Cell-surface heparan sulfate proteoglycans are essential components of the unconventional export machinery of FGF-2

Abstract

FGF-2 is an unconventionally secreted lectin that transmits proangiogenic signals through a ternary complex with high-affinity FGF receptors and heparan sulfate proteoglycans (HSPGs). Although FGF-2 signal transduction is understood in great detail, its mechanism of release from cells, which is independent of the classical secretory pathway, remains elusive. To test the hypothesis that FGF-2 secretion is linked to its cell-surface ligands, we studied FGF-2 release using mutants defective for HSPG binding and cells with impaired HSPG biosynthesis. Here, we report that a functional interaction between FGF-2 and HSPGs is required for net export of FGF-2 from mammalian cells. FGF-2 release requires extracellular, membrane-proximal HSPGs. We propose that extracellular HSPGs form a molecular trap that drives FGF-2 translocation across the plasma membrane.

Fig. 1.

FGF-2 mutants deficient in binding to HSPGs fail to get exported from mammalian cells. (A) CHO cells (7 × 10⁴) were induced with doxycycline for 16 h at 37°C to achieve similar expression levels of the reporter molecules indicated. Cells were decorated with anti-GFP primary and APC-coupled secondary antibodies and analyzed for both total FGF-2-GFP protein (GFP fluorescence) and FGF-2-GFP cell-surface localization (APC-derived fluorescence). To quantify the cell surface-bound fraction in relation to total FGF-2-GFP, defined amounts of cell-free supernatants (percentage of totally expressed material) prepared from 7 × 10⁴ CHO_FGF-2-GFP cells were incubated with equal numbers of nontransduced CHO cells for 30 min at 4°C (n = 3). (B) CHO cells (1.5 × 10⁵) were cultivated as described above. Cells were incubated with a membrane-impermeable biotinylation reagent. After detergent-mediated cell lysis, biotinylated material was purified by using streptavidin beads. Aliquots from the cell lysate (10%), the cell surface-biotinylated fractions (45%), and defined amounts of recombinant FGF-2-GFP (lanes 9–11) were analyzed by Western blotting. Antigens were quantified with an Odyssey infrared imaging system (LI-COR Biosciences). (C) The biotinylation signals in B were quantified by using the Odyssey infrared imaging system (LI-COR Biosciences) and graphed as the percentage of wild-type FGF-2, which was set to 100%. (D) CHO cells were cultivated for 16 h at 37°C to a confluency of ≈80%. Growth medium was centrifuged (500 × g for 20 min at 4°C), and cells attached to the culture dish were used to prepare total lysates. Both medium and defined amounts of cell lysates were incubated with Ni-NTA-agarose for 1 h at 4°C. Bound material was analyzed by Western blotting as described in Experimental Procedures.

Fig. 2.

Full-length FGF-2 fails to get exported from cells lacking surface HSPGs. (A) CHO cells (7 × 10⁴) were cultivated for 16 h at 37°C in the presence of varying doxycycline concentrations to achieve similar expression levels of FGF-2-GFP. Where indicated, the growth medium was supplemented with 75 mM sodium chlorate. Cells were decorated with anti-GFP primary and APC-conjugated secondary antibodies. Total fusion protein (GFP fluorescence) and cell surface-localized protein (APC-derived fluorescence) were determined by flow cytometry (n = 4). (B) CHO cells were grown to a confluency of ≈80%. The medium was centrifuged (500 ×g for 20 min at 4°C), and cells attached to the culture dish were used to prepare total lysates. Medium and defined amounts of cell lysates (as indicated) were incubated with heparin Sepharose for 1 h at 4°C. Bound material was analyzed by Western blotting as described in Materials and Methods. (C) CHO cells were grown and processed as in B. Where indicated, growth medium was supplemented with 125 μg/ml heparin (Hep.). Both medium and defined amounts of cell lysates (as indicated) were subjected to immunoprecipitation using affinity-purified anti-GFP antibodies. Bound material was eluted with SDS sample buffer, followed by Western blotting. Antigen detection was conducted as described in Materials and Methods. (D) CHO cells (7 × 10⁴) were cultivated as described above. Cell culture supernatants were centrifuged (500 × g for 20 min at 4°C) and incubated with 7 × 10⁴ nontransduced CHO-K1 cells for 30 min at 4°C. Cells were decorated with anti-GFP primary and APC-coupled secondary antibodies and analyzed by flow cytometry. To estimate the quantity of cell surface-bound material, defined amounts of cell-free supernatant prepared from 7 × 10⁴ CHO-K1_FGF-2-GFP cells were added as indicated to the same number of CHO-K1 cells and analyzed the same way. In addition, the APC-derived signal at the surface of CHO-K1_FGF-2-GFP cells was determined (n = 4).

Fig. 3.

Reconstitution of FGF-2 secretion from HSPG-deficient cells by cocultivation with HSPG-expressing acceptor cells. (Aa–Af) CHO-K1_FGF-2-GFP or CHO-745_FGF-2-GFP cells (7 × 10⁴) (both shown in green), were grown for 16 h at 37°C in 24-well plates either separately or in a mixed culture (1:1 ratio) with wild-type cells CHO (shown in red). Cells were decorated with anti-GFP primary and APC-conjugated secondary antibodies. Total FGF-2-GFP (GFP fluorescence) and cell surface-localized fusion proteins (APC-derived fluorescence) were analyzed by flow cytometry. (Ba–Bf) CHO-K1_FGF-2-GFP or CHO-745_FGF-2-GFP cells were mixed with CHO-K1 cells in a 1:1 ratio and grown on glass coverslips for 16 h at 37°C. After fixation with 3% paraformaldehyde, nonpermeabilized cells were processed with anti-GFP primary and Alexa Fluor 546-coupled secondary antibodies. Subsequently, specimens were embedded in Fluoromount G (Southern Biotechnology Associates, Birmingham, AL) and analyzed by confocal microscopy. Nontransduced CHO-K1 cells that express cell-surface HSPGs are labeled with an asterisk.

Fig. 4.

Spatial requirements for rescue of FGF-2 secretion from HSPG-deficient cells by wild-type acceptor cells. CHO-K1, CHO-K1_FGF-2-GFP, or CHO-745_FGF-2-GFP cells (8 × 10⁴ cells per experimental condition) were seeded on glass coverslips either alone or mixed together (1:1 ratio), as indicated. Cells were cultivated at 37°C for ≈6 h. Coverslips were placed in 24-well plates, as indicated, and induced with doxycycline for 16 h at 37°C. Cells were incubated with anti-GFP primary and APC-coupled secondary antibodies and analyzed for total FGF-2-GFP (GFP fluorescence) and FGF-2-GFP cell-surface localization (APC-derived fluorescence) by using flow cytometry (n = 5).