Identification of Glypican as a Dual Modulator of the Biological Activity of Fibroblast Growth Factors
1997; Elsevier BV; Volume: 272; Issue: 19 Linguagem: Inglês
10.1074/jbc.272.19.12415
ISSN1083-351X
AutoresDafna Bonneh‐Barkay, Meir Shlissel, Bluma Berman, Ester Shaoul, Arie Admon, Israël Vlodavsky, David J. Carey, Vinod Asundi, Ronit Reich‐Slotky, Dina Ron,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoHeparan sulfate moieties of cell-surface proteoglycans modulate the biological responses to fibroblast growth factors (FGFs). We have reported previously that cell-associated heparan sulfates inhibit the binding of the keratinocyte growth factor (KGF), but enhance the binding of acidic FGF to the KGF receptor, both in keratinocytes, which naturally express this receptor, and in rat myoblasts, which ectopically express it (Reich-Slotky, R., Bonneh-Barkay, D., Shaoul, E., Berman, B., Svahn, C. M., and Ron, D. (1994) J. Biol. Chem. 269, 32279–32285). The proteoglycan bearing these modulatory heparan sulfates was purified to homogeneity from salt extracts of rat myoblasts by anion-exchange and FGF affinity chromatography and was identified as rat glypican. Affinity-purified glypican augmented the binding of acidic FGF and basic FGF to human FGF receptor-1 in a cell-free system. This effect was abolished following digestion of glypican by heparinase. Addition of purified soluble glypican effectively replaced heparin in supporting basic FGF-induced cellular proliferation of heparan sulfate-negative cells expressing recombinant FGF receptor-1. In keratinocytes, glypican strongly inhibited the mitogenic response to KGF while enhancing the response to acidic FGF. Taken together, these findings demonstrate that glypican plays an important role in regulating the biological activity of fibroblast growth factors and that, for different growth factors, glypican can either enhance or suppress cellular responsiveness. Heparan sulfate moieties of cell-surface proteoglycans modulate the biological responses to fibroblast growth factors (FGFs). We have reported previously that cell-associated heparan sulfates inhibit the binding of the keratinocyte growth factor (KGF), but enhance the binding of acidic FGF to the KGF receptor, both in keratinocytes, which naturally express this receptor, and in rat myoblasts, which ectopically express it (Reich-Slotky, R., Bonneh-Barkay, D., Shaoul, E., Berman, B., Svahn, C. M., and Ron, D. (1994) J. Biol. Chem. 269, 32279–32285). The proteoglycan bearing these modulatory heparan sulfates was purified to homogeneity from salt extracts of rat myoblasts by anion-exchange and FGF affinity chromatography and was identified as rat glypican. Affinity-purified glypican augmented the binding of acidic FGF and basic FGF to human FGF receptor-1 in a cell-free system. This effect was abolished following digestion of glypican by heparinase. Addition of purified soluble glypican effectively replaced heparin in supporting basic FGF-induced cellular proliferation of heparan sulfate-negative cells expressing recombinant FGF receptor-1. In keratinocytes, glypican strongly inhibited the mitogenic response to KGF while enhancing the response to acidic FGF. Taken together, these findings demonstrate that glypican plays an important role in regulating the biological activity of fibroblast growth factors and that, for different growth factors, glypican can either enhance or suppress cellular responsiveness. Proteoglycans are proteins bearing glycosaminoglycan side chains that exist in the extracellular matrix and on the surface of many cell types. These molecules are thought to play an important role in cell growth, morphogenesis, and cancer (1Hardingham T.E. Fosang A.J. FASEB J. 1992; 6: 861-870Crossref PubMed Scopus (1013) Google Scholar, 2Ruoslahti E. J. Biol. Chem. 1989; 264: 13369-13372Abstract Full Text PDF PubMed Google Scholar). The most abundant proteoglycans are those that bear glycosaminoglycan chains consisting of heparan sulfate (HS). 