FGF3 Attached to a Phosholipid Membrane Anchor Gains a High Transforming Capacity
2002; Elsevier BV; Volume: 277; Issue: 36 Linguagem: Inglês
10.1074/jbc.m204661200
ISSN1083-351X
AutoresRoman Ko ̈hl, Marianne Antoine, Kerstin Reimers, Paul Kiefer,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoNIH3T3 cells transformed by mouse FGF3-cDNA (DMI cells) selected for their ability to grow as anchorage-independent colonies in soft agar and in defined medium lacking growth factors exhibit a highly transformed phenotype. We have used dominant negative (DN) fibroblast growth factor (FGF) receptor 2 (FGFR2) isoforms to block the FGF response in DMI cells. When the DN-FGFR was expressed in DMI cells, their transformed phenotype can be reverted. The truncated FGFR2(IIIb), the high affinity FGFR for FGF3, is significantly more efficient at reverting the transformed phenotype as the IIIc isoform, reaffirming the notion that the affinity of the ligand to the DN-FGFR2 isoform determines the effect. Heparin or heparan sulfate displaces FGF3 from binding sites on the cell surface inhibiting the growth of DMI cells and reverts the transformed phenotype (1Kiefer P. Peters G. Dickson C. Mol. Cell. Biol. 1991; 11: 5929-5936Crossref PubMed Scopus (26) Google Scholar). However, the presence of heparin is necessary to induce a mitogenic response in NIH3T3 cells when stimulated with soluble purified mouse FGF3. We have investigated the importance of cell surface binding of FGF3 for its ability to transform NIH3T3 cells by creating an FGF3 mutant anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI anchor renders the cell surface association of FGF3 independent from binding to heparan sulfate-proteoglycan of the cell surface membrane. Attachment of a GPI anchor to FGF3 also confers a much higher transforming potential to the growth factor. Even more, the purified GPI-attached FGF3 is as much transforming as the secreted protein acting in an autocrine mode. Because NIH3T3 cells do not express the high affinity tyrosine kinase FGF receptors for FGF3, these findings suggest that FGF3 attached to GPI-linked heparan sulfate-proteoglycan may have a broader biological activity as when bound to transmembrane or soluble heparan sulfate-proteoglycan. NIH3T3 cells transformed by mouse FGF3-cDNA (DMI cells) selected for their ability to grow as anchorage-independent colonies in soft agar and in defined medium lacking growth factors exhibit a highly transformed phenotype. We have used dominant negative (DN) fibroblast growth factor (FGF) receptor 2 (FGFR2) isoforms to block the FGF response in DMI cells. When the DN-FGFR was expressed in DMI cells, their transformed phenotype can be reverted. The truncated FGFR2(IIIb), the high affinity FGFR for FGF3, is significantly more efficient at reverting the transformed phenotype as the IIIc isoform, reaffirming the notion that the affinity of the ligand to the DN-FGFR2 isoform determines the effect. Heparin or heparan sulfate displaces FGF3 from binding sites on the cell surface inhibiting the growth of DMI cells and reverts the transformed phenotype (1Kiefer P. Peters G. Dickson C. Mol. Cell. Biol. 1991; 11: 5929-5936Crossref PubMed Scopus (26) Google Scholar). However, the presence of heparin is necessary to induce a mitogenic response in NIH3T3 cells when stimulated with soluble purified mouse FGF3. We have investigated the importance of cell surface binding of FGF3 for its ability to transform NIH3T3 cells by creating an FGF3 mutant anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI anchor renders the cell surface association of FGF3 independent from binding to heparan sulfate-proteoglycan of the cell surface membrane. Attachment of a GPI anchor to