Membrane Heparan Sulfate Proteoglycan-supported FGF2-FGFR1 Signaling (2001)

Fibroblast growth factor 2 (FGF2)-initiated FGF receptor (FGFR)-signaling requires the assistance of heparin/heparan sulfate. Here, we evaluated the effects of different heparan sulfate proteoglycan (HSPG)-expressing cell lines and HSPGs derived from these cells on FGF2-induced FGFR1-phosphorylation in heparan sulfate-negative BaF3 cells. HSPGs supplied in membrane-associated form, by presenting cells, were all effective promotors of FGF2-initiated FGFR1 phosphorylation, independently of their nature (syndecan/glypican) or cellular origin (human lung fibroblasts, transfected Namalwa cells, or transfected K562 cells). A treatment with heparitinase initially stimulated, but finally completely inhibited, the activity of these presenting cells. In comparison, equivalent amounts of soluble HSPGs, obtained by trypsinization of these cells or by immunopurification from cell extracts, did not promote FGF2-induced FGFR1-phosphorylation, yet removal of the less anionic species or a further treatment with heparitinase converted these soluble fractions into potent activators of FGF2/FGFR1 signaling. Extrapolating from current structural models, we suggest that FGFR dimerization and autophosphorylation is supported by cooperative "heparin-like end structures," and that cell surface association and concentration compensate for the relative scarcity of such end structures in native HSPGs. In this model, "proteolytic" shedding of heparan sulfate would act as a diluting, down-regulatory mechanism, while "heparanolytic" shedding might act as an up-regulatory mechanism, by increasing the concentration of these end structures. Fibroblast growth factor 2 (FGF2)-initiated FGF receptor (FGFR)-signaling requires the assistance of heparin/heparan sulfate. Here, we evaluated the effects of different heparan sulfate proteoglycan (HSPG)-expressing cell lines and HSPGs derived from these cells on FGF2-induced FGFR1-phosphorylation in heparan sulfate-negative BaF3 cells. HSPGs supplied in membrane-associated form, by presenting cells, were all effective promotors of FGF2-initiated FGFR1 phosphorylation, independently of their nature (syndecan/glypican) or cellular origin (human lung fibroblasts, transfected Namalwa cells, or transfected K562 cells). A treatment with heparitinase initially stimulated, but finally completely inhibited, the activity of these presenting cells. In comparison, equivalent amounts of soluble HSPGs, obtained by trypsinization of these cells or by immunopurification from cell extracts, did not promote FGF2-induced FGFR1-phosphorylation, yet removal of the less anionic species or a further treatment with heparitinase converted these soluble fractions into potent activators of FGF2/FGFR1 signaling. Extrapolating from current structural models, we suggest that FGFR dimerization and autophosphorylation is supported by cooperative "heparin-like end structures," and that cell surface association and concentration compensate for the relative scarcity of such end structures in native HSPGs. In this model, "proteolytic" shedding of heparan sulfate would act as a diluting, down-regulatory mechanism, while "heparanolytic" shedding might act as an up-regulatory mechanism, by increasing the concentration of these end structures. fibroblast growth factor FGF receptor heparan sulfate proteoglycan heparan sulfate phosphate-buffered saline hemagglutinin A bovine serum albumin monoclonal antibody polyacrylamide gel electrophoresis proteoglycan polyvinylidene difluoride Fibroblast growth factors (FGFs)1 bind not only to their cognate receptors (FGFRs) but also to heparan sulfate proteoglycans (HSPGs). HSPGs are associated with the cell surface of many, if not most, cell types. Most known HSPG functions are contributed by the interactions of the heparan sulfate (HS) chains of these molecules. Besides growth factors, these HSPGs interact with various adhesion molecules, protease inhibitors, and enzymes, modifying the spatial distributions and activities of these ligands. Among FGFs, the interaction with FGF2 has been studied most intensively, and it is now generally accepted that HSPGs play important roles in FGF2 signaling. Initially, the association of FGF2 with HS has been proposed to protect this FGF from proteolysis and thermal denaturation (1Saksela O. Rifkin D.B. J. Cell Biol. 1990; 110: 767-775Crossref PubMed Scopus (433) Google Scholar, 2Vlodavsky I. Miao H.Q. Medalion B. Danagher P. Ron D. Cancer Metastasis Rev. 1996; 15: 177-186Crossref PubMed Scopus (270) Google Scholar) and to serve as a reservoir of growth factor that can be released by enzymes that degrade the proteoglycans (1Saksela O. Rifkin D.B. J. Cell Biol. 1990; 110: 767-775Crossref PubMed Scopus (433) Google Scholar). Later, HSPGs were identified as co-receptors for FGF2, strongly promoting FGF-FGFR binding and the subsequent activation of the receptor (3Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Abstract Full Text PDF PubMed Scopus (2094) Google Scholar, 4Steinfeld R. Van Den Berghe H. David G. J. Cell Biol. 1996; 133: 405-416Crossref PubMed Scopus (232) Google Scholar). Recently, genetic studies in Drosophila provided compelling evidence that HSPGs are essential for FGF signaling in vivo (5Lin X. Buff E.M. Perrimon N. Michelson A.M. Development. 1999; 126: 3715-3723Crossref PubMed Google Scholar). Although the importance of HSPG in FGF-signaling is well documented, the nature of the "co-receptor" and the precise mechanisms at work are less well characterized. Distinctive core protein structures define two major families of cell surface-associated HSPGs: syndecans and glypicans (6David G. FASEB J. 1993; 7: 1023-1030Crossref PubMed Scopus (375) Google Scholar). Prior work from our laboratory showed that syndecans and glypican-1 stimulate FGF2-FGFR1 interaction and signaling in K562 cells, at least when co-expressed with receptor in these cells (4Steinfeld R. Van Den Berghe H. David G. J. Cell Biol. 1996; 133: 405-416Crossref PubMed Scopus (232) Google Scholar). This agrees with the work of other groups, showing that cell surface syndecan-1 from Raji cells acts as a positive regulator of FGF2 binding and signaling (7Filla M.S. Dam P. Rapraeger A.C. J. Cell. Physiol. 1998; 174: 310-321Crossref PubMed Scopus (122) Google Scholar), that syndecan-2 on human macrophages promotes FGF2-mediated proliferation (8Clasper S. Vekemans S. Fiore M. Plebanski M. Wordsworth P. David G. Jackson D.G. J. Biol. Chem. 1999; 274: 24113-24123Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), and that glypican-1 can stimulate FGF2 signaling (9Bonneh-Barkay D. Shlissel M. Berman B. Shaoul E. Admon A. Vlodavsky I. Carey D.J. Asundi V.K. Reich-Slotky R. Ron D. J. Biol. Chem. 1997; 272: 12415-12421Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 10Kleeff J. Ishiwata T. Kumbasar A. Friess H. Buchler M.W. Lander A.D. Korc M. J. Clin. Invest. 1998; 102: 1662-1673Crossref PubMed Scopus (316) Google Scholar). Meanwhile, there are also other, contradictory reports. Syndecans and glypican-1 purified from human lung fibroblast extracts are unable to promote high affinity binding of FGF2 to FGFR1 (11Aviezer D. Levy E. Safran M. Svahn C. Buddecke E. Schmidt A. David G. Vlodavsky I. Yayon A. J. Biol. Chem. 1994; 269: 114-121Abstract Full Text PDF PubMed Google Scholar), overexpression of syndecan-1 in NIH 3T3 cells inhibits FGF2-induced proliferation (12Mali M. Elenius K. Miettinen H.M. Jalkanen M. J. Biol. Chem. 1993; 268: 24215-24222Abstract Full Text PDF PubMed Google Scholar), and HSPGs purified from endothelial secretions prevent FGF2 binding to vascular smooth muscle cells and inhibit FGF2-induced mitogenesis (13Forsten K.E. Courant N.A. Nugent M.A. J. Cell. Physiol. 