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

TY - JOUR

T1 - Biosynthetic FGF-2 is targeted to non-lipid raft microdomains following translocation to the extracellular surface of CHO cells

AU - Engling, André

AU - Backhaus, Rafael

AU - Stegmayer, Carolin

AU - Zehe, Christoph

AU - Seelenmeyer, Claudia

AU - Kehlenbach, Angelika

AU - Schwappach, Blanche

AU - Wegehingel, Sabine

AU - Nickel, Walter

PY - 2002/9/15

Y1 - 2002/9/15

N2 - 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 non-classical 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 rate-limiting.

AB - 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 non-classical 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 rate-limiting.

KW - Animals

KW - CHO Cells/cytology

KW - Cell Communication/genetics

KW - Cell Membrane/genetics

KW - Cricetinae

KW - Eukaryotic Cells/cytology

KW - Extracellular Space/genetics

KW - Fibroblast Growth Factor 2/biosynthesis

KW - Genetic Vectors

KW - Green Fluorescent Proteins

KW - Heparan Sulfate Proteoglycans/metabolism

KW - Luminescent Proteins

KW - Membrane Glycoproteins/genetics

KW - Membrane Microdomains/genetics

KW - Microscopy, Confocal

KW - Protein Structure, Tertiary/physiology

KW - Protein Transport/genetics

KW - Recombinant Fusion Proteins/metabolism

KW - Transduction, Genetic

U2 - 10.1242/jcs.00036

DO - 10.1242/jcs.00036

M3 - SCORING: Journal article

C2 - 12186948

VL - 115

SP - 3619

EP - 3631

JO - J CELL SCI

JF - J CELL SCI

SN - 0021-9533

IS - Pt 18

ER -