FGF3 also confers a much higher transforming potential to the growth factor. Even more, the purified GPI-attached FGF3 is as much transforming as the secreted protein acting in an autocrine mode. Because NIH3T3 cells do not express the high affinity tyrosine kinase FGF receptors for FGF3, these findings suggest that FGF3 attached to GPI-linked heparan sulfate-proteoglycan may have a broader biological activity as when bound to transmembrane or soluble heparan sulfate-proteoglycan. fibroblast growth factor receptor extracellular matrix glycosylphosphatidylinositol dominant negative heparan sulfate-proteoglycans decay accelerating factor phosphate-buffered saline Dulbecco's modified Eagle's medium fetal calf serum The fibroblast growth factor (FGF)1 family presently constitutes 22 structurally related polypeptides that show a wide range of biological activities. They modulate growth, differentiation, migration, and survival depending on cell type and biological context (reviewed in Refs. 2Baird A. Klagsbrun M. Cancer Cells. 1991; 3: 239-243PubMed Google Scholar, 3Basilico C. Moscatelli D. Adv. Cancer Res. 1992; 59: 115-165Crossref PubMed Scopus (1053) Google Scholar, 4Bikfalvi A. Klein S. Pintucci G. Rifkin D.B. Endocr. Rev. 1997; 18: 26-45Crossref PubMed Scopus (851) Google Scholar, 5Goldfarb M. Cell Growth & Differ. 1990; 1: 439-445PubMed Google Scholar, 6Klagsbrun M. Prog. Growth Factor Res. 1989; 1: 207-235Abstract Full Text PDF PubMed Scopus (320) Google Scholar, 7Ornitz D.M. Itoh N. Genome Biol. 2001; 2: 3005.1-3005.2Crossref Google Scholar, 8Szebenyi G. Fallon J. Int. Rev. Cytol. 1999; 185: 45-106Crossref PubMed Google Scholar). In vivo the FGFs have been implicated in embryonic development, neuronal survival, wound repair, and angiogenesis, but also in a number of pathological responses such as neovascularization and tumor growth. The mouse FGF3/int-2 gene is one of several cellular oncogenes identified at sites of proviral integration in retrovirus-induced breast carcinomas associated with infection by mouse mammary tumor virus (MMTV). In cell culture it proved to be a weak transforming gene, but under selective pressures it causes the morphological transformation of NIH3T3 cells (1Kiefer P. Peters G. Dickson C. Mol. Cell. Biol. 1991; 11: 5929-5936Crossref PubMed Scopus (26) Google Scholar). FGFs signal by binding to and activating high affinity cell surface tyrosine kinase receptors. Four high affinity receptor genes (designated FGFR1–FGFR4) have been identified that encode a cytoplasmic tyrosine kinase domain, an extracellular region composed of two (β-form) or three (α-form) immunoglobulin-like domains, depending on the choice of splice sites. The ligand binding site involves the two Ig loops located adjacent to the cell membrane. Alternative splicing of the membrane proximal loop generates isoforms (termed IIIb and IIIc) that have different ligand binding specificities (9Chellaiah A. McEwen D. Werner S., Xu, J. Ornitz D. J. Biol. Chem. 1994; 269: 11620-11627Abstract Full Text PDF PubMed Google Scholar, 10Bottaro D. Rubin J. Ron D. Finch P. Florio C. Aaronson S. J. Biol. Chem. 1990; 265: 12767-12770Abstract Full Text PDF PubMed Google Scholar, 11Dell K. Williams L. J. Biol. Chem. 1992; 267: 21225-21229Abstract Full Text PDF PubMed Google Scholar, 12Johnson D. Williams L. Adv. Cancer Res. 1993; 60: 1-41Crossref PubMed Scopus (1176) Google Scholar, 13Ornitz D.M., Xu, J. Colvin J.S. McEwen D.G. MacArthur C.A. Coulier F. Gao G. Goldfarb M. J. Biol. Chem. 1996; 271: 15292-15297Abstract Full Text Full Text PDF PubMed Scopus (1423) Google Scholar, 14Ornitz D.M. Bioessays. 