1997; 172: 209-220Crossref PubMed Scopus (66) Google Scholar). One possible explanation for this discrepancy is that the HSPGs were from different sources and might have had different compositions. It is well known, indeed, that the fine structure of HS is cell- and differentiation-specific, and highly diverse (14Lyon M. Deakin J.A. Gallagher J.T. J. Biol. Chem. 1994; 269: 11208-11215Abstract Full Text PDF PubMed Google Scholar, 15Kato M. Wang H. Bernfield M. Gallagher J.T. Turnbull J.E. J. Biol. Chem. 1994; 269: 18881-18890Abstract Full Text PDF PubMed Google Scholar). Another possibility is that the membrane association of HSPG might play a role in promoting FGF2 signaling, since most of the inhibitory effects of HSPGs reported so far relate to soluble forms. Syndecans and glypicans are constitutively shed from cultured cells (16Mertens G. Van der Schueren B. van den Berghe H. David G. J. Cell Biol. 1996; 132: 487-497Crossref PubMed Scopus (101) Google Scholar, 17Fitzgerald M.L. Wang Z. Park P.W. Murphy G. Bernfield M. J. Cell Biol. 2000; 148: 811-824Crossref PubMed Scopus (348) Google Scholar), and shed soluble syndecan ectodomains can also be found in inflammatory fluid (18Subramanian S.V. Fitzgerald M.L. Bernfield M. J. Biol. Chem. 1997; 272: 14713-14720Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar), where they appear to act as inhibitors of FGF2 (19Kato M. Wang H. Kainulainen V. Fitzgerald M.L. Ledbetter S. Ornitz D.M. Bernfield M. Nat. Med. 1998; 4: 691-697Crossref PubMed Scopus (287) Google Scholar). Finally, sometimes different end point analyses were used as a measure of receptor activity. In the present study, we attempted to define the relative importance of these variables. We used FGF2-induced FGFR1 autophosphorylation as an end point and tested the activities of HSPGs from different origins, both in soluble and in membrane-associated form. Clones encoding FGFR1 were isolated from a human embryonic lung fibroblast λ ZAPII phage library (4Steinfeld R. Van Den Berghe H. David G. J. Cell Biol. 1996; 133: 405-416Crossref PubMed Scopus (232) Google Scholar). The cDNA of the IgII/IIIc isoform of FGFR1 was used as a polymerase chain reaction template. The primer 5′-CGGGATCCAGGCCGTCCCCGACCTTGCCT was designed to introduce a BamHI restriction site at the 5′-end. The BamHI-SpeI fragment of this FGFR1 cDNA was blunted at the SpeI site and cloned into the eukaryotic expression vector pDisplay (Invitrogen, San Diego, CA), using the compatible BglII and SmaI sites, to produce an N-terminal hemagglutinin A (HA) epitope-tagged version of this receptor. The cDNAs for syndecan-1 (20Mali M. Jaakkola P. Arvilommi A.M. Jalkanen M. J. Biol. Chem. 1990; 265: 6884-6889Abstract Full Text PDF PubMed Google Scholar) and glypican-1 (21David G. Lories V. Decock B. Marynen P. Cassiman J.J. Van den Berghe H. J. Cell Biol. 1990; 111: 3165-3176Crossref PubMed Scopus (221) Google Scholar) were cloned into the episomal expression vector pREP4 (Invitrogen), using the KpnI and NheI sites and the HindIII and NotI sites, respectively. The same cDNAs were also cloned in pcDNA3/neo, using the KpnI and NotI and HindIII and XbaI sites, respectively. Chain valence mutants of glypican-1 were generated with the Transformer site-directed mutagenesis kit (CLONTECH Laboratories, Palo Alto, CA). The