Formation of disulfide bridges drives oligomerization, membrane pore formation, and translocation of fibroblast growth factor 2 to cell surfaces

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.

Keywords: Fibroblast Growth Factor (FGF); Fibroblast Growth Factor 2/FGF2; Inositol Phospholipid; Membrane Pore Formation; Membrane Recruitment and Translocation; Phosphoinositides; Plasma Membrane; Protein Sorting; Secretion; Unconventional Protein Secretion.

FIGURE 1.

Structural comparison of FGF2 with FGF4, a signal-peptide-containing FGF family member that is secreted through the classical, ER/Golgi-dependent pathway. In panels A and B, a combination of space-filling and secondary structure-based models of FGF2 (PDB: 1BFF) and FGF4 (PDB: 1IJT) are shown from a similar perspective emphasizing the overall structural similarity between FGF2 and FGF4. In panel A, two surface cysteines in positions 77 and 95 of FGF2 are highlighted in yellow that are absent from FGF4 (panel B). The corresponding positions in FGF4 are shown in gray. In addition, in panel A, important cis elements in FGF2 are highlighted in red (tyrosine 81) and blue (basic residues of the PI(4,5)P₂ binding pocket). In FGF4 (panel B), a phenylalanine residue is present in position 81 (shown in gray). The region in FGF2 forming the PI(4,5)P₂ binding pocket is highlighted in blue (panel A). The corresponding region in FGF4 also contains basic residues that are shown in blue as well (panel B). In panel C, a sequence alignment between FGF2 and FGF4 is shown with the two surface cysteines in positions 77 and 95, tyrosine 81 and the PI(4,5)P₂ binding pocket of FGF2 being highlighted in the colors used in panels A and B. Note that amino acid numbering was adjusted to the FGF2 sequence starting with the residue A, which represents the first amino acid of FGF2 after removal of the N-terminal methionine. The N-terminal signal peptide of FGF4 has been omitted from the sequence analysis shown in panel C. In panel D, a multiple sequence alignment (Clustal Omega) spanning residues 32–105 (numbering based on the 18 kDa form of FGF2) of the human FGF family illustrates that the two surface cysteines of FGF2 are not present in any other member of the FGF family secreted by the ER/Golgi-dependent pathway.

FIGURE 2.

Oligomerization of FGF2 variant forms on the surface of giant unilamellar vesicles analyzed by scanning cross-correlation fluorescence spectroscopy. GUVs were prepared with a plasma-membrane-like lipid composition including 2 mol % PI(4,5)P₂. Three variant forms of FGF2, FGF2-Y81pCMF with wild-type cysteines, FGF2-Y81pCMF with alkylated cysteines (treatment with N-ethylmaleimide), and FGF2-Y81pCMF-C77A/C95A, were C-terminally labeled with Atto488 and Atto655 dyes employing sortagging (see “Experimental Procedures” for details). All three variants of FGF2 were incubated with GUVs as mixtures of two different FGF2 populations based upon the two different dyes. Cross-correlation curves were derived from a two-color analysis and used to compare the relative abundance of FGF2 oligomers under the conditions indicated (panel A). In addition, the data were used to calculate diffusion constants of FGF2 oligomers formed under the experimental conditions indicated (panel B). To test whether observed differences between experimental conditions were statistically significant, an unpaired two-tailed t test was performed (ns = not significant; * = p value ≤ 0.05; *** = p value ≤ 0.001). For further details see “Experimental Procedures.”

FIGURE 3.

Analysis of membrane-associated FGF2 oligomers by native gel electrophoresis as well as non-reducing and reducing SDS-PAGE. FGF2-Y81pCMF was incubated either in the absence of liposomes (panels A, C, E, and F) or with liposomes containing a plasma-membrane (PM)-like lipid composition including 2 mol % PI(4,5)P₂ (panels B, D, F, and H) for the times indicated. After detergent extraction, membrane-bound material was analyzed either on a native gel system developed for the separation of basic proteins (panels A and B; see “Experimental Procedures” for details), on non-reducing SDS gels (panels C and D), or on reducing SDS gels (panels E and F). In addition, FGF2 samples pretreated with 10 mm DTT before incubation under the conditions indicated were analyzed on reducing SDS gels (panels G and H). The molecular weight markers (MWM) used on SDS gels correspond to 15, 25, 30, 40, 50, 70 (dark blue), 80, 115, and 140 kDa, respectively. In all experiments, before membrane extraction with detergent, samples were treated with NEM to block free thiols, preventing disulfide formation during sample processing. All gels were stained with Coomassie InstantBlue (Expedeon).

FIGURE 4.

