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（FGF）蛋白是分泌蛋白，其作为同源受体的配体 （FGFRs）在其他细胞上表达。然而，我们假设FGF具有额外的功能。的 这里提出的实验将研究FGF在支持配体反应中的非经典作用， 表达细胞，以确定FGF是否也“反向信号”。反向信号已经与其他几个 信号通路，包括肝配蛋白，脑信号蛋白和TNF α，但以前没有证明的FGF。我们 在果蝇中研究FGF信号，我们认为这一模型系统有望为FGF提供新的见解 信号果蝇的FGF信号系统不太复杂，只有3个受体-配体 与脊椎动物中超过100种组合相比，然而，即使在更简单的 果蝇系统中，仍然不清楚是否个别配体具有不同的活动。提供 果蝇FGF蛋白Pyramus（Pyr）和Thisbe（Ths）结构与功能初步分析 进行了。我们发现蛋白质拓扑结构的差异表明这些蛋白质具有不同的活性 具体地包括一个配体起到“反向信号”的作用。果蝇是一个极好的模型系统 以分析FGF的反向信号传导是否存在，并且还确定其在以下方面是否具有功能性作用： 支持胚胎正常发育。为了提供洞察力，我们提出的研究有三个具体目标。在 目的1、对FGF蛋白进行结构-功能分析，并对定位进行研究 和胚胎内FGF蛋白结构域的功能，以提供对N-和C- 末端分别位于细胞外和细胞内空间。在目标2中，提出实验来识别 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 使用质谱分析法来提供对Pyr细胞内C-末端结构域的功能的深入了解。在目标3中， 提出实验来检验无情FGFR和/或诱饵受体FGFRL 1 作为Pyr的“配体”，以支持其反向信号传导。果蝇FGF信号在 相反，这表明一些脊椎动物FGF也表现出这种活性。更好地了解全面 FGF蛋白功能的能力有可能为先天性心脏病的基因治疗提供见解。 作为靶向特定FGF相关活性的候选药物， 副作用会更少由于FGF信号在发育过程中起着重要和普遍的作用， 所有后生动物，包括人类，深入了解FGF活性的调节，从这个建议， 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.