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

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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.