primer (and corresponding antisense primer) 5′-GACGACGGCAGCGGCTCGGGCGCCGGTGATGGCTG was used to generate the two-chain form (changing Ser488 into Ala488). The primer (and corresponding antisense primer) 5′-GACGACGGCAGCGGCGCGGGCGCCGGTGATGGCTG was used to generate the single chain form (changing Ser488 into Ala488 and Ser490 into Ala490). The HindIII–XbaI fragments of the mutant cDNAs, blunted at the XbaI sites, were cloned into the episomal expression vector pREP4 (Invitrogen), using the HindIII and blunted XhoI site. BaF3 cells (generously provided by Dr. D. M. Ornitz) were routinely cultured in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Hyclone, Logan, Utah) and 10% WEHI cell-conditioned medium (as a source of murine interleukin-3). Before transfection, cells were washed with calcium/magnesium-free phosphate-buffered saline (PBS). For transfection, 1 × 107 cells were incubated with 30 μg of linearized HA-FGFR1-pDisplay or pDisplay plasmid in 0.5 ml of calcium/magnesium-free PBS at 4 °C for 10 min. Cells were then electroporated at 350 V and 960 microfarads (Gene Pulser; Bio-Rad). After 24–48 h of cell culture, selection was started with 600 μg/ml G418 (Life Technologies). Two weeks later, stable clones were obtained, and subclones were established after 1 month. Individual clones were tested for HA-FGFR1 expression as described under "Western Blotting." One of the highest expressors, clone B6, was selected for further experiments. Namalwa cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum. For transfection, 1 × 107 cells were incubated with 30 μg of linearized syndecan-1-pcDNA/neo, glypican-1-pcDNA/neo, or pcDNA/neo plasmid in 0.5 ml of calcium/magnesium-free PBS at 4 °C for 10 min. Cells were then electroporated at 240 V and 960 microfarads. Selection was started 24–48 h later, with 1.5 mg/ml G418. Stable transfection was achieved after 2 weeks. The selected population was characterized by quantitative immunofluorescence flow cytometry, using HS and core protein-specific antibodies (see below). K562 cells were routinely cultured in Dulbecco's modified Eagle's medium/F-12 medium (Life Technologies) supplemented with 10% fetal calf serum. For transfection, 5 × 106 cells were incubated with 30 μg of syndecan-1-pREP4, glypican-1-pREP4, or pREP4 plasmid in 0.5 ml of calcium/magnesium-free PBS for 10 min at 4 °C. Cells were then electroporated at 250 V and 960 microfarads. Selection with hygromycin B (Roche Molecular Biochemicals) at 200 μg/ml resulted in stable cell populations that were not further subcloned. The population was characterized by quantitative immunofluorescence flow cytometry, using HS and core protein-specific antibodies. Transfections with pRep4 vectors encoding one-chain (SAA), two-chain (SSA), and wild type (SSS) forms of glypican-1 into Namalwa cells were performed in similar ways. Namalwa cells or K562 cells were incubated with 5 μg/ml 10E4 (monoclonal antibody (mAb) recognizing HS) or the core protein-specific antibodies BB4 (mAb recognizing syndecan-1; Serotec Ltd., Oxford, United Kingdom) and S1 (mAb recognizing glypican-1). After 1 h at 4 °C, the cells were washed with PBS plus 2% bovine serum albumin (BSA) and incubated for another 1 h at 4 °C with fluorescein isothiocyanate-labeled goat anti-mouse antibodies (Nordic Immunology, Tilburg, The Netherlands). Cells were washed again with PBS plus 2% BSA and fixed with formaldehyde. The fluorescence was measured with a FACSort (Becton Dickinson, Mountain View, CA). The value obtained for cells that were incubated with fluorescein isothiocyanate-labeled goat anti-mouse antibodies only was taken as background fluorescence. Proteins were extracted from cells with lysis