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Abstract

Fibroblast growth factor 2 (FGF2) is a potent mitogen involved in many physiological processes such as cell proliferation, differentiation as well as angiogenesis and, therefore, has a prominent function in tumor growth. FGF2 is secreted via an unconventional pathway using a route that is independent from the endoplasmic reticulum and the Golgi apparatus. Secretion of FGF2 occurs by direct translocation across the plasma membrane in a folded state, which is initiated by binding to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) at the inner leaflet of the plasma membrane. In the extracellular space, membrane proximal heparan sulfate proteoglycans (HSPGs) are required to complete translocation as well as to store and protect FGF2 on cell surfaces. The interaction with PI(4,5)P2 was shown to facilitate oligomerization of FGF2 leading to the formation of a lipidic membrane pore that has been interpreted as an intermediate structure in FGF2 membrane translocation. In addition, Tec kinase was discovered as a factor regulating FGF2 secretion by phosphorylating tyrosine 82 in FGF2. Recent evidence suggests that phosphorylation stabilizes the formation of FGF2 oligomers, which favors the arrangement of a transient hydrophilic pore constituted by FGF2 molecules in the plasma membrane. Based on a genome-wide ribonucleic acid interference (RNAi) screen this study aimed for the identification of further proteinaceous components that regulate or facilitate FGF2 secretion. Pharmacological studies previously implicated the sodium potassium pump (Na+/K+-ATPase) as a potential factor involved in FGF2 secretion, which indeed was a major hit in the genome-wide RNAi screen described in this thesis. Therefore, the main goal of this study was to validate these findings and to obtain mechanistic insight into the role of the Na+/K+-ATPase in the overall process of FGF2 secretion. Since the Na+/K+-ATPase is formed of a heterodimeric complex that consists of the α- and β-subunit, cell-based secretion experiments identified the α1-subunit (ATP1A1) as the component involved in FGF2 secretion. Furthermore, a direct interaction between the cytoplasmic domains of ATP1A1 with FGF2 could be demonstrated. In addition, both proteins were shown to co-localize at the plasma membrane in HeLa cells. Therefore, it is highly likely that a direct interaction of FGF2 and ATP1A1 occurs at the cytoplasmic leaflet facilitating export of FGF2 from cells. Based on an alpha screening setup analyzing a large library of small molecules, compounds were identified that block the interaction between FGF2 and ATP1A1. Therefore, this study provides new tools for future investigations on the mechanism of FGF2 secretion as well as lead compounds with a high potential for the development of novel anti-angiogenic drugs.