1The abbreviations used are: HS, heparan sulfate(s); HSPG, heparan sulfate proteoglycan; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; KGF, keratinocyte growth factor; KGFR, keratinocyte growth factor receptor; FCS, fetal calf serum; PAGE, polyacrylamide gel electrophoresis; GAG, glycosaminoglycan; HPLC, high pressure liquid chromatography. Heparan sulfate proteoglycans (HSPGs) interact with a variety of heparin-binding proteins such as extracellular matrix components and growth factors (1Hardingham T.E. Fosang A.J. FASEB J. 1992; 6: 861-870Crossref PubMed Scopus (1013) Google Scholar, 2Ruoslahti E. J. Biol. Chem. 1989; 264: 13369-13372Abstract Full Text PDF PubMed Google Scholar, 3Ruoslahti E. Yamaguchi Y. Cell. 1991; 64: 867-869Abstract Full Text PDF PubMed Scopus (1171) Google Scholar). Studies in recent years have strongly indicated that HSPGs are important modulators of the activity of heparin-binding growth factors (3Ruoslahti E. Yamaguchi Y. Cell. 1991; 64: 867-869Abstract Full Text PDF PubMed Scopus (1171) Google Scholar), an issue that has been particularly well studied for fibroblast growth factors (FGFs). FGFs constitute a large family of polypeptides that are important in the control of cell growth and differentiation and play a key role in oncogenesis and developmental processes including limb formation, mesoderm induction, and neuronal development (4Basilico C. Moscatelli D. Adv. Cancer Res. 1992; 59: 115-165Crossref PubMed Scopus (1057) Google Scholar). FGFs elicit their biological activities by interaction with four distinct cell-surface tyrosine kinase receptors (FGFR1–FGFR4) that display overlapping affinities for the various FGFs (5Johnson D.E. Williams L.T. Adv. Cancer Res. 1993; 60: 1-41Crossref PubMed Scopus (1178) Google Scholar). Several members of the receptor family also exist in alternatively spliced forms that display altered ligand binding properties (5Johnson D.E. Williams L.T. Adv. Cancer Res. 1993; 60: 1-41Crossref PubMed Scopus (1178) Google Scholar). For example, the KGF receptor (KGFR) is a splice variant of FGFR2. Whereas FGFR2 interacts with aFGF and bFGF, but not with KGF, KGFR binds aFGF and KGF and exhibits a significantly reduced affinity for bFGF (6Miki T. Bottaro D.P. Fleming T.P. Smith C.L. Burgess W.H. Chan A.M.-L. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 246-250Crossref PubMed Scopus (662) Google Scholar). Heparan sulfates or heparin can modulate the activities of FGFs by several mechanisms. They can stabilize FGFs by protecting them from proteolysis and thermal denaturation (7Saksela O. Rifkin D.B. J. Cell Biol. 1990; 110: 767-775Crossref PubMed Scopus (433) Google Scholar, 8Vlodavsky I. Miao H.-Q. Medalion B. Danagher P. Ron D. Cancer Metastasis Rev. 1996; 15: 177-186Crossref PubMed Scopus (270) Google Scholar). They can also increase the affinity of FGFs for their signaling receptors (8Vlodavsky I. Miao H.-Q. Medalion B. Danagher P. Ron D. Cancer Metastasis Rev. 1996; 15: 177-186Crossref PubMed Scopus (270) Google Scholar, 9Roghani M. Mansukhani A. Dell'Era P. Bellosta P. Basilico C. Rifkin D.B. Moscatelli D. J. Biol. Chem. 1994; 269: 3976-3984Abstract Full Text PDF PubMed Google Scholar, 10Rapraeger A.C. Guimond S. Krufka A. Olwin B.B. Methods Enzymol. 1994; 245: 219-240Crossref PubMed Scopus (96) Google Scholar) and facilitate receptor dimerization and subsequent signaling (10Rapraeger A.C. Guimond S. Krufka A. Olwin B.B. Methods Enzymol. 