buffer (0.5% Triton X-100 in Tris-buffered saline, supplemented with 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin, 5 μg/ml leupeptin, and 50 mm NaF). Cell lysates were clarified by centrifugation at 14,000 × gfor 15 min. Supernatants were subjected to 4–12% SDS-polyacrylamide gel electrophoresis (PAGE) (Bio-Rad). Proteins were then transferred for 3 h at 0.5 A to PVDF membranes (Millipore, Bedford, MA). Membranes were blocked with Tris-buffered saline (pH 7.4) containing 0.2% I-block (Tropix, Bedford, MA) and 0.01% Tween, at 37 °C for 1 h and incubated with the anti-HA mAb 3F10 (Roche Molecular Biochemicals). HA-FGFR1 was visualized by chemiluminescence, using peroxidase-conjugated goat anti-rat antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and ECL Western blotting detection reagents (PerkinElmer Life Sciences). HA-FGFR1 was extracted from BaF3 B6 cells with lysis buffer. Total cell lysate was presorbed with protein A-Sepharose beads (Amersham Pharmacia Biotech). The supernatant was incubated with the anti-HA mAb 12CA5 (10 μg/ml; Roche Molecular Biochemicals) at 4 °C for 2 h. The immune complex was collected by further incubation with protein A beads at 4 °C overnight. After extensive washing with lysis buffer and PBS, the beads were incubated with 0.5 milliunits of endoglycosidase H, 1 unit of N-glycosidase F (Roche Molecular Biochemicals), or only assay buffer at 37 °C overnight. Then the protein A beads were boiled in 2× SDS sample buffer in the presence of 10 mmdithiothreitol. Samples were fractionated by SDS-PAGE and transferred to PVDF membranes. mAb 3F10 was used to detect the HA-FGFR1 in Western blotting. HA-FGFR1-transfected or sham-transfected BaF3 cells were washed with cold PBS and incubated with 0.5 mg/ml sulfo-N-hydroxysuccinimide-biotin (Pierce) at 4 °C for 20 min. Cells were washed and incubated with 0.5 mg/ml sulfo-N-hydroxysuccinimide-biotin for another 20 min at 4 °C. After three washes with cold PBS, cells were lysed with lysis buffer. Streptavidin beads were added to the cell lysate to capture all biotinylated proteins. After 2 h of incubation at 4 °C, the streptavidin beads were washed and boiled in 2× SDS sample buffer in the presence of dithiothreitol. Both the biotinylated and nonbiotinylated fractions were subjected to SDS-PAGE and then transferred to PVDF membranes. mAb 3F10 was used to detect the HA-FGFR1 in Western blotting. BaF3 B6 cells were serum-starved in RPMI medium supplemented with 1 mg/ml BSA and 2% WEHI cell-conditioned medium. After 2 days of starvation, 2 ng/ml FGF2 (Roche Molecular Biochemicals) was added to cells in HEPES-buffered medium (RPMI medium, 25 mm HEPES, pH 7.5, 1 mg/ml BSA, 0.1 mm orthovanadate), in the presence or absence of 100 ng/ml heparin (Calbiochem), at 4 °C for 1.5 h. Then the cells were warmed up at 37 °C for 5–30 min and extracted with lysis buffer in the presence of tyrosine phosphatase inhibitor (1 mmorthovanadate). Cell lysates were clarified by centrifugation at 14,000 × g for 15 min. The supernatants were incubated with PY-20 (anti-phosphotyrosine antibody)-conjugated protein A-agarose beads (Santa Cruz Biotechnology) overnight at 4 °C. The beads were washed with lysis buffer and boiled in 2× SDS sample buffer in the presence of dithiothreitol. Samples were fractionated by SDS-PAGE and then transferred to PVDF membranes. HA-FGFR1 was detected with mAb 3F10. For testing the effects of membrane-associated HSPGs on FGF2-induced HA-FGFR1 phosphorylation, 3 × 106 B6 cells were incubated with 1 × 106 HSPG-presenting cells (fetal human lung fibroblasts; syndecan-1-transfected, glypican-1-transfected, or sham-transfected K562 cells; or transfected Namalwa cells) in the