2000; 22: 108-112Crossref PubMed Scopus (623) Google Scholar, 15Schlessinger J. Plotnikov A.N. Ibrahimi O.A. Eliseenkova A.V. Yeh B.K. Yayon A. Linhardt R.J. Mohammadi M. Mol. Cell. 2000; 6: 743-750Abstract Full Text Full Text PDF PubMed Scopus (965) Google Scholar). FGF signaling can be blocked by dominant negative mutant FGFRs (DN-FGFR) that have a cytoplasmic domain with the kinase domain removed. The mutant receptors form non-functional heterodimers with the wild type receptors through binding to a common ligand. The effectiveness of DN-FGFRs has been used to demonstrate a requirement for FGF signaling in mesoderm formation in Xenopus embryos, as well as in skin wound repair, development of the mouse lung, and lobulo-alveolar development of the mammary gland. Furthermore, expression of a DN-FGFR mutant is able to revert the transformed phenotype of NIH3T3 cells transformed by FGF4 (16Celli G. LaRochelle W.J. Mackem S. Sharp R. Merlino G. EMBO J. 1998; 17: 1642-1655Crossref PubMed Scopus (338) Google Scholar, 17Jackson D. Bresnick J. Rosewell I. Crafton T. Poulsom R. Stamp G. Dickson C. J. Cell Sci. 1997; 110: 1261-1268Crossref PubMed Google Scholar, 18Li Y. Basilico C. Mansukhani A. Mol. Cell. Biol. 1994; 14: 7660-7669Crossref PubMed Google Scholar, 19Peters K. Werner S. Liao X. Wert S. Whitsett J. Williams L. EMBO J. 1994; 13: 3296-3301Crossref PubMed Scopus (378) Google Scholar, 20Werner S. Weinberg W. Liao X. Peters K.G. Blessing M. Yuspa S.H. Weiner R.L. Williams L.T. EMBO J. 1993; 12: 2635-2643Crossref PubMed Scopus (218) Google Scholar, 21Ueno H. Gunn M. Dell K. Tseng A., Jr. Williams L. J. Biol. Chem. 1992; 267: 1470-1476Abstract Full Text PDF PubMed Google Scholar). Recent reports have demonstrated that besides the FGFR isoforms present on the cell surface, the binding of FGFs and FGFRs to heparan sulfate-proteoglycans (HSPGs) act as cofactors to regulate FGF signaling (22Klagsbrun M. Baird A. Cell. 1991; 67: 229-231Abstract Full Text PDF PubMed Scopus (498) Google Scholar, 23Guimond S.E. Turnbull J.E. Curr. Biol. 1999; 9: 1343-1346Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 24Berman B. Ostrovsky O. Shlissel M. Lang T. Regan D. Vlodavsky I. Ishai-Michaeli R. Ron D. J. Biol. Chem. 1999; 274: 36132-36138Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). FGF3 exhibits ∼10× higher affinity for the IIIb isoforms of FGFR1 and FGFR2 compared with the IIIc variant of FGFR2. Previous studies (25Mathieu M. Chatelain E. Ornitz D. Bresnick J. Mason I. Kiefer P. Dickson C. J. Biol. Chem. 1995; 270: 24197-24203Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) revealed that heparin progressively increased the potency of FGF3 on HC11 cells expressing the IIIb variants of FGFR1 and FGFR2 but decreased it on C57MG cells that express the corresponding IIIc variants. This different modulation of FGF3 activity is not easily explained by the expression of different FGFR variants because NIH3T3 cells express the same FGFR isoforms as C57MG cells, but they show a dependence on heparin for FGF3-mediated mitogenic response. Although heparin is essential for FGF3-induced mitogenic activity on NIH3T3 cells, heparin inhibits the growth of FGF3-transformed NIH3T3 cells and reverts their transformed phenotype (1Kiefer P. Peters G. Dickson C. Mol. Cell. Biol. 1991; 11: 5929-5936Crossref PubMed Scopus (26) Google Scholar). Secreted FGF3 is preferentially associated with the cell surface and is displaced by heparin and soluble heparan sulfate (26Kiefer P. Mathieu M. Close M.J. Peters G. Dickson C. EMBO J. 1993; 12: 4159-4168Crossref PubMed Scopus (27) Google Scholar, 27Kiefer P. Mathieu M. Mason I. Dickson C. Oncogene. 