Biosynthetic FGF-2 is targeted to non-lipid raft microdomains following translocation to the extracellular surface of CHO cells
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Biosynthetic FGF-2 is targeted to non-lipid raft microdomains following translocation to the extracellular surface of CHO cells. / Engling, André; Backhaus, Rafael; Stegmayer, Carolin; Zehe, Christoph; Seelenmeyer, Claudia; Kehlenbach, Angelika; Schwappach, Blanche; Wegehingel, Sabine; Nickel, Walter.
In: J CELL SCI, Vol. 115, No. Pt 18, 15.09.2002, p. 3619-31.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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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 -