Mechanisms of mammalian Wnt5a-Ror signaling

项目摘要/摘要 Wnt5a-Ror信号通路是胚胎组织形态发生所必需的。Wnt5a-RoR中断 信号会导致出生缺陷，如Robinow综合征和B型短指畸形，并且经常 与癌症转移和炎症性疾病有关。Wnt5a蛋白是棕榈油酰化的 保守的丝氨酸，这是所有Wnt配体共有的独特特征，需要GPCR样蛋白Wnless 将其脂质修饰的成熟形式从内质网运输到质膜，以便 分泌物。与其机制被相对较好地描述的规范WNTT相比，WNT5a信号 非规范地通过ROR受体酪氨酸激酶家族(ROR1/2)，该家族独立于 转录因子β-连环蛋白。尽管Wnt5a在生理和病理上具有重要意义，但其机制-- RoR信号仍然知之甚少。主要悬而未决的问题包括WNT5a是如何分泌的，如何 Wnt5A激活Rors，以及激活的Rors如何将Wnt5a信号转导到细胞质。在前期工作中，我们 确定了Wnt5a在与Wnless的复合体中的冷冻EM结构，揭示了Wnt5a的一个意想不到的特征 对于它的非正则函数具有重要的意义。我们发现纯化的胞外区(ECD) ROR2促进Wnt5a的分泌，以及与果蝇ROR2 ECD结合的最新晶体结构 棕榈油酸，表明ROR2直接识别WNT5a脂类。此外，我们还开发了一种 过氧化物酶APEX2催化的邻近标记方法，并通过定量多重蛋白质组学，我们 证明了第一次，下游效应蛋白的募集混乱(DVL)到 ROR受体完全依赖于Wnt5a的刺激。基于这些结果，我们建议将 生物化学、结构、细胞生物学和蛋白质组学研究以实现以下目标：a)表征 Wnless如何运输Wnt5a，以及Wnless如何在Wnt5a分泌后被回收；B)确定Wnt5a如何 结合并激活ROR受体；以及C)阐明激活的ROR受体如何触发下游 细胞质信号事件。这些研究将产生重大影响，原因如下：1)它们将 促进我们对Wnt5a-Ror信号的机制的理解；2)它们将有助于解释 由Wnt5a-Ror信号异常引起的人类疾病；3)它们将通过以下方式促进治疗发现 为治疗Wnt5a相关癌症提供新的靶点和策略；以及4)APEX2催化的邻近 我们开发的标记和蛋白质组学方法将在研究信号转导方面有广泛的应用 Wnt5a-Ror信号以外的通路。

PROJECT SUMMARY/ABSTRACT The Wnt5a-Ror signaling pathway is essential for embryonic tissue morphogenesis. Disruption of Wnt5a-Ror signaling results in birth defects such as Robinow syndrome and Brachydactyly Type B, and is frequently implicated in cancer metastasis and inflammatory disorders. The Wnt5a protein is palmitoleoylated at a conserved serine, a unique feature shared by all the Wnt ligands, which require the GPCR-like protein Wntless to transport their lipid-modified mature forms from the endoplasmic reticulum to the plasma membrane for secretion. In contrast to the canonical Wnts whose mechanisms are relatively well characterized, Wnt5a signals noncanonically through the ROR family of receptor tyrosine kinases (ROR1/2), which is independent of the transcriptional factor β-catenin. Despite its physiological and pathological importance, the mechanisms of Wnt5a- Ror signaling remain poorly understood. The major unanswered questions include how Wnt5a is secreted, how Wnt5a activates RORs and how active RORs transduce Wnt5a signals to the cytoplasm. In preliminary work, we determined a Cryo-EM structure of Wnt5a in complex with Wntless that reveals an unanticipated feature of Wnt5a with important implications for its non-canonical function. We showed that purified extracellular domain (ECD) of ROR2 promotes Wnt5a secretion, together with the recent crystal structure of Drosophila ROR2 ECD bound with palmitoleic acid, suggesting that ROR2 directly recognizes the Wnt5a lipid. In addition, we developed a peroxidase APEX2-catalyzed proximity labeling approach, and by quantitative multiplexed proteomics, we demonstrated that for the first time, the recruitment of the downstream effector protein Dishevelled (DVL) to the ROR receptors is exclusively dependent on Wnt5a stimulation. Based on these results, we propose to combine biochemistry, structural, cell biology and proteomic studies to accomplish the following aims: A) To characterize how Wntless transports Wnt5a, and how Wntless is recycled after Wnt5a secretion; B) To determine how Wnt5a binds and activates the ROR receptors; and C) To elucidate how active ROR receptors trigger the downstream cytoplasmic signaling events. These studies will have high impacts for the following reasons: 1) They will advance our mechanistic understanding of Wnt5a-Ror signaling; 2) They will help explain the pathogenesis of human diseases caused by abnormal Wnt5a-Ror signaling; 3) They will facilitate the therapeutic discoveries by providing novel targets and strategies to treat Wnt5a-associated cancers; and 4) The APEX2-catalyzed proximity labeling and proteomic approach that we developed will have broad applications to study signal transduction pathways beyond Wnt5a-Ror signaling.