1996; 12: 1503-1511PubMed Google Scholar). Therefore, cell surface-bound FGF3 protein appears to be essential in the morphological transformation of NIH3T3 cells. The present study was undertaken to characterize the dependence of the FGF3 cell surface localization on its ability to transform NIH3T3 cells. In this report we were able to show that the phenotype of FGF3 transformed cells could be reverted by expressing DN-FGFR2 mutants and that the truncated FGFR2(IIIb) variant was significantly more efficient at reverting the phenotype than the IIIc isoform. We created a GPI-anchored FGF3 mutant to insert the ligand in the plasma membrane via a GPI anchor sequence. The GPI anchor renders the cell surface association of FGF3 independent of binding to the cell surface membrane via HSPGs, suggesting that localization on the cell surface is crucial for FGF3 transformation and presumably not a requirement for interacting with specific cell surface HSPGs. COS-1, NIH3T3, and DMI-1 cells were maintained as previously described (28Kiefer P. Peters G. Dickson C. Mol. Cell. Biol. 1993; 13: 5781-5793Crossref PubMed Scopus (30) Google Scholar). For transient DNA transfections, plasmid DNA as indicated was introduced into 5 × 105 COS-1 cells by electroporation (450 V/250 μF) using a BioRad Gene-Pulser. Between 48 and 72 h after transfection, the cells were harvested for immunoblot analysis or processed for immunofluorescence. For stable DNA transfection purified plasmid DNA was introduced by using the transfection reagent FuGENE 6 (Roche) as recommended by the manufacturer. pBabepuroDN-FGFR2(IIIb) was constructed by inserting a 1.2-kb EcoRI-BamHI fragment of DN-FGFR2(IIIb) cDNA, kindly provided by J. Bresnick (17Jackson D. Bresnick J. Rosewell I. Crafton T. Poulsom R. Stamp G. Dickson C. J. Cell Sci. 1997; 110: 1261-1268Crossref PubMed Google Scholar), into the expression plasmid pBabepuro. To create the DN-FGFR2(IIIc) isoform, the mutant FGFR2(IIIb) cDNA was used to exchange its extracellular domain to the IIIc variant via twoEcoRI sites. The C terminal GPI anchor signal sequence of FGF3-DAF was generated by PCR using specific primers to amplify the 108 base pairs of DAF C terminal (29Caras I.W. Weddell G.N. Davitz M.A. Nussenzweig V. Martin D.W., Jr. Science. 1987; 238: 1280-1283Crossref PubMed Scopus (145) Google Scholar). The 5′ primer included a XhoI site, and a 3′EcoRI site was introduced by the 3′ primer. TheXhoI-EcoRI fragment was fused downstream to aSacI-XhoI sequence encoding the cytoplasmic isoform of FGF3 (pKC3.2) and inserted into the vector pGem7 (Promega) via a SacI and EcoRI site. The plasmid pKC3.2-DAF was constructed by exchanging an XbaI-EcoRI fragment of pKC3.2 with the appropriate sequence of 3.2-DAF cDNA. The subcloning of 3.2-DAF cDNA into the expression vector pBabeneo was carried out by filling in the 5′-end of theXbaI-EcoRI fragment and inserting into the vector via SnabI sites. COS-1 cells grown on glass coverslips were transfected with the appropriate plasmids, and 48 h later the cells were fixed and processed as previously described (28Kiefer P. Peters G. Dickson C. Mol. Cell. Biol. 1993; 13: 5781-5793Crossref PubMed Scopus (30) Google Scholar). For surface immunostaining, the cells were incubated with antibodies without permeablizing, or alternatively, cells were incubated with the antibodies at 4 °C in the presence of 0.05% sodium azide prior to fixation. After washing in PBS, the stained cells were mounted in 90% glycerol containing p-phenylenediamine and viewed with a ×63 oil immersion lens on a Zeiss microscope equipped with barrier filters for fluorescein or Texas red. Rabbit antiserum directed to the C terminal of mouse FGF3 was diluted 1:200 in PBS. The procedures used for preparing cell lysates and ECM have been described in detail elsewhere (1Kiefer P. Peters G. Dickson C. Mol. Cell. Biol. 1991; 