presence of FGF2 at 4 °C for 1.5 h. To remove the cell surface HS, cells were treated with 0.01 units/ml heparinase (Seikagaku Corp., Tokyo, Japan) at 37 °C for 40 min. Treated cells were incubated with B6 cells and FGF2 and analyzed as described above. For analyzing the time course of the effect of a heparitinase digestion, cells were incubated with 0.006 units/ml heparitinase (Seikagaku) for 2, 5, 10, or 40 min at 37 °C. Cells were washed twice with assay buffer (HEPES-buffered medium) and then mixed with B6 cells in the presence of FGF2. The phosphorylation of HA-FGFR1 was assayed as described above. To test the soluble forms of these HSPGs, HSPG-presenting cells were treated with 100 μg/ml trypsin (Sigma) at 4 °C for 10 min. Trypsin inhibitor (Sigma) was then added to quench the effect of trypsin. Further treatment with 0.006 units/ml heparitinase was performed at 37 °C for 40 min. Soluble HS chains or chain clusters prepared from immunopurified syndecan-1 or glypican-1 (see below) were incubated with B6 cells in the presence of FGF2, and tested as described above. Cell surface proteoglycans were extracted from fetal human lung fibroblasts, syndecan-1- or glypican-1-transfected K562 cells, and transfected Namalwa cells as described previously (4Steinfeld R. Van Den Berghe H. David G. J. Cell Biol. 1996; 133: 405-416Crossref PubMed Scopus (232) Google Scholar, 22Lories V. Cassiman J.J. Van den Berghe H. David G. J. Biol. Chem. 1989; 264: 7009-7016Abstract Full Text PDF PubMed Google Scholar). Briefly, cells were labeled with [35S]sulfate and lysed with a Triton X-100 buffer in the presence of protease inhibitors. The cell extract was then centrifuged and concentrated on a DEAE-Trisacryl M column (Life Technologies). Proteoglycans were immunopurified with specific mAb, immobilized on CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech). After further by on a column (Amersham Pharmacia in these HSPGs were treated with (Seikagaku) 3 h at 37 Soluble HS fractions were obtained from these HSPGs by trypsin 30 min at 37 or treatment overnight at 4 treated proteoglycan fractions were on was at 37 °C for 3 h. To test the effects of heparin or HSPGs on receptor a to background of HSPG expression is BaF3 cells of cells also be to heparan sulfate transfection with core protein cDNAs, their for the of receptor and HSPG experiments. suggest that cell surface HSPGs can FGF2 binding to an FGFR that is on cells and the signaling of this receptor (7Filla M.S. Dam P. Rapraeger A.C. J. Cell. Physiol. 1998; 174: 310-321Crossref PubMed Scopus (122) Google Scholar, C. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We for such of the using as BaF3 cells transfected with an epitope-tagged human FGFR1 IgII/IIIc isoform were to act as cells. Namalwa and K562 cells, transfected with syndecan-1 or glypican-1 cDNAs, and fetal human lung fibroblasts, high of syndecans and were to act as HSPG-presenting cells. The expression of HA-FGFR1 in the BaF3 cells was by Western blotting. HA-FGFR1 as two 100 and The of this HA-FGFR1 is and that the and forms two different of receptor N-glycosidase 1 Cell surface that the of the cell of the isoform 1 HA-FGFR1-transfected BaF3 cells were and of the highest expressors, clone B6, was for further experiments. B6 and sham-transfected BaF3 cells were then incubated with or FGF2 in the presence or absence of and the phosphorylation of HA-FGFR1 was analyzed as described under blotting with the phosphorylation of HA-FGFR1 in BaF3 cells was the of both and heparin 2 A time course showed that FGFR1 autophosphorylation 15 min of to at 37 °C 2 in BaF3 cells. FGF2 and heparin and sham-transfected BaF3 cells were of 3 × 106 cells were incubated