1994; 245: 219-240Crossref PubMed Scopus (96) Google Scholar, 11Spivak-Kroizman T. Lemmon M.A. Dikic I. Landbury J.E. Pinchasi D. Huang J. Jaye M. Crumley G. Schlessinger J. Lax I. Cell. 1994; 79: 1015-1024Abstract Full Text PDF PubMed Scopus (596) Google Scholar, 12Pantoliano M.W. Horlick R.A. Springer B.A. Van-Dyk D.E. Tobery T. Wetmore D.R. Lear J.D. Nahapetian A.T. Bradley J.D. Sisk W.P. Biochemistry. 1994; 33: 10229-10248Crossref PubMed Scopus (229) Google Scholar, 13Schlessinger J. Lax I. Lemon M. Cell. 1995; 93: 357-360Abstract Full Text PDF Scopus (453) Google Scholar). On the other hand, these molecules can also inhibit the activities of FGFs (14Mali M. Elenius K. Miettinen H.M. Jalkanen M. J. Biol. Chem. 1993; 268: 24215-24222Abstract Full Text PDF PubMed Google Scholar). The mechanisms by which HS exert these multiple effects are not very well understood. The modulatory effects of the low affinity binding sites were studied mainly with HS extracted from cells or with heparin, which shares structural similarity with HS and thus can mimic the action of cell- or matrix-associated HSPGs (15Spillmann D. Lindahl U. Curr. Opin. Struct. Biol. 1994; 4: 677-682Crossref Scopus (133) Google Scholar). Because the level of expression of HSPGs and the ability of cells to synthesize HS side chains of a defined structure are developmentally regulated (16David G. FASEB J. 1993; 7: 1023-1030Crossref PubMed Scopus (375) Google Scholar, 17Kato M. Wang H. Bernfield M. Gallagher J.T. Turnbull J.E. J. Biol. Chem. 1994; 269: 18881-18890Abstract Full Text PDF PubMed Google Scholar, 18Nurcombe V. Ford M.D. Wildschut J. Bartlett P.F. Science. 1993; 260: 103-106Crossref PubMed Scopus (374) Google Scholar), it is critical to identify the core proteins bearing such modulatory side chains, to elucidate whether the cores influence the structure and function of the glycosaminoglycans attached to them, and to determine the structural requirements for growth factor interaction with the HS moiety. Until now, most of the attempts were concentrated on the identification of native HSPGs that modulate interaction of bFGF with FGFR1. Perlecan, the large basal lamina proteoglycan, was identified as a major candidate for the bFGF low affinity accessory receptor (19Aviezer D. Hecht M. Safran M. Eisinger G. Yayon A. Cell. 1994; 79: 1005-1013Abstract Full Text PDF PubMed Scopus (493) Google Scholar). In addition, syndecans and glypican can either inhibit or stimulate bFGF/FGFR1 interactions and signaling depending on the cell type in which they are expressed or their level of expression (14Mali M. Elenius K. Miettinen H.M. Jalkanen M. J. Biol. Chem. 1993; 268: 24215-24222Abstract Full Text PDF PubMed Google Scholar, 20Aviezer 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, 21Steinfeld R. Van Der Berghe H. David G. J. Cell Biol. 1996; 133: 405-416Crossref PubMed Scopus (232) Google Scholar). Little is known about the identity of HSPGs that bind and modulate the activities of other members of the FGF family. In a previous study, we reported that cell-associated HSPGs exert a differential effect on the binding of KGF and aFGF to KGFR (22Reich-Slotky R. Bonneh-Barkay D. Shaoul E. Bluma B. Svahn C.M. Ron D. J. Biol. Chem. 1994; 269: 32279-32285Abstract Full Text PDF PubMed Google Scholar), which binds both growth factors equally well (23Bottaro D.P. Rubin J.S. Ron D. Finch P.W. Florio C. Aaronson S.A. J. Biol. Chem. 1990; 265: 12767-12770Abstract Full Text PDF PubMed Google Scholar). Thus, treatment of cells with a metabolic inhibitor of sulfation or with HS-degrading enzymes reduced the binding of aFGF to KGFR, but enhanced the binding of KGF. Addition of heparin reversed the effect (22Reich-Slotky R. Bonneh-Barkay D. Shaoul E. Bluma B. Svahn C.M. Ron