11: 5929-5936Crossref PubMed Scopus (26) Google Scholar, 28Kiefer P. Peters G. Dickson C. Mol. Cell. Biol. 1993; 13: 5781-5793Crossref PubMed Scopus (30) Google Scholar). Samples from equivalent numbers of cells were fractionated by SDS-PAGE in 12.5% gels, transferred to nitrocellulose membranes (Schleicher and Schuell), then probed with rabbit antiserum against the C terminal of FGF3. Immunoreactive proteins were detected by enhanced chemiluminescence (Amersham Biosciences). Total cellular RNA was extracted from cultured cell lines and mouse tissues by guanidium isothiocyanate and cesium trifluoroacetate gradient purification. For Northern blot analysis, 20 mg of total RNA were fractionated in denaturing glyoxal gels, transferred to Hybond N (Amersham Biosciences), and hybridized with 32P-labeled probes under stringent conditions (30Reimers K. Antoine M. Zapatka M. Blecken V. Dickson C. Kiefer P. Mol. Cell. Biol. 2001; 21: 4996-5007Crossref PubMed Scopus (28) Google Scholar). DMI cells or K-ras-transformed NIH3T3 were labeled for 10 min with 0.5 mCi of carrier-free Na125I in a final volume of 0.5 ml in PBS. The cells were rinsed three times in PBS, and the monolayer was removed by incubation in PBS containing 0.5% Triton X-100 at room temperature for 20 min. Then the cells were lysed in ice-cold lysis buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.1% Nonidet P-40, 0.02% sodium azide, 0.1% SDS, 0.5% sodium deoxycholate, 1 mm phenylmethylsulfonyl fluoride, and 100 μg/ml aprotinin) for 30 min. COS-1 cells transiently expressing FGF3-DAF- and FGF3-related proteins were plated in 100-mm dishes and incubated with [3H]ethanolamine (0.2 mCi/dish) in Dulbecco’s modified Eagle medium (DMEM) medium, 5% FCS for 24 h. After removing the medium the monolayers were washed twice with ice-cold PBS before subsequent immunoprecipitation analysis. COS-1 cells transfected with vectors containing the constructs 3.2 and 3.2-DAF were washed twice with PBS and lysed by incubating the cells in 1 ml of ice-cold lysis buffer for 30 min. The lysates were vortexed and cetrifuged by 20,000 ×g for 15 min. The supernatents were incubated for 1 h at 4 °C with a rabbit antiserum directed to the C terminal of mouse FGF3. Through the subsequent addition of 100 μl of a 50% slurry protein A-Sepharose (Amersham Biosciences) and incubation at 4 °C overnight the antibody-protein complexes were precipitated. The precipitates were washed four times in NET buffer (50 mmTris-HCl, pH 7.5, 150 mm NaCl, 0.1% Nonidet P-40, 1 mm EDTA, 0.25% gelatin, and 0.02% sodium azide) and eluted in Laemmli loading buffer and subjected to SDS-PAGE and autoradiography. NIH3T3 cells were maintained in DMEM plus 10% FCS. The test plasmid DNAs were introduced by using the transfection reagent FuGENE 6 (Roche), and after 24 h, 8 × 105 cells were plated into 10-cm diameter dishes. In the delayed focus formation assay, the cells were cultured in DMEM, 10% FCS plus 1 mg/ml neomycin until a cytotoxic effect could be visualized (normally 1–2 days). Within 7 or 8 days the G418-resistant colonies grew to near confluence, the medium was changed to DMEM plus 3% FCS, and cells were cultured another 6 days. Cell foci were scored microscopically. For the agar colony assay, transfected cells were maintained in G418 as above, after which duplicate samples of 5 × 104 cells were suspended in 1.5 ml of DMEM with 10% FCS containing 0.3% agar (Difco). The plates were incubated at 37 °C for a minimum of 10 days, and the cell foci were counted. COS-1 cells were transfected with pKC3.2 and pKC3.2-DAF plasmid DNA by electroporation (see above) and cultured for 48 h. All protein purification steps were carried out at 4 °C. The cells were lysed by