症开辟了新的治疗机会。特别是，这里发现的机制为靶向治疗人类中枢神经系统神经血管疾病提供了机会。用于精心设计的 Wnt7 特定细胞响应的任务共享。（上）Gpr124 和 Reck 通过选择性地增强 Wnt7 信号（青色点）来共同改变细胞对其 Wnt 微环境的感知。（底部）Reck 通过与高度不同的 Wnt7 接头域建立单特异性接触来解码 Wnt 配体。Gpr124 将 Reck-bound Wnt7 链接到 Dishevelled。通过与 Gpr124 和 Fz 同时相互作用的蓬乱聚合物组装富含 Wnt7 的信号小体，通过 Fz 受体和 Lrp5/6 共受体触发信号传导。Wnt 信号传导是许多发育、生理和疾病过程的关键，在这些过程中，细胞似乎能够区分多个 Wnt 配体。这种选择性 Wnt 识别或“解码”能力仍然是个谜，因为 Wnt/Frizzled 相互作用在很大程度上与单特异性识别不相容。Gpr124 和 Reck 使脑内皮细胞能够选择性地响应 Wnt7。我们表明 Reck 以低微摩尔亲和力与 Wnt7 的固有无序链接器区域结合。用于 Frizzled 信号的 Reck-bound Wnt7 的可用性依赖于 Gpr124 和 Dishevelled 之间的相互作用。通过聚合，Disheveled 将 Gpr124 和相关的 Reck-bound Wnt7 募集到动态 Wnt/Frizzled/Lrp5/6 信号小体中，导致可用于 Frizzled 信号传导的 Wnt7 局部浓度增加。这项工作提供了对脊椎动物细胞 Wnt 解码能力的机械见解，并揭示了 Wnt 家族成员功能多样化的结构决定因素。