Chemical alkylation and substitution of surface cysteines by alanines impairs formation of FGF2 oligomers higher than dimers. Native gel electrophoresis was employed to analyze FGF2 oligomer formation under the conditions indicated. Three variant forms of FGF2, FGF2-Y81pCMF, FGF2-Y81pCMF with alkylated surface cysteines, and FGF2-Y81pCMF-C77A/C95A, were incubated with liposomes containing a plasma-membrane-like lipid composition including 2 mol % PI(4,5)P₂. After 6 h of incubation, samples were subjected to protein extraction with detergent. Where indicated, samples were treated with DTT to break disulfide bonds before native gel electrophoresis. All gels were stained with Coomassie InstantBlue (Expedeon). Panel A provides a representative example for the analysis on native gels of FGF2-Y81pCMF, FGF2-Y81pCMF with alkylated surface cysteines, and FGF2-Y81pCMF-C77A/C95A under the conditions indicated. Panels B, C, and D provide quantification of monomers, dimers, trimers, and tetramers under all experimental conditions using the LI-COR imaging platform. The statistical analysis is based on three independent experiments. Standard deviations are shown.

FIGURE 5.

Membrane pore formation mediated by FGF2-Y81pCMF depends on functional surface cysteines. Carboxyfluorescein (CF) was sequestered in liposomes containing a plasma-membrane-like lipid composition containing 2 mol % PI(4,5)P₂. Liposomes were incubated with either FGF2-Y81pCMF (panels A and B), FGF2-Y81pCMF-C77A/C95A (panels A and B), or FGF2-Y81pCMF with alkylated surface cysteines (NEM treatment; panel B) as indicated. At the highest concentration of 4 μm NEM, the ratio of the two surface cysteines at 2 μm FGF2 to NEM equals 1. Membrane pore formation was analyzed by measuring the release of luminal carboxyfluorescein quantified by fluorescence dequenching as described under “Experimental Procedures.” The results shown are representative of five independent experiments.

FIGURE 6.

FGF2 secretion from cells depends on functional surface cysteines. CHO cells stably expressing either FGF2-GFP, FGF2-C77A/C95A-GFP, FGF2-K127Q/R128Q/K133Q-GFP, or GFP in a doxycycline-dependent manner were induced for protein expression for 16 h at 37 °C. Cells were incubated with a membrane-impermeable biotinylation reagent targeting primary amines of cell surface proteins. After quenching and detergent-mediated cell lysis, biotinylated material was purified using streptavidin beads. Aliquots from the non-biotinylated fractions (10%; corresponding to intracellular population) and the biotinylated fractions (50%); corresponding to the secreted population) were subjected to SDS-PAGE and Western blotting using anti-GFP primary antibodies. In addition, all fractions were analyzed for an endogenous marker, GAPDH (panel A). Antigens were detected and quantified using an Odyssey infrared imaging system (LI-COR Biosciences). Signals derived from biotinylated FGF2 variant forms corresponding to the secreted cell surface population were normalized based on FGF2 wild type that was set to 100% (panel B).

FIGURE 7.

Localization of various FGF-GFP fusion proteins as analyzed by confocal microscopy. Constructs encoding various kinds of FGF-GFP fusion proteins were used to generate stable CHO cell lines by retroviral transduction (25). Upon protein expression in the presence of doxycycline subcellular localization of the FGF fusion proteins indicated was analyzed by confocal microscopy (panels A, E, I, M, Q, and U). For colocalization experiments, either the Golgi marker GM-130 (panel B) or wheat germ agglutinin (panels F, J, N, R, and V) as a plasma membrane marker was used. In addition, nuclei were stained with DAPI (panels C, G, K, O, S, and W), and merged images were generated (panels D, H, L, P, T, and X). The following constructs were analyzed: SP-FGF4-GFP (authentic FGF4 with a signal peptide; panels A–D), FGF4-GFP without signal peptide (panels E–H), a FGF4/2-GFP hybrid protein (panels I–L), FGF4-(+Cys)-GFP (panels M-P), FGF4/2-(−Cys)-GFP (panels Q–T), and FGF2-GFP (panels U-X). Scale bars = 100 nm.

FIGURE 8.

Cis elements required for FGF2 membrane translocation are transplantable and redirect a signal peptide-lacking variant form of FGF4 to the unconventional secretory pathway of FGF2. The efficiency of secretion of the various fusion proteins indicated was compared by a combination of cell surface biotinylation (as introduced in Fig. 6) and heparin affinity purification from cellular supernatants. Stable CHO cell lines were cultivated for 24 h in the presence of 1 μg/ml doxycycline to induce the expression of the fusion proteins indicated. Heparin affinity chromatography was used to collect FGF fusion proteins present in cellular supernatants. Cells were surface-biotinylated as described under “Experimental Procedures” followed by affinity purification using streptavidin beads. For each cell line, the amounts of the FGF fusion proteins indicated present in cells (10% loaded), on cell surfaces (33% loaded), and in cellular supernatants (33% loaded) were analyzed by SDS-PAGE and quantitative Western blotting using the LI-COR imaging platform. Panel A shows the results from a representative experiment with GAPDH as an intracellular control protein not present on cell surfaces or in cellular supernatants. Panel B provides a statistical analysis with standard deviations calculated from three independent experiments with the amount of FGF2-GFP on cell surfaces set as a reference point (=100%).