ultrasonication in ice-cold 0.5% Triton X-100 in PBS containing 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 100 μg/ml phenylmethylsulfonyl fluoride. Subsequent to a 20,000 ×g centrifugation step, the protein extracts were loaded onto heparin-Sepharose beads (Amersham Biosciences) for retention of the FGF3-related proteins and poured onto a Poly-Prep column (Bio-Rad). After extensive washing with PBS, bound proteins were eluted with 50 mm Tris/HCl, pH 8.0 plus 2 m NaCl in 1-ml fractions. For reducing the salt concentration, fractions were dialyzed against PBS. Fractions were tested for FGF3-related proteins by Western blot using a polyclonal antipeptide serum. NIH3T3 cells grown in a 6-cm diameter dish to a confluency of 80% were washed three times with PBS. The incorporation mixture contained 500 μl of a positive FGF3/FGF3-DAF fraction. This corresponds to 10 μg of FGF3-related protein in 2 ml of DMEM without FCS but with 0.01% Triton X-100, and the cells were incubated for 2 h at 37 °C. Subsequently, the incorporation mixture was replaced with DMEM, 10% FCS, and the cells were cultivated for 2 days before the microscopic analysis of their morphology. FGF3 presumably transforms NIH3T3 cells by establishing an autocrine loop mediated by endogenous FGFRs. DMI-1 cells were isolated as a transformed colony following transfection of a cDNA encoding secreted FGF3. The 32.5-kDa secreted FGF3 protein (gp32.5) is predominately bound to proteoglycosaminoglycans of the cell surface from where it can be quantitatively displaced by heparin as demonstrated by FGF3 cell surface iodination, and the labeled cells were recovered as monolayer by incubating in PBS, 0.5% Triton X-100 leaving the ECM on the culture dish (Fig.1). Using receptor binding competition assays, FGF3 was shown to bind with high affinity to the IIIb isoforms of FGFR1 and FGFR2 but with 10× lower affinity to the IIIc variant of FGFR2. No binding affinity could be demonstrated for FGFR1(IIIc), the isoforms of FGFR3 or for FGFR4. NIH3T3 cells predominantly express the IIIc isoforms of both FGFR1 and FGFR2 and low levels of FGFR4. A reverse transcriptase-PCR analysis showed that the DMI-1 cells had retained the same FGFR expression pattern as the parental NIH3T3 cells (25Mathieu M. Chatelain E. Ornitz D. Bresnick J. Mason I. Kiefer P. Dickson C. J. Biol. Chem. 1995; 270: 24197-24203Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). To show that the transformed phenotype of DMI-1 cells was due to FGF3 expression, a DN-FGFR was used to block FGF3 signaling. DMI cells were stably transfected with DN-FGFR cDNAs inserted into a MuLV-based retrovirus vector containing a puromycin-resistant gene to enable selection of transformants (Fig.2A). DMI-1 cells expressing high levels of DN-FGF receptor 2 isoform IIIb and IIIc were identified by Northern blot analysis (Fig. 2B). Overexpression of the DN-FGFR results in an FGF-mediated sequestration of the wild type receptor by the DN-FGFR to form an inactive complex. Although the DN-FGFR2(IIIc) isoform resulted in a partial reversion of the transformed DMI phenotype (Fig.3E), overexpression of the DN-FGFR2 isoform IIIb resulted in the morphological reversion of most colonies to an untransformed phenotype (Fig. 3D). The greater effect of the DN-FGFR2(IIIb) isoform is consistent with its 10-fold higher affinity for FGF3. To exclude the possibility that revertants were due to the selection procedure, recombinant retroviruses were used to infect rather than transfect DMI cells to express mutant FGF receptors. This resulted in most cells showing a non-transformed phenotype. These results confirmed the presumption that DMI-1 cells were transformed by FGF3.Figure 3Morphologies of fibroblast cell lines expressing dominant negative FGFR2 isoforms.Cells were fixed and stained with Giemsa. Photographs of untransfected NIH3T3 fibroblast cells (A) with their flat epitheloid appearance, the FGF3-transformed spindle-shaped DMI-1 cells (B), and vector-transfected DMI-1 cells (C) are shown comparatively. Stably transfected DMI-1 cell lines expressing the dominant negative FGFR2(IIIb) isoform (D) showing a revertant phenotype and FGFR2(IIIc) isoform (E) representing an intermediate reversion of the transformed morphology.