with or 2 ng/ml FGF2, in the absence or presence of 100 ng/ml heparin at 4 °C. Cells were warmed up at 37 °C and then The mAb PY-20 was used to all of the present in the The and supernatants were then subjected to SDS-PAGE and transferred to PVDF membranes. HA-FGFR1 was detected with mAb 3F10. The autophosphorylation of HA-FGFR1 is on the of both FGF2 and FGF2 plus of FGF2-induced FGFR1 After incubation with FGF2 and heparin at 4 °C, the cells were warmed up at 37 °C for 5–30 as and analyzed as above. FGFR1 autophosphorylation the highest 15 human lung amounts of and on their cell V. Cassiman J.J. Van den Berghe H. David G. J. Biol. Chem. 1989; 264: 7009-7016Abstract Full Text PDF PubMed Google Scholar). Namalwa and K562 cells, in to of cell surface glypican-1 Namalwa cells) and syndecans K562 cells) (4Steinfeld R. Van Den Berghe H. David G. J. Cell Biol. 1996; 133: 405-416Crossref PubMed Scopus (232) Google Scholar). K562 cells amounts of into their S. A.D. I. Cell PubMed Scopus Google Scholar), but under only of this proteoglycan can be detected on the of these cells not To cells that only or a single major form of both the Namalwa and K562 cell lines were transfected with syndecan-1 or glypican-1 The expression of cell surface syndecan-1 and glypican-1 proteoglycan were by quantitative immunofluorescence flow cytometry, using the mAb 10E4 and protein-specific antibodies. In all cells, the expression of cell surface HS was by at least 1 of The protein-specific antibodies BB4 and S1 the expression of cell surface syndecan-1 and HA-FGFR1-transfected BaF3 cells were then incubated with HSPG-presenting cells in the presence of FGF2 and for autophosphorylation of independently of their all membrane-associated HSPGs syndecan-1 and glypican-1 in the of Namalwa or K562 cells and cell surface HSPGs in the of fetal human lung the phosphorylation of In K562 or Namalwa cells that had been treated with heparinase or transfected with vectors had effects on FGFR1 This that the effects of these HSPG-presenting cells on FGFR1 phosphorylation were the presenting cells with heparitinase of for increasing of and then the of these cells to FGF2-induced receptor autophosphorylation of heparitinase on HSPG-presenting cells. Namalwa cells were treated with heparitinase at 37 °C for different of as of 1 × 106 cells were washed with assay buffer and mixed with of 3 × 106 B6 cells in the presence of FGF2. HA-FGFR1 was detected as described under activity. are as in had that purified syndecans and glypican-1 from cultured fetal human lung not FGF2 in high affinity receptor binding (11Aviezer D. Levy E. Safran M. Svahn C. Buddecke E. Schmidt A. David G. Vlodavsky I. Yayon A. J. Biol. Chem. 1994; 269: 114-121Abstract Full Text PDF PubMed Google Scholar). in the present forms of these HSPGs were to promote FGF2-induced receptor To test this membrane association a the HSPG-presenting cells were treated with trypsin. Trypsin would be to the core of these proteoglycans and to the and fragments that the HS chains into medium. of the HSPG expression by quantitative immunofluorescence flow that after HSPG on the cell surface not The supernatants of these trypsin the released were supplemented with trypsin inhibitor and then added to the HA-FGFR1-transfected B6 cells in the presence of FGF2. The soluble forms of these HSPGs all to FGF2-induced HA-FGFR1 phosphorylation To that HS had been the of the HSPG-presenting cells and supernatants were also supplemented with trypsin and added to the B6 cells. of these FGF2-induced receptor phosphorylation not when HSPG-presenting cells were mixed with soluble HS, obtained by the equivalent of 10 cells, phosphorylation of HA-FGFR1 be detected in the