D. J. Biol. Chem. 1994; 269: 32279-32285Abstract Full Text PDF PubMed Google Scholar). This differential effect was observed both in keratinocytes, which naturally express KGFR, and in the rat myoblast cell line L6E9, which ectopically expresses this receptor (22Reich-Slotky R. Bonneh-Barkay D. Shaoul E. Bluma B. Svahn C.M. Ron D. J. Biol. Chem. 1994; 269: 32279-32285Abstract Full Text PDF PubMed Google Scholar). This study was carried out to identify the proteoglycan that may be responsible for this effect. Here, we report the purification of such a proteoglycan from rat myoblasts and its identification as glypican (24Karthikeyan L. Maurel P. Rauch U. Margolis R.K. Margolis R.U. Biochem. Biophys. Res. Commun. 1992; 188: 395-401Crossref PubMed Scopus (46) Google Scholar). We show that glypican exerts either a stimulatory or an inhibitory activity that is dependent on the type of growth factor. Recombinant aFGF, bFGF, and KGF were produced in bacteria as described (25Ron D. Bottaro D.P. Finch P.W. Morris D. Rubin J.S. Aaronson S.A. J. Biol. Chem. 1993; 268: 2984-2988Abstract Full Text PDF PubMed Google Scholar, 26Gitay-Goren H. Soker S. Vlodavsky I. Neufeld G. J. Biol. Chem. 1992; 267: 6093-6098Abstract Full Text PDF PubMed Google Scholar). Bovine brain aFGF was purchased from R&D Systems. Carrier-free Na125I and Na235SO4 were purchased from DuPont NEN. Fetal and newborn calf sera and media were purchased from Life Technologies, Inc. Heparin from bovine lung and all other chemicals were purchased from Sigma. Chondroitinase ABC was from Seikagaku, and heparinases I and III were from Ibex Technologies. Fibronectin was purchased from Upstate Biotechnology, Inc. The rat myoblast cell line L6E9 and L6E9 cells transfected with KGFR (27Ron D. Reich R. Chedid M. Lengel C. Cohen O.E. Chan A.M.-L. Neufeld G. Miki T. Tronick S.R. J. Biol. Chem. 1993; 268: 5388-5394Abstract Full Text PDF PubMed Google Scholar) were grown in Dulbecco's modified Eagle's medium containing 10% FCS. Balb/MK cells were grown in low calcium medium containing 5 ng/ml epidermal growth factor and 10% dialyzed FCS as described previously (28Weissman B. Aaronson S.A. Cell. 1983; 32: 599-606Abstract Full Text PDF PubMed Scopus (160) Google Scholar). The lymphocytic cell line BaF3 transfected with mouse FGFR1 (designated F32 (29Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (562) Google Scholar)) was grown in RPMI 1640 medium supplemented with 10% FCS and 10% conditioned medium from WEHI-3B cells (30Treardy D.J. Morel P.A. Herberman R.B. Sakurai M. Cancer Res. 1991; 51: 4355-4359PubMed Google Scholar). A soluble extracellular domain of the short isoform of human FGFR1 (hR1 (31Eisemann A. Ahn J.A. Graziani G. Tronick S.R. Ron D. Oncogene. 1991; 6: 1195-1202PubMed Google Scholar)) was cloned into the APtag vector to produce an in-frame fusion of hR1 with secreted placental alkaline phosphatase (32Flanagan J.G. Leder P. Cell. 1990; 63: 185-194Abstract Full Text PDF PubMed Scopus (633) Google Scholar). This plasmid was cotransfected with the selectable NeoR marker into NIH/3T3 cells. Conditioned medium from G418-resistant colonies was screened for alkaline phosphatase activity (32Flanagan J.G. Leder P. Cell. 1990; 63: 185-194Abstract Full Text PDF PubMed Scopus (633) Google Scholar). The clone that produced the highest level of activity was expanded and used to purify the hR1/alkaline phosphatase fusion protein using bFGF affinity chromatography. Purified glypican and FGFs were radioiodinated using chloramine T (33Hunter W.M. Greenwood F.C. Nature. 1962; 194: 495-496Crossref PubMed Scopus (5861) Google Scholar) as described previously (27Ron D. Reich R. Chedid M. Lengel C. Cohen O.E. Chan A.M.