Abstract
Fibroblast growth factor 2 (FGF2) is a key signaling molecule in tumor-induced angiogenesis. FGF2 is secreted by an unconventional secretory mechanism that involves phosphatidylinositol 4,5-bisphosphate-dependent insertion of FGF2 oligomers into the plasma membrane. This process is regulated by Tec kinase-mediated tyrosine phosphorylation of FGF2. Molecular interactions driving FGF2 monomers into membrane-inserted FGF2 oligomers are unknown. Here we identify two surface cysteines that are critical for efficient unconventional secretion of FGF2. They represent unique features of FGF2 as they are absent from all signal-peptide-containing members of the FGF protein family. We show that phosphatidylinositol 4,5-bisphosphate-dependent FGF2 oligomerization concomitant with the generation of membrane pores depends on FGF2 surface cysteines as either chemical alkylation or substitution with alanines impairs these processes. We further demonstrate that the FGF2 variant forms lacking the two surface cysteines are not secreted from cells. These findings were corroborated by experiments redirecting a signal-peptide-containing FGF family member from the endoplasmic reticulum/Golgi-dependent secretory pathway into the unconventional secretory pathway of FGF2. Cis elements known to be required for unconventional secretion of FGF2, including the two surface cysteines, were transplanted into a variant form of FGF4 without signal peptide. The resulting FGF4/2 hybrid protein was secreted by unconventional means. We propose that the formation of disulfide bridges drives membrane insertion of FGF2 oligomers as intermediates in unconventional secretion of FGF2.
Highlights
Fibroblast growth factor 2 (FGF2) translocation across plasma membranes depends on phosphoinositide-dependent oligomerization and membrane pore formation
To test whether the presence of Cys-77 and Cys-95 in FGF2 relates to its ability to reach the extracellular space, we analyzed whether these residues are critical for (i) PI[4,5]P2-dependent oligomerization of FGF2 on membrane surfaces, (ii) membrane insertion of FGF2 oligomers as intermediates in translocation, and (iii) FGF2 secretion from cells
Membrane-inserted FGF2 oligomers have been interpreted as translocation intermediates that are resolved by cell surface heparan sulfate proteoglycans [11]
Summary
FGF2 translocation across plasma membranes depends on phosphoinositide-dependent oligomerization and membrane pore formation. Various kinds of unconventional secretory pathways from eukaryotic cells have been described [7, 8] with FGF2 being the classical example for the type I pathway of unconventional secretion [7] This secretory mechanism is based upon direct translocation of cargo across the plasma membrane mediated by PI[4,5]P2-dependent membrane insertion of FGF2 oligomers [3, 9, 10]. With the results from the current study, three cis elements have been identified in FGF2 that are required for unconventional secretion These are the basic residues that form the binding pocket for PI[4,5]P2 [4, 9, 19], the tyrosine residue that is targeted by Tec kinase [3, 7, 16], and the two surface cysteines involved in disulfide formation (this study). These findings establish the three cis elements in FGF2 including the two surface cysteines described in the current study to represent critical molecular determinants of unconventional secretion
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