Nanodomain organization of the FGF2 secretory machinery

Fibroblast growth factor 2 (FGF2) is a potent mitogen involved in angiogenesis and tumor cell survival. Although a secretory protein involved in autocrine and paracrine signaling, FGF2 does not contain a signal peptide and bypasses the classical ER-Golgi-dependent route of protein secretion. In fact, FGF2 is secreted unconventionally (type I UPS) via direct translocation across the plasma membrane by self-sustained pores. FGF2 is recruited to the plasma membrane via interaction with the ⍺1 subunit of the Na,K-ATPase. Thereafter FGF2 interacts with Tec kinase, which phosphorylates FGF2. FGF2 binds to the phosphoinositide PI(4,5)P2, which drives its dimerization and oligomerization into membrane-spanning complexes. FGF2 is then captured on the cell surface through binding to heparan sulfate proteoglycans (HSPGs). The GPI-anchored HSPG glypican-1 (GPC1) was identified in our laboratory via a genome-wide BioID screen. In the here presented thesis, GPC1 CRISPR-Cas9 knockout (KO) cell lines were shown to secrete significantly less FGF2 in cell surface biotinylation assays. GPC1 reintroduction and stable overexpression did not only restore secretion to wild-type levels but even further increased secretion. FGF2 endocytosis was not affected by GPC1 KO or overexpression. As I could correlate GPC1 levels to FGF2 secretion in TIRF microscopy, GPC1 can be considered a rate-limiting factor for FGF2 secretion. GPC5, another endogenously expressed glypican in HeLa cells, did not reduce FGF2 secretion when knocked out. Also, GPC5 could not compensate for loss of GPC1 in the here demonstrated data. GPC6 knockout in U2OS cells only led to a modest decrease in FGF2 secretion, yet further decreased FGF2 secretion in GPC1 KO cells in my experiments. Organization of FGF2 and the components needed for secretion into nanodomains would facilitate fast FGF2 secretion from cells. FGF2, ⍺1 and GPC1 were indeed shown to be in proximity to each other in the here conducted proximity ligation assays. Also, I found all three components in detergent resistant membrane (DRM) fractions, whereby GPC1 was the determining factor for FGF2 DRM localization. Super resolution STED experiments showed a homogenous distribution of ⍺1 and GPC1 throughout the membrane. Interestingly, I found FGF2 and PI(4,5)P2 in areas enriched in cholesterol detected via EGFP-Gram1b G187L transfection, which is supported by recent findings demonstrating that cholesterol promotes FGF2 secretion. Caveolins, cholesterol-binding proteins that assemble into detergent resistant membrane fractions, were analyzed regarding their effect on FGF2 secretion. In my experiments caveolin-1 (Cav1) and -2 (Cav2) affected FGF2 secretion in HeLa S3 and U2OS. In HeLa cells, caveolin-2 KO cells showed reduced FGF2 secretion in biotinylation experiments. Intriguingly, caveolin-1 failed to localize into DRM fractions in this context and also ⍺1 and FGF2 levels were reduced in liquid ordered detergent resistant fractions. Cav2 KO in U2OS on the other hand, did not impact FGF2 secretion in my hands, although TIRF data demonstrated Cav1 to be involved in FGF2 secretion. Caveolin function in FGF2 nanodomain organization remained unresolved. Pulldown experiments I conducted using trifunctional lipid probes demonstrated an interaction between PI(3,4,5)P3 and the ⍺1 subunit of the Na,K-ATPase, further supporting the hypothesis of specialized membrane domains involved in FGF2 membrane translocation into the extracellular space