Understanding Wnt signaling through Ror and Ryk family receptor tyrosine kinases

描述（由申请人提供）：本提案的目的是了解两种在其胞外区具有Wnt结合结构域的受体酪氨酸激酶（RTK）如何介导和/或促进Wnt信号传导。Wnt是由大约330个氨基酸组成的分泌型配体，在胚胎发育中起关键作用，并控制成年人的一系列过程，从器官再生到干细胞维持，从神经回路生成到性别决定。它们通过调节许多细胞过程来实现这一点，包括细胞增殖、迁移、极性、凋亡、分化和存活。不出所料， 因此，现在已知Wnt信号传导中的异常与许多疾病有关，包括骨疾病、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（人类的Ryk，果蝇的Derailed，Derailed-2和Doughnut）和Ror家族（人类的Ror 1和Ror 2，果蝇的DRor和DNrk）。两者都是受体酪氨酸激酶（RTK）家族-通常不与Wnt信号传导相关的受体类别，但已在其他信号传导系统中用激酶抑制剂和抗体治疗剂成功靶向治疗的类别。Ryk和Ror家族RTK的胞外区含有通常与结合Wnt配体相关的结构域。Ryks含有Wnt抑制因子-1（WIF）结构域，Rors含有细胞外富含半胱氨酸的结构域（CRD），类似于Frizzled受体中的Wnt结合CRD。 我们建议在这里调查Ryk和Ror家族RTK的信号转导机制，使用一系列的方法，从在非洲爪蟾胚胎和细胞研究的体内测定的生化，生物物理和结构的方法。我们的目标是确定Ryk和Ror受体在其信号传导模式上是否类似于其他众所周知的RTK，或者作为Wnt与Frizzleds或其他分子的共受体发挥作用。在我们的具体目标中，我们将解决以下问题：1。Ryk家族成员如何识别它们的Wnt配体，Ryk在对细胞外配体结合的反应中与其他RTK相似吗？2i. Ror家族成员在对配体结合的反应中是否类似于其他RTK，Wnt通过其富含半胱氨酸的结构域（CRD）的结合是否类似于Frizzleds？2二. Ryks和Rors异源二聚化吗？Wnt能否同时结合WIF结构域和CRD以驱动共受体异源二聚化？这些研究将为这些新型Wnt受体如何发出信号提供重要的基础性新见解，这对于理解Wnt信号传导特异性和梳理其多重作用至关重要。此外，我们的研究结果应该为治疗抑制开辟潜在的新途径-因为这些Wnt结合RTK在疾病中的作用越来越清楚。

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.