Understanding Wnt signaling through Ror and Ryk family receptor tyrosine kinases

描述(申请人提供)：这项建议的目标是了解两个受体酪氨酸激酶(RTK)如何在其胞外区与Wnt结合区域介导和/或贡献Wnt信号。WNTs是由大约330个氨基酸组成的分泌型配体，在胚胎发育中发挥关键作用，并控制着成人的一系列过程，从器官再生到干细胞维持，再到神经回路的产生，再到性别决定。它们通过调节许多细胞过程来做到这一点，包括细胞增殖、迁移、极性、凋亡、分化和生存。这并不出人意料， 因此，Wnt信号的异常现在被认为与许多疾病有关，包括骨骼疾病、癌症、糖尿病、神经发育障碍和几种先天性畸形。除了最著名的WnT受体(Frizzleds)外，还发现了另外两个WnT受体家族：Ryk家族(人类中的Ryk，果蝇中的Ryk，脱轨，脱轨-2和甜甜圈)和Ror家族(人类中的Ror1和Ror2，果蝇中的Dror和DNrk)。两者都是受体酪氨酸激酶(RTK)家族-一种受体类别，通常与Wnt信号无关，但这一类别已成功地在其他信号系统中作为治疗靶点，使用激酶抑制剂和抗体疗法。Ryk和Ror家族RTK的胞外区含有典型的与Wnt配体结合的结构域。RYKs含有Wnt抑制因子-1(WIF)结构域，而RORs含有一个胞外富含半胱氨酸的结构域(CRD)，类似于Frizzled型受体中Wnt结合的CRD。我们建议使用一系列的方法来研究Ryk和Ror家族RTK的信号机制，从非洲爪哇胚胎的体内测试和细胞研究到生化、生物物理和结构方法。我们的目标是确定Ryk和Ror受体是否在信号模式上与其他众所周知的RTK相似，或者作为Wnts与Frizzleds或其他分子的辅助受体发挥作用。在我们的具体目标中，我们将解决以下问题：1.Ryk家族成员如何识别他们的Wnt配体，以及Ryk与其他RTK一样对细胞外配体结合的反应吗？Ror家族成员对配体结合的反应是否与其他RTK相似，Wnt通过其富含半胱氨酸的结构域(CRD)结合是否类似Frizzleds？2ii。Ryks和Rors是否异二聚化，以及WNTs能否同时与WIF结构域和CRD结合来驱动共同受体的异二聚化？这些研究将为这些新类别的Wnt受体如何信号提供重要的基础新见解，这对于理解Wnt信号的特异性和梳理其多重作用至关重要。此外，随着这些与Wnt结合的RTK在疾病中的作用变得越来越清楚，我们的发现应该会为治疗抑制开辟潜在的新途径。

DESCRIPTION (provided by applicant): The goal of this proposal is to understand how two receptor tyrosine kinases (RTKs) with Wnt-binding domains in their extracellular regions mediate and/or contribute to Wnt signaling. Wnts are secreted ligands of approximately 330 amino acids that play key roles in embryonic development and control a spectrum of processes in the adult, ranging from organ regeneration to stem cell maintenance to neural circuit generation to sex determination. They do this by regulating many cellular processes including cell proliferation, migration, polarity, apoptosis, differentiation and survival. Not unexpectedly, therefore, aberrations in Wnt signaling are now known to be involved in many diseases, including bone diseases, cancers, diabetes, neurodevelopment disorders, and several congenital malformations. In addition to the best known Wnt receptors (the Frizzleds), two additional Wnt receptor families have been identified: the Ryk family (Ryk in humans, Derailed, Derailed-2 and Doughnut in Drosophila) and the Ror family (Ror1 and Ror2 in human, DRor and DNrk in Drosophila). Both are receptor tyrosine kinase (RTK) families - a receptor class not typically associated with Wnt signaling, but a class that has been successfully targeted therapeutically in other signaling systems with kinase inhibitors and antibody therapeutics. The extracellular regions of Ryk and Ror family RTKs contain domains typically associated with binding to Wnt ligands. Ryks contain a Wnt Inhibitory Factor-1 (WIF) domain, and Rors contain an extracellular cysteine-rich domain (CRD) that resembles the Wnt-binding CRDs in the Frizzled receptors. We propose here to investigate the signaling mechanisms of Ryk and Ror family RTKs using an array of approaches from in vivo assays in Xenopus embryos and cellular studies to biochemical, biophysical and structural approaches. Our goal is to determine whether Ryk and Ror receptors resemble other well known RTKs in their mode of signaling, or instead function as co-receptors for Wnts with Frizzleds or other molecules. In our Specific Aims, we will address the following questions: 1. how do Ryk family members recognize their Wnt ligands, and do Ryks resemble other RTKs in their response to extracellular ligand binding? 2i. Do Ror family members resemble other RTKs in responses to ligand binding, and does Wnt binding by their cysteine-rich domains (CRDs) resemble that of Frizzleds? 2ii. Do Ryks and Rors heterodimerize, and can Wnts bind simultaneously to both WIF domains and CRDs to drive co-receptor heterodimerization? These studies will provide important fundamental new insight into how these novel classes of Wnt receptors signal, which is crucial for understanding Wnt signaling specificity and teasing out its multiple roles. In addition, our findings should open potential new avenues for therapeutic inhibition - as the roles of these Wnt-binding RTKs in disease become increasingly clear.