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although mitogenic stimulation of NIH3T3 cells by FGF3 is dependent on the presence of soluble heparin, heparin at the same dose inhibits the morphological transformation in DMI-1 cells (1Kiefer P. Peters G. Dickson C. Mol. Cell. Biol. 1991; 11: 5929-5936Crossref PubMed Scopus (26) Google Scholar, 25Mathieu M. Chatelain E. Ornitz D. Bresnick J. Mason I. Kiefer P. Dickson C. J. Biol. Chem. 1995; 270: 24197-24203Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 28Kiefer P. Peters G. Dickson C. Mol. Cell. Biol. 1993; 13: 5781-5793Crossref PubMed Scopus (30) Google Scholar). Furthermore, purified FGF3 from DMI cells induces mitogenesis of NIH3T3, but even at higher concentrations this does not lead to the transformation of NIH3T3 cells. Therefore, the localization of FGF3 at the cell surface through binding to HSPGs could be seen as the essential prerequisite for its transforming potential. To test this hypothesis, a chimeric FGF3 protein was created and attached to the cell surface via a glycophospholipid anchor, which renders the FGF3 cell surface localization independent of its binding to HSPGs. To generate a chimeric protein of FGF3 linked to a GPI, the signal sequence of decay accelerating factor (CD55; DAF), which directs attachment of a GPI anchor, was fused to the C terminal of FGF3 (Fig.4A). The previously described plasmid pKC3.2 (28Kiefer P. Peters G. Dickson C. Mol. Cell. Biol. 1993; 13: 5781-5793Crossref PubMed Scopus (30) Google Scholar) contains a FGF3 cDNA where the AUG initiation codon was optimized for efficient translation of an FGF3, which is exclusively directed into the secretory pathway. COS-1 cells transiently transfected with pKC3.2 express several FGF3-related products that can be detected by immunoblotting with specific antisera raised against the C terminal peptide of mouse FGF3. Two major intracellular species of 31.5 and 30.5 kDa (gp31.5 and gp30.5) and two much less abundant non-glycosylated forms, 28.5 and 27.5 kDa, can be observed (Ref. 28Kiefer P. Peters G. Dickson C. Mol. Cell. Biol. 1993; 13: 5781-5793Crossref PubMed Scopus (30) Google Scholar and Fig.5A). As previously shown (28Kiefer P. Peters G. Dickson C. Mol. Cell. Biol. 1993; 13: 5781-5793Crossref PubMed Scopus (30) Google Scholar,31Dixon M. Deed R. Acland P. Moore R. Whyte A. Peters G. Dickson C. Mol. Cell. Biol. 1989; 9: 4896-4902Crossref PubMed Scopus (37) Google Scholar), each pair differs with respect to the presence or absence of the signal peptide. The last 37 amino acids of DAF were fused in-frame to the C terminal of FGF3 to create a fusion protein.Figure 5The effect of GPI-anchored FGF3 on its secretion.Immunoblot analysis of cell extracts, ECM, and culture medium from COS-1 cell transfected with pKC3.2, pKC3.2-DAF, or the control vector pKC4. A, extracts of COS-1 cells transfected with pKC3.2, pKC3.2-DAF, or the empty vector pKC4 were separated by SDS-PAGE, and the FGF3-related proteins were detected by immunoblotting with a rabbit polyclonal antiserum against FGF3. B, COS-1 cells transfected with pKC3.2, pKC3.2-DAF, or the empty vector pKC4 were harvested after 48 h, and the culture fluid