Dissecting the Role of ATP1A1 in Unconventional Secretion of Fibroblast Growth Factor 2

多种机械上不同的途径调节哺乳动物细胞的蛋白质分泌。经典的分泌途径包括信号肽依赖的蛋白质转运到内质网的管腔，然后囊泡通过高尔基体转运到质膜。当分泌囊泡与质膜融合时，运往细胞外间隙的货物蛋白被发送到细胞表面。长期以来，信号肽的存在一直被认为是蛋白质退出细胞能力的必要条件。然而，许多胞外蛋白缺乏信号肽的例子已经被鉴定出来。这一过程被称为非常规蛋白质分泌，成纤维细胞生长因子2(FGF2)就是一个突出的例子。详细了解细胞分泌FGF2的机制对生物医学研究具有特别重要的意义。这是因为FGF2是肿瘤诱导血管生成的关键介质。此外，FGF2是肿瘤细胞的关键生存因子，通过自分泌-信号环路阻止细胞凋亡，使肿瘤细胞对抗癌药物产生耐药性。我们以前已经证明，FGF2是通过直接跨膜转位从肿瘤细胞中分泌出来的。这一过程依赖于PI(4，5)P2依赖的膜募集，PI(4，5)P2是一种富含在质膜内叶中的肌醇磷脂。膜上的FGF2寡聚并在质膜上形成脂质孔，这一过程受Tec激酶介导的FGF2酪氨酸磷酸化的调节。FGF2低聚体形成的膜孔形成动态结构，被认为允许FGF2单体的膜转位。这一过程依赖于细胞表面的硫酸乙酰肝素蛋白多糖将FGF2捕获到细胞表面，从而将FGF2定向运输到细胞外空间。在最近的一项研究中，我们已经鉴定并验证了FGF2分泌机制的一个新组件，即完整的膜蛋白ATP1A1。ATP1A1是一种质膜驻留蛋白，其胞浆结构域与FGF2形成直接接触。这种相互作用是细胞分泌FGF2所必需的。利用生化和结构方法以及基于细胞的实验，本研究方案旨在剖析ATP1A1作为调节肿瘤细胞分泌FGF2的机制的分子机制。

Multiple, mechanistically distinct pathways mediate protein secretion from mammalian cells. The classical secretory pathway involves signal peptide dependent protein translocation into the lumen of the endoplasmic reticulum followed by vesicular transport via the Golgi to the plasma membrane. Upon membrane fusion of secretory vesicles with the plasma membrane, cargo proteins destined for the extracelluar space are dispatched on cell surfaces. For long, the presence of a signal peptide has been considered obligatory for the ability of a protein to exit cells. However, many examples of extracellular proteins lacking signal peptides have been identified. This process has been termed unconventional protein secretion with Fibroblast Growth Factor 2 (FGF2) being a prominent example. A detailed understanding of the mechanism of FGF2 secretion from cells is of extraordinary relevance for biomedical research. This is because FGF2 is a key mediator of tumor-induced angiogenesis. In addition, FGF2 is a key survival factor of tumor cells preventing apoptosis by an autocrine secretion-signaling loop that confers resistance of tumor cells against anti-cancer drugs. We have previously demonstrated that FGF2 is secreted from tumor cells by direct translocation across plasma membranes. This process depends on PI(4,5)P2 dependent membrane recruitment, a phosphoinositide enriched in the inner leaflet of plasma membranes. Membrane recruited FGF2 oligomerizes and forms lipidic pores in the plasma membrane, a process that is regulated by Tec kinase mediated tyrosine phosphorylation of FGF2. Membrane pores formed by FGF2 oligomers form dynamic structures that are believed to allow for membrane translocation of FGF2 monomers. This process depends on cell surface heparan sulfate proteoglycans required to trap FGF2 on cell surfaces resulting in directional transport of FGF2 into the extracellular space. In a recent study, we have identified and validated a new component of the secretory machinery of FGF2, the integral membrane protein ATP1A1. ATP1A1 is a plasma membrane resident protein whose cytoplasmic domain forms a direct contact with FGF2. This interaction is required for FGF2 secretion from cells. Using biochemical and structural methods as well as cell-based experiments this research proposal aims at dissecting the molecular mechanism by which ATP1A1 acts as a component of the machinery mediating FGF2 secretion from tumor cells.