Biosynthetic FGF-2 is targeted to non-lipid raft microdomains following translocation to the extracellular surface of CHO cells.
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.
Biochemie-Zentrum Heidelberg (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.
André
A
Engling
Rafael
R
Backhaus
Carolin
C
Stegmayer
Christoph
C
Zehe
Claudia
C
Seelenmeyer
Angelika
A
Kehlenbach
Blanche
B
Schwappach
Sabine
S
Wegehingel
Walter
W
Nickel
D000818:Animals; D016466:CHO Cells / Q000166:cytology / Q000378:metabolism*; D002450:Cell Communication / Q000235:genetics; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D005057:Eukaryotic Cells / Q000166:cytology / Q000378:metabolism*; D005110:Extracellular Space / Q000235:genetics / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D021962:Membrane Microdomains / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014161:Transduction, Genetic
D019812:Heparan Sulfate Proteoglycans; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D016222:Fibroblast Growth Factor 2; D049452:Green Fluorescent Proteins