-L. Neufeld G. Miki T. Tronick S.R. J. Biol. Chem. 1993; 268: 5388-5394Abstract Full Text PDF PubMed Google Scholar). Radiolabeled glypican was separated from free iodine by chromatography on DEAE-Sephacel, and radiolabeled FGFs were purified by heparin-Sepharose affinity chromatography (34Yuen S. Methods Enzymol. 1991; 198: 91-95Crossref PubMed Scopus (5) Google Scholar). Specific activities of iodinated proteins were in the range of 1–2.5 × 105 cpm/ng. Subconfluent cultures in 24-well plates were subjected to a binding assay as described previously (22Reich-Slotky R. Bonneh-Barkay D. Shaoul E. Bluma B. Svahn C.M. Ron D. J. Biol. Chem. 1994; 269: 32279-32285Abstract Full Text PDF PubMed Google Scholar,27Ron D. Reich R. Chedid M. Lengel C. Cohen O.E. Chan A.M.-L. Neufeld G. Miki T. Tronick S.R. J. Biol. Chem. 1993; 268: 5388-5394Abstract Full Text PDF PubMed Google Scholar). Binding sites were distinguished as low or high affinity by salt washes at neutral or low pH, respectively (0.5 m NaCl for KGF and 1 m NaCl for aFGF) (22Reich-Slotky R. Bonneh-Barkay D. Shaoul E. Bluma B. Svahn C.M. Ron D. J. Biol. Chem. 1994; 269: 32279-32285Abstract Full Text PDF PubMed Google Scholar). Binding of FGFs to soluble FGFR1 (hR1/alkaline phosphatase) was assessed following covalent cross-linking. Affinity-purified hR1/alkaline phosphatase (0.15 alkaline phosphatase A units/min) was incubated for 1.5 h at room temperature in 50 μl of HEPES binding buffer (100 mm HEPES, 150 mm NaCl, 5 mm KCl, 1.2 mm MgCl2, 8.8 mm dextrose, and 0.1% bovine serum albumin) containing 2 ng/ml radioiodinated FGF. Cross-linking was performed with 0.2 mm disuccinimidyl suberate for 30 min. The reaction was quenched by adding 20 mm glycine, and ligand-receptor complexes were visualized following SDS-PAGE and autoradiography. Coupling of the growth factors to Sepharose CL-2B (Sigma) was done as described previously (35Wilchek M. Miron T. Biochem. Int. 1982; 4: 629-635Google Scholar). Briefly, 1 mg of growth factor (aFGF, bFGF, or KGF) was coupled to 1 ml of packed beads in the presence of 1 mg of heparin. Following coupling, the column was extensively washed with 3 m NaCl to remove heparin and was stored in 10 mm Tris, pH 7.0, 0.5m NaCl, and 10 mm dithiothreitol. Coupling efficiency in different preparations was between 35 and 80%. Subconfluent cultures of L6E9 cells (100–200 tissue culture dishes of 150-mm diameter) were maintained for 16 h in nutrient mixture F-12 containing 10% dialyzed FCS. A portion of the cultures was metabolically labeled with Na235SO4 (30 μCi/ml). Peripheral membrane proteins were extracted with HEPES binding buffer containing 1.5 m NaCl, and the extracts were pooled and concentrated 5–10 times in dialysis tubing with dry polyethylene glycol and dialyzed overnight against 10 mm Tris, pH 7.0, and 0.2m NaCl. The dialyzed material was applied to a 20-ml DEAE-Sephacel column (Sigma) pre-equilibrated with 10 mmTris, pH 7.0, and 0.2 m NaCl, and the column was washed with 10–20 volumes of Tris buffer containing 0.3 m NaCl. Elution of the bound material was performed with a linear gradient of NaCl (0.4–1.2 m NaCl in 10 mm Tris, pH 7.0) at a flow rate of 24 ml/h. Radiolabeled proteoglycan peaks were identified by measuring the radioactivity in a liquid scintillation counter, by SDS-PAGE and autoradiography, or by safranin O staining (36Lammi M. Tammi M. Anal. Biochem. 1988; 168: 352-357Crossref PubMed Scopus (76) Google Scholar). Samples containing HSPGs were identified following nitrous acid deamination and solid-phase assay on cationic nylon (Zeta-probe) as described (37Rapraeger A. Yeaman C. Anal. Biochem. 