Biosynthetic FGF-2 is targeted to non-lipid raft microdomains following translocation to the extracellular surface of CHO cells

Abstract

Basic fibroblast growth factor (FGF-2) is a secretory protein that lacks a signal peptide. Consistently, FGF-2 has been shown to be secreted by an ER-Golgi-independent mechanism; however, the machinery mediating this process remains to be established at the molecular level. Here we introduce a novel experimental system based on flow cytometry that allows the quantitative assessment of nonclassical FGF-2 secretion in living cells. Stable cell lines have been created by retroviral transduction that express various kinds of FGF-2-GFP fusion proteins in a doxicyclin-dependent manner. Following induction of protein expression, biosynthetic FGF-2-GFP is shown to translocate to the outer surface of the plasma membrane as determined by both fluorescence activated cell sorting (FACS) and confocal microscopy. Both N-and C-terminal GFP tagging of FGF-2 is compatible with FGF-2 export, which is shown to occur in a controlled fashion rather than through unspecific release. The experimental system described has strong implications for the identification of both FGF-2 secretion inhibitors and molecular components involved in FGF-2 secretion.In the second part of this study we made use of the FGF-2 export system described to analyze the fate of biosynthetic FGF-2-GFP following export to the extracellular space. We find that secreted FGF-2 fusion proteins accumulate in large heparan sulfate proteoglycan (HSPG)-containing protein clusters on the extracellular surface of the plasma membrane. These microdomains are shown to be distinct from caveolae-like lipid rafts known to play a role in FGF-2-mediated signal transduction. Since CHO cells lack FGF high-affinity receptors (FGFRs), it can be concluded that FGFRs mediate the targeting of FGF-2 to lipid rafts. Consistently, FGF-2-GFP-secreting CHO cells do not exhibit increased proliferation activity. Externalization and deposition of biosynthetic FGF-2 in HSPG-containing protein clusters are independent processes, as a soluble secreted intermediate was demonstrated. The balance between intracellular FGF-2 and HSPG-bound secreted FGF-2 is shown not to be controlled by the availability of cell surface HSPGs, indicating that the FGF-2 secretion machinery itself is ratelimiting.

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

“…Under steady-state conditions, Ϸ10% of wild-type FGF-2-GFP was associated with the cell surface, as measured by flow cytometry and comparison with defined amounts of FGF-2-GFP from cell-free supernatants (Fig. 1A) (21). By contrast, consistent with the data shown in Fig.…”

“…5 and 6 and Supporting Experimental Procedures, which are published as supporting information on the PNAS web site). The mutants were stably introduced into CHO cells by using a retroviral vector system that allows for their doxycycline-dependent expression (21). As shown in Fig.…”

“…1D, 1% of the total population of each FGF-2-GFP was easily detectable, which is at least one order of magnitude more sensitive than the amount of cell-surface FGF-2 observed under steady-state conditions ( Fig. 1 A and B) (21). When the media of cells expressing wild-type FGF-2 or the FGF-2 mutants deficient for HSPG binding were analyzed, none of the fusion proteins were detectable (Fig.…”

“…These include the proangiogenic lectins FGF-1 and FGF-2 (13)(14)(15)(16)(17)(18)(19)(20)(21), the inflammatory cytokines interleukin 1␤ (22)(23)(24) and macrophage migration inhibitory factor (MIF) (25), and galectins, which are ␤-galactoside-specific lectins of the extracellular matrix that function in development and immune regulation (26)(27)(28)(29)(30)(31)(32)(33)(34). Consistent with their critical functions, unconventional secretory processes are, in most cases, specifically regulated (reviewed in refs.…”

A Novel Nonsynonymous Variant of Matrix Metalloproteinase-7 Confers Risk of Liver Cirrhosis†

“…These data suggest that the MMP-7(Asp-137) variant associated with the detergentresistant microdomain may enhance the activation of MMP-7. In addition, because HSPG was demonstrated to be detergent-soluble, 21 the possibility that the increased proportion of cell surface localization and enhanced enzyme activity of the detergent-resistant MMP-7(Asp-137) was mediated by HSPG docking can be ruled out.…”