Investigating reverse signaling by FGFs using an animal model system

项目摘要 大多数成纤维细胞生长因子(成纤维细胞生长因子)蛋白是分泌蛋白，作为同源受体的配体。 (FGFRs)在其他细胞上表达。然而，我们假设FGFs有额外的功能。这个 这里提出的实验将研究FGFs在支持配体反应中的非正则作用- 表达细胞，以确定FGFs是否也“反向信号”。反向信令已经链接到其他几个 信号转导途径包括eaffin、symaphorin和TNFa，但以前没有对FGFs进行过演示。我们 在果蝇中研究成纤维细胞生长因子信号，我们争辩说，一个模型系统有望为成纤维细胞生长因子提供新的见解 发信号。果蝇的成纤维细胞生长因子信号系统比较简单，只有3个受体-配体 与脊椎动物的100多种组合相比，这些组合具有功能性。然而，即使在更简单的情况下 在果蝇系统中，目前尚不清楚个别配体是否具有不同的活性。要提供 果蝇成纤维细胞生长因子两种蛋白Pyramus和Thisbe的洞察力及初步结构功能分析 都进行了测试。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 具体地说，包括这一配体起到“反向信号”的作用。果蝇是一个很好的模型系统 分析FGFs的反向信号是否存在，并确定它是否在 支持正常的胚胎发育。为了提供洞察力，我们提议的研究有三个具体目标。在……里面 目的1、对成纤维细胞生长因子蛋白的结构和功能进行分析，并对其定位进行研究。 以及胚胎中成纤维细胞生长因子蛋白结构域的功能，以深入了解N-和C-蛋白的不同活性 Termini分别位于胞外间隙和胞内间隙。在目标2中，建议进行实验以识别 FGFs在体内逆转信号的作用机制。我们将研究成纤维细胞生长因子相互作用的蛋白质 使用质谱学来洞察PYR细胞内C-末端结构域的功能。在《目标3》中， 实验是为了检验无情的FGFR和/或诱饵受体FGFRL1的特定假设 充当PYR的“配体”，支持其反向信号。果蝇成纤维细胞生长因子信号在 相反，这表明一些脊椎动物的FGFs也表现出这种活动。更好地理解完整的 成纤维细胞生长因子蛋白的功能能力有可能为先天性疾病的基因治疗提供线索 与异常的成纤维细胞生长因子信号有关的疾病，作为靶向特定成纤维细胞生长因子相关活动的候选药物 可能会有更少的副作用。AS成纤维细胞生长因子信号在发育过程中起着重要而普遍的作用 在包括人类在内的所有后生动物中，从这项提议中获得了对成纤维细胞生长因子活性调节的见解 工作可能会产生深远的影响。

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.