1989; 179: 361-365Crossref PubMed Scopus (48) Google Scholar). Fractions containing HSPGs were pooled, diluted to reduce the salt concentration to 0.2 m, and applied to a 4-ml aFGF affinity column. The column was extensively washed with 0.2 m NaCl, and elution was performed by a stepwise increase in the NaCl concentration (see below). Enzymatic deglycosylation was carried out in Dulbecco's phosphate-buffered saline containing 125I-HSPG and 0.5 unit/ml heparinases I and III or chondroitinase ABC. Incubation was for 2 h at 37 °C. Deaminitive scission of HS with HNO2 was performed as described (38Soroka C.J. Farquhar M.G. J. Cell Biol. 1991; 113: 1231-1241Crossref PubMed Scopus (38) Google Scholar). Anhydrous trifluoromethanesulfonic acid was used to strip the peripheral sugars (39Edge A.S.B. Spiro R.G. J. Biol. Chem. 1987; 262: 6893-6898Abstract Full Text PDF PubMed Google Scholar). The core protein of the HSPG was digested overnight with proteinase K (0.5 mg/ml). To ensure that digestion was complete, a parallel incubation was carried out in the presence of radioiodinated HSPG, and digestion was monitored by SDS-PAGE and autoradiography. The GAG side chains were then separated from the protein and degradation products by DEAE-Sephacel chromatography. Affinity-purified HSPG was digested with modified trypsin (Promega). The peptides were loaded onto DEAE-Sephacel in the presence of 0.2 m NaCl to absorb GAG-containing peptides. Non-absorbed peptides were resolved by reverse-phase HPLC on a 1-mm diameter Vydac C18 column. The peptides were sequenced using standard chemistry on an Applied Biosystems sequencer (Model 476A). Mass spectrometry was done using a matrix-assisted laser desorption/ionization time-of-flight apparatus (Fisons Instruments, Inc.). Safranin O dye, which reacts with carboxyl and sulfate groups and can detect nanogram amounts of sulfated GAGs (36Lammi M. Tammi M. Anal. Biochem. 1988; 168: 352-357Crossref PubMed Scopus (76) Google Scholar), was occasionally used to monitor GAG content during purification. For the biological assays, we normalized the concentration of glypican HS relative to known concentrations of heparin on the basis of their sulfate content using the dimethylmethylene blue assay (40Farndale R.W. Buttle D.J. Barrett A.J. Biochem. Biophys. Acta. 1986; 883: 173-177Crossref PubMed Scopus (2907) Google Scholar). We estimated that glypican HS contain ∼50% of the sulfate content of heparin. Balb/MK cells, plated on fibronectin (1 μg/cm2)-coated 96-well microtiter plates, were serum-starved for 48 h and incubated with growth factors for 16 h. BaF3 cells were washed three times with Dulbecco's phosphate-buffered saline, placed in 96-well microtiter plates (2 × 104 cells/well) in RPMI 1640 medium containing 10% FCS, and incubated with growth factors for 36 h. [3H]Thymidine incorporation was assayed as described previously (29Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (562) Google Scholar, 41Reich-Slotky R. Shaoul E. Berman B. Graziani G. Ron D. J. Biol. Chem. 1995; 270: 29813-29818Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Each point in the experiment was done in duplicate or triplicate, and the results are representative of at least four different experiments. HSPGs exist either as integral or peripheral membrane proteins or as part of the extracellular matrix (1Hardingham T.E. Fosang A.J. FASEB J. 1992; 6: 861-870Crossref PubMed Scopus (1013) Google Scholar, 42Vlodavsky I. Bar-Shavit R. Ishai-Michaeli R. Bashkin P. Fuks Z. Trends Biochem. Sci. 1991; 16: 268-271Abstract Full Text PDF PubMed Scopus (254) Google Scholar). To determine the mode of association of the HSPG with parental L6E9 cells, we treated L6E9 cells expressing KGFR (designated L6/KGFR cells) with increasing concentrations of salt, a condition that is known to remove peripheral membrane proteins. L6/KGFR cells were then assayed for binding of radioiodinated