was recovered. The cells were washed with PBS and removed from the culture dish with 0.5% Triton X-100 in PBS. The material remaining on the dish was operationally defined as ECM and recovered in dissociation buffer as described under “Experimental Procedures.” Samples of ECM and culture medium were fractionated by SDS-PAGE on a 12.5% gel and immunoblotted with the antiserum against FGF3. The + and − indicate whether the cells were grown in the presence or absence of 10 μg/ml heparin. The immunocomplexes were visualized by ECL using a specific anti-rabbit secondary antibody (for details see “Experimental Procedures”). The protein sizes were calculated relative to prestained protein standards.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To confirm the presence of the GPI anchor, COS-1 cells were transfected with pKC3.2-DAF and metabolically labeled with [3H]ethanolamine, a component of the GPI anchor.3H-labeled FGF3-related proteins were immunoprecipitated with FGF3-specific antisera only from pKC3.2-DAF-transfected cells encoding the fusion protein but not from pKC3.2-transfected control cells. Two products of 32.5 and 33.5 kDa, analogous to the major glycosylated FGF3 species of 30.5 and 31.5 kDa, could be identified by autoradiography (Fig. 4B). The function of GPI modification is to direct proteins efficiently to the cell surface where they stay integrated into the membrane by their phospholipid moiety. To test the ability of the GPI anchor to stably attach FGF3 at the cell surface, COS-1 cells transfected with pKC3.2 and pKC3.2-DAF cells were analyzed for cell-associated and secreted products in the presence or absence of heparin. The apparent molecular mass of the three major intracellular FGF3-DAF-related proteins were increased by ∼2 kDa consistent with the presence of the GPI moiety (Fig.5A). These isoforms are analogous to the FGF3 products gp31.5 and gp30.5, which are both glycosylated but with and without the signal peptide respectively, and gp28.5 the non-glycosylated form with a retained signal peptide. The secreted 32.5-kDa form associates with the cell surface and ECM by binding to HSPGs, which can be reversed by incubation with soluble heparin. Hence, in the presence of heparin the concentration of FGF3 in the culture medium increases (Fig.5B). In contrast, the exported chimeric FGF3-DAF could not be displaced from its binding to the cell surface by the addition of heparin. The results are consistent with the conclusion that the chimeric FGF3-DAF contains a functional GPI anchor, which leads to a stable integration of a substantial amount of FGF3 into the cell membrane. To investigate further the influence of the GPI tag on the subcellular distribution of FGF3, COS-1 cells were transfected with pKC3.2 and pKC3.2-DAF, grown in the presence or absence of heparin, and examined by immunofluorescence. The staining patterns of the intracellular FGF3 and FGF3-DAF proteins displayed a typical juxtanuclear distribution characteristic for proteins that are located in the Golgi complex, confirming that both wild type FGF3 and mutant proteins accumulate in the Golgi stacks. The GPI tag appears not to change the primary intracellular distribution of FGF3 (Fig. 6, A andB). The cell surface staining analysis of fixed and non-permeabilized COS-1 cells expressing FGF3 and FGF3-DAF demonstrated in both cases FGF3-related protein at the cell surface with a clear, more intense staining when the cells have been transfected with the FGF3-DAF cDNA (Fig. 6, C and E). In the presence of heparin, cell surface staining for FGF3 was significantly reduced in cells transfected with FGF3 cDNA (Fig. 6D), whereas the cell sur
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