A Direct Role for Phosphatidylinositol‐4,5‐bisphosphate in Unconventional Secretion of Fibroblast Growth Factor 2

Abstract

Fibroblast growth factor 2 (FGF-2) is a mitogen that is exported from cells by an endoplasmic reticulum/Golgiindependent secretory pathway. Recent findings have shown that FGF-2 export occurs by direct translocation from the cytoplasm across the plasma membrane into the extracellular space. Here, we report that FGF-2 contains a binding site for phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ], the principal phosphoinositide species associated with plasma membranes. Intriguingly, in the context of a lipid bilayer, the interaction between FGF-2 and PI(4,5)P 2 is shown to depend on a lipid background that resembles plasma membranes. We show that the interaction with PI(4,5)P 2 is critically important for FGF-2 secretion as experimental conditions reducing cellular levels of PI(4,5)P 2 resulted in a substantial drop in FGF-2 export efficiency. Likewise, we have identified FGF-2 variant forms deficient for binding to PI(4,5)P 2 that were found to be severely impaired with regard to export efficiency. These data show that a transient interaction with PI(4,5)P 2 associated with the inner leaflet of plasma membranes represents the initial step of the unconventional secretory pathway of FGF-2.

Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2)-dependent Oligomerization of Fibroblast Growth Factor 2 (FGF2) Triggers the Formation of a Lipidic Membrane Pore Implicated in Unconventional Secretion

“…Thus, the reconstitution experiments of this study suggest a novel self-sustained mechanism of membrane translocation with a lipidic membrane pore being a transient translocation intermediate. These findings explain the behavior of FGF2 in living cells where secretion depends on PI(4,5)P 2 -mediated membrane recruitment (21,22,28), tyrosine phosphorylation (25,29), and proper folding of FGF2 (26,27).…”

“…Biochemical Analysis of Protein Binding to Liposomes-Liposomes with a plasma membrane-like lipid composition containing either 2 mol % PI(4,5)P 2 or 2 mol % of a Ni-NTA lipid were generated as described earlier (22,32). Membranes were blocked with 3% (w/v) fatty acid-free BSA for 1 h at 25°C and washed with buffer B (25 mM HEPES, 150 mM KCl, pH 7.4).…”

“…At the outer leaflet, membrane-proximal heparan sulfate proteoglycans are involved in a late step of FGF2 secretion resulting in its exposure on cell surfaces (23,24). These sequential interactions are essential, as experimental conditions that prevent the ability of FGF2 to interact with either PI(4,5)P 2 or heparan sulfates cause inhibition of FGF2 secretion (22,24,25). Both of these interactions require FGF2 to be folded properly (26), and FGF2 also remains folded during membrane translocation (26,27).…”

“…FGF2 was shown to translocate across plasma membranes (5,6), a process that is initiated by its recruitment at the inner leaflet mediated by the phosphoinositide PI(4,5)P 2 3 (21,22). At the outer leaflet, membrane-proximal heparan sulfate proteoglycans are involved in a late step of FGF2 secretion resulting in its exposure on cell surfaces (23,24).…”

6-Hydroxydopamine induces secretion of PARK7/DJ-1 via autophagy-based unconventional secretory pathway

“…There are a smaller number of proteins that lack the ER-targeting signal sequence which are secreted from the cell by what is referred to as the unconventional secretion mechanism [1–3]. Unconventionally secreted proteins include DBI (diazepam binding inhibitor, acyl-CoA binding protein), IL1B (interleukin 1 beta), IL18 (interleukin 18), HMGB1 (high mobility group box 1), and FGF2 (fibroblast growth factor 2) [4–7]. Although it is known that these unconventionally secreted proteins are released from the cell via an ER-/Golgi-independent pathway, the specific mechanism by which the soluble protein is able to translocate across the hydrophobic plasma membrane still remains to be discussed.…”