aFGF and KGF. We reasoned that if the HSPG is salt-extractable, its removal should differentially affect the binding of aFGF and KGF to KGFR. As shown in Fig. 1, extraction of L6/KGFR cells with increasing salt concentrations resulted in a progressive reduction of the binding of aFGF to both low (Fig.1 A) and high (Fig. 1 B) affinity receptors. In samples that were extracted at 1.5 m NaCl, the binding of aFGF to low and high affinity receptors was reduced by 60 and 80%, respectively. By contrast, salt extraction enhanced the binding of KGF to KGFR by up to 1.6-fold (Fig. 1 B), whereas little or no effect was observed with respect to binding to low affinity receptors (Fig. 1 A). These results are in accordance with our previous observations obtained following treatment of L6/KGFR cells with heparan sulfate-degrading enzymes or with a metabolic inhibitor of sulfation (22Reich-Slotky R. Bonneh-Barkay D. Shaoul E. Bluma B. Svahn C.M. Ron D. J. Biol. Chem. 1994; 269: 32279-32285Abstract Full Text PDF PubMed Google Scholar) and further suggest that the differential effect on the binding of aFGF and KGF to KGFR is mediated by a peripheral membrane HSPG. The lack of reduction of the binding of KGF to low affinity receptors in the salt-extracted cells is probably due to the presence of non-heparan sulfate-binding sites, which account for >90% of the low affinity sites for KGF in L6E9 cells (22Reich-Slotky R. Bonneh-Barkay D. Shaoul E. Bluma B. Svahn C.M. Ron D. J. Biol. Chem. 1994; 269: 32279-32285Abstract Full Text PDF PubMed Google Scholar). The putative HSPG was purified from salt extracts of parental L6E9 cells that were metabolically labeled with [35S]sulfate. Purification was carried out by anion-exchange chromatography on DEAE-Sephacel followed by affinity chromatography on an aFGF column. The elution profile from DEAE-Sephacel is shown in Fig. 2 A. Over 99% of the material was bound to the column as judged by the negligible amount of radioactivity in the flow-through fraction, and the material eluted at salt concentrations of up to 0.4 m NaCl. A major peak of 35S eluted at ∼0.6 m NaCl. This peak contained predominantly heparan sulfates as judged by its sensitivity to deaminitive cleavage with nitrous acid (Fig. 2 A). The eluted material migrated on SDS-PAGE as a broad band of ∼200 kDa (Fig. 2 A, inset). The fractions containing heparan sulfates were pooled and subjected to aFGF affinity chromatography. About 80% of the pooled material bound to the column and could be eluted by 0.75–1 m NaCl (Fig. 2 B). SDS-PAGE and silver staining of the eluted material revealed the presence of a broad high molecular mass band similar to that observed with the 35S-labeled DEAE fractions (Fig. 2 C). The material purified by aFGF affinity chromatography also bound to bFGF and KGF columns (data not shown). This ability to interact with all three growth factors is in agreement with the previously described ligand binding characteristics of L6E9 HSPG (22Reich-Slotky R. Bonneh-Barkay D. Shaoul E. Bluma B. Svahn C.M. Ron D. J. Biol. Chem. 1994; 269: 32279-32285Abstract Full Text PDF PubMed Google Scholar). Enzymatic and chemical deglycosylations were carried out to characterize the GAG side chain of the purified material and to determine the molecular mass of the core protein. To increase the sensitivity of detection, the core protein was radioiodinated. Fig. 3 A shows the results of enzymatic deglycosylation carried out with heparinase I/III or chondroitinase ABC treatment. Heparinase, which specifically cleaves heparan sulfates, shifted the molecular mass of the broad band to a single band of ∼64 kDa (Fig. 3 A, lane 3). No shift in molecular mass was detected following treatment with chondroitinase ABC of either