Investigating reverse signaling by FGFs using an animal model system

使用动物模型系统研究 FGF 的反向信号传导

• 批准号: 10212438
• 负责人: Angelike Stathopoulos
• 金额: $ 20.81万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-10 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10212438
• 关键词: Animal Model; Animals; Biological Assay; Biological Models; C-terminal; Cell Adhesion; Cell Culture Techniques; Cell Nucleus; Cells; Complex; Data; Development; Disease; Drosophila genus; Drosophila inturned protein; Dyspnea; Embryo; Embryonic Development; Ephrins; Event; Exhibits; Extracellular Space; FGFRL1 gene; Fibroblast Growth Factor; Fibroblast Growth Factor Receptors; Genes; Growth Factor; Growth Factor Gene; Human; Individual; Intracellular Space; Length; Ligands; Link; MAP Kinase Gene; Mammals; Mass Spectrum Analysis; Mediating; Membrane; Membrane Proteins; Mitogen-Activated Protein Kinases; Pathway interactions; Play; Positioning Attribute; Property; Proteins; Receptor Protein-Tyrosine Kinases; Regulation; Role; Semaphorins; Signal Pathway; Signal Transduction; Sodium Channel; Specificity; Structure; System; Tertiary Protein Structure; Testing; Vertebrates; Work; cell motility; cofactor; drug candidate; experimental study; extracellular; gene therapy; in vivo; insight; novel; protein function; receptor; response; side effect; voltage

Project Summary Most fibroblast growth factor (FGF) proteins are secreted proteins that act as ligands for cognate receptors (FGFRs) expressed on other cells. However, we hypothesize that FGFs have additional functions. The experiments proposed here will investigate a noncanonical role for FGFs in supporting a response in the ligand- expressing cell, to determine if FGFs also “reverse signal”. Reverse signaling has been linked to several other signaling pathways including ephrin, semaphorin, and TNFa, but not previously demonstrated for FGFs. We study FGF signaling in Drosophila, a model system that we contend is poised to provide novel insights into FGF signaling. The Drosophila FGF signaling system of Drosophila is less complex, with only 3 receptor-ligand combinations functional compared to over 100 combinations in vertebrates. Nevertheless, even in the simpler Drosophila system, it has remained unclear whether individual ligands have different activities. To provide insight, preliminary structure-function analyses of two Drosophila FGF proteins Pyramus (Pyr) and Thisbe (Ths) were conducted. We uncovered differences in protein topology that suggest these proteins have distinct activities including, specifically, that one ligand functions to “signal in reverse”. Drosophila is an excellent model system to analyze whether reverse signaling by FGFs exists and also to determine whether it has a functional role in support of proper embryonic development. To provide insight, our proposed study has three specific aims. In Aim 1, a structure-function analysis of FGF proteins will be undertaken, in which we will investigate localization and function of FGF protein domains within embryos to provide insight into distinct activities of the N- and C- termini in the extracellular and intracellular spaces, respectively. In Aim 2, experiments are proposed to identify the mechanism of action used by FGFs to reverse signaling in vivo. We will investigate FGF-interacting proteins using mass spectrometry to provide insight into function of the Pyr intracellular C-terminal domain. In Aim 3, experiments are proposed to test the specific hypothesis that Heartless FGFR and/or decoy receptor FGFRL1 act as “ligands” for Pyr to support its reverse signaling. The demonstration that a Drosophila FGF signals in reverse would suggest that some vertebrate FGFs also exhibit this activity. A better understanding of the full capacity of FGF protein function has the potential to provide insight towards gene therapies for congenital diseases that relate to aberrant FGF signaling, as drug candidates that target particular FGF-associated activities will likely have fewer side effects. As FGF signaling plays an important and pervasive role during development of all metazoan animals including humans, insights gained into the regulation of FGF activity from this proposed work are likely to have far-reaching impact.

项目概要 大多数成纤维细胞生长因子 (FGF) 蛋白是分泌蛋白，充当同源受体的配体 (FGFR) 在其他细胞上表达。然而，我们假设 FGF 还有其他功能。这 这里提出的实验将研究 FGF 在支持配体反应中的非典型作用 表达细胞，以确定 FGF 是否也“反向信号”。反向信号已与其他几个相关联 信号通路包括肝配蛋白、信号蛋白和 TNFa，但之前未针对 FGF 进行证实。我们 研究果蝇中的 FGF 信号传导，我们认为该模型系统有望为 FGF 提供新的见解 发信号。果蝇的FGF信号系统不太复杂，只有3个受体-配体 与脊椎动物中 100 多种组合相比，这些组合具有功能性。尽管如此，即使在更简单的情况下 在果蝇系统中，单个配体是否具有不同的活性仍不清楚。提供 两种果蝇 FGF 蛋白 Pyramus (Pyr) 和 Thisbe (Ths) 的初步结构功能分析 进行了。我们发现了蛋白质拓扑结构的差异，表明这些蛋白质具有不同的活性 具体而言，包括一种配体发挥“反向信号”的功能。果蝇是一个优秀的模型系统 分析 FGF 的反向信号传导是否存在，并确定其是否在 支持适当的胚胎发育。为了提供见解，我们提出的研究有三个具体目标。在 目标 1，将进行 FGF 蛋白的结构功能分析，其中我们将研究定位 胚胎内 FGF 蛋白结构域的功能和功能，以深入了解 N- 和 C- 的不同活性 末端分别位于细胞外和细胞内空间。在目标 2 中，建议进行实验来确定 FGF 用于逆转体内信号传导的作用机制。我们将研究 FGF 相互作用蛋白 使用质谱分析深入了解 Pyr 胞内 C 末端结构域的功能。在目标 3 中， 提出实验来检验 Heartless FGFR 和/或诱饵受体 FGFRL1 的具体假设 充当 Pyr 的“配体”以支持其反向信号传导。果蝇 FGF 信号的证明 相反，表明一些脊椎动物 FGF 也表现出这种活性。更全面地了解 FGF 蛋白功能的能力有可能为先天性遗传病的基因治疗提供见解 与异常 FGF 信号传导相关的疾病，作为针对特定 FGF 相关活性的候选药物 可能会有更少的副作用。由于 FGF 信号在发育过程中发挥着重要而普遍的作用 在包括人类在内的所有后生动物中，从该提议中获得了对 FGF 活性调节的见解 工作可能会产生深远的影响。