Exploring the structure-function relationship of membrane-pore-forming FGF2 oligomers - a single molecule approach

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Fibroblast Growth Factor 2 (FGF2) is a cell survival factor mediating tumor-induced angiogenesis. However, as opposed to the majority of extracellularly localized proteins that travel along the ER/Golgi dependent secretory pathway, FGF2 lacks a signal peptide and was shown to be secreted from cells by direct protein translocation across the plasma membrane. Several key aspects of the molecular mechanism behind this unconventional secretory pathway as well as the molecular components involved have been revealed. FGF2 secretion from cells is characterized by (i) sequential interactions of FGF2 with ATP1A1, Tec kinase and PI(4,5)P2 at the inner leaflet of the plasma membrane, (ii) Tec kinase mediated tyrosine phosphorylation of FGF2, (iii) PI(4,5)P2-dependent formation and membrane insertion of FGF2 oligomers and (iv) disassembly of oligomers and extracellular trapping of FGF2 mediated by cell surface heparan sulfate proteoglycans. Several lines of evidence suggest that PI(4,5)P2-induced oligomerization of FGF2 triggers the formation of lipidic membrane pores with a toroidal architecture. As part of this arrangement, the headgroups of PI(4,5)P2 contribute a hydrophilic surface in the periphery physically interacting with the FGF2 oligomer that is accommodated in the center of the lipidic membrane pore. Based upon the addition of FGF2 units at the cytoplasmic leaflet and removal of FGF2 units at the outer leaflet, an assembly/disassembly mechanism has been proposed to mediate directional transport of FGF2 across the plasma membrane. The latter process is driven by cell surface heparan sulfates that outcompete PI(4,5)P2 for the interaction with FGF2 at the outer leaflet resulting in accumulation of FGF2 on cell surfaces. In a previous funding round of the German-Czech Cooperation Programme, we obtained the first insights into the structure-function relationship of membrane-inserted FGF2 oligomers analyzed at the single molecule level. Here, we build upon this work aiming at (i) the determination of the precise oligomeric state of individual FGF2 translocation intermediates, (ii) the analysis of a potential role of ATP1A1 in initiating oligomerization of FGF2 and (iii) studying the role of heparan sulfates in facilitating the formation of FGF2 translocation intermediates. Throughout this study, we will use state-of-the art single molecule techniques and expect to gain substantial new insight into the molecular mechanism of FGF2 membrane translocation at an unprecedented level of detail. Therefore, our studies will make a significant contribution to our understanding of the molecular mechanism by which tumor cells secrete FGF2 in an ER/Golgi-independent manner.