the intact HSPG or the preparation that had been treated with heparan sulfate-degrading enzymes (Fig.3 A, lanes 2 and 4). Fig. 3 B shows the results of chemical deglycosylation. Deaminitive cleavage with nitrous acid resulted in a shift in the molecular mass of the HSPG that was similar to that observed following treatment with heparinase (Fig. 3 B, lane 2). Treatment with trifluoromethanesulfonic acid further shifted the molecular mass of the HSPG to 54 kDa (Fig. 3 B, lane 3). We conclude that the purified HSPG does not carry chondroitin sulfate and that heparan sulfates account for about two-thirds of the mass of the protein. The results obtained with trifluoromethanesulfonic acid indicate that other types of glycosylations account for ∼10 kDa of the molecular mass. The purified HSPG was digested with modified trypsin; GAG-containing peptides were removed by absorption to DEAE-Sephacel; and GAG-free peptides were resolved by reverse-phase HPLC. Analysis of two peptides gave the sequences LSDVPQAEISGEHLR and QAEALRPFGDAPR. A search in the GenBank™ using the Blast program revealed that the above sequences are identical to residues 46–60 and 195–207 of rat glypican, respectively (24Karthikeyan L. Maurel P. Rauch U. Margolis R.K. Margolis R.U. Biochem. Biophys. Res. Commun. 1992; 188: 395-401Crossref PubMed Scopus (46) Google Scholar). It is noteworthy that even though the peptide encompassing residues 46–60 contains the consensus sequence for GAG attachment (24Karthikeyan L. Maurel P. Rauch U. Margolis R.K. Margolis R.U. Biochem. Biophys. Res. Commun. 1992; 188: 395-401Crossref PubMed Scopus (46) Google Scholar), this peptide is apparently not glycosylated. Mass spectrometry of several additional peptides confirmed that the purified HSPG is glypican (data not shown). Furthermore, the purified HSPG was immunoreactive with antibodies directed against bacterially expressed rat glypican, whereas no recognition was observed using antibodies against syndecan-1 and fibroglycan (Fig. 4). It is well established that heparin potentiates the binding of FGFs to FGFR1 and is required for receptor-mediated signaling (8Vlodavsky I. Miao H.-Q. Medalion B. Danagher P. Ron D. Cancer Metastasis Rev. 1996; 15: 177-186Crossref PubMed Scopus (270) Google Scholar, 9Roghani M. Mansukhani A. Dell'Era P. Bellosta P. Basilico C. Rifkin D.B. Moscatelli D. J. Biol. Chem. 1994; 269: 3976-3984Abstract Full Text PDF PubMed Google Scholar, 10Rapraeger A.C. Guimond S. Krufka A. Olwin B.B. Methods Enzymol. 1994; 245: 219-240Crossref PubMed Scopus (96) Google Scholar, 11Spivak-Kroizman T. Lemmon M.A. Dikic I. Landbury J.E. Pinchasi D. Huang J. Jaye M. Crumley G. Schlessinger J. Lax I. Cell. 1994; 79: 1015-1024Abstract Full Text PDF PubMed Scopus (596) Google Scholar, 12Pantoliano M.W. Horlick R.A. Springer B.A. Van-Dyk D.E. Tobery T. Wetmore D.R. Lear J.D. Nahapetian A.T. Bradley J.D. Sisk W.P. Biochemistry. 1994; 33: 10229-10248Crossref PubMed Scopus (229) Google Scholar, 13Schlessinger J. Lax I. Lemon M. Cell. 1995; 93: 357-360Abstract Full Text PDF Scopus (453) Google Scholar). We therefore compared the ability of affinity-purified glypican and heparin to promote FGF receptor binding and mitogenic activity. Binding of bFGF and aFGF to FGFR1 was assayed in a cell-free system utilizing a soluble extracellular domain of human FGFR1 fused to alkaline phosphatase. As shown in Fig. 5, glypican augmented the binding of bFGF and aFGF to FGFR1 at concentrations as low as 10 and 25 ng/ml, respectively. Quantitation of the results from several experiments showed that both glypican and heparin gave a similar 4–6-fold augmentation of bFGF binding, whereas heparin was somewhat more effectiv
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