Requirement for Anticoagulant Heparan Sulfate in the Fibroblast Growth Factor Receptor Complex
1999; Elsevier BV; Volume: 274; Issue: 31 Linguagem: Inglês
10.1074/jbc.274.31.21511
ISSN1083-351X
AutoresWallace L. McKeehan, Xiaochong Wu, Mikio Kan,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoA divalent cation-dependent association between heparin or heparan sulfate and the ectodomain of the fibroblast growth factor (FGF) receptor kinase (FGFR) restricts FGF-independent trans-phosphorylation between self-associated FGFR and determines specificity for and mediates binding of activating FGF. Here we show that only the fraction of commercial heparin or rat liver heparan sulfate which binds to immobilized antithrombin formed an FGF-binding binary complex with the ectodomain of the FGFR kinase. Conversely, only the fraction of heparin that binds to immobilized FGFR inhibited Factor Xa in the presence of antithrombin. Only the antithrombin-bound fraction of heparin competed with 3H-heparin bound to FGFR in absence of FGF, whereas both antithrombin-bound and unretained fractions competed with radiolabeled heparin bound independently to FGF-1 and FGF-2. The antithrombin-bound fraction of heparin was required to support the heparin-dependent stimulation of DNA synthesis of endothelial cells by FGF-1. The requirement for divalent cations and the antithrombin-binding motif distinguish the role of heparan sulfate as an integral subunit of the FGFR complex from the wider range of effects of heparan sulfates and homologues on FGF signaling through FGFR-independent interactions with FGF. A divalent cation-dependent association between heparin or heparan sulfate and the ectodomain of the fibroblast growth factor (FGF) receptor kinase (FGFR) restricts FGF-independent trans-phosphorylation between self-associated FGFR and determines specificity for and mediates binding of activating FGF. Here we show that only the fraction of commercial heparin or rat liver heparan sulfate which binds to immobilized antithrombin formed an FGF-binding binary complex with the ectodomain of the FGFR kinase. Conversely, only the fraction of heparin that binds to immobilized FGFR inhibited Factor Xa in the presence of antithrombin. Only the antithrombin-bound fraction of heparin competed with 3H-heparin bound to FGFR in absence of FGF, whereas both antithrombin-bound and unretained fractions competed with radiolabeled heparin bound independently to FGF-1 and FGF-2. The antithrombin-bound fraction of heparin was required to support the heparin-dependent stimulation of DNA synthesis of endothelial cells by FGF-1. The requirement for divalent cations and the antithrombin-binding motif distinguish the role of heparan sulfate as an integral subunit of the FGFR complex from the wider range of effects of heparan sulfates and homologues on FGF signaling through FGFR-independent interactions with FGF. The FGF 1The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor kinase; FGFR1–4, type 1 through 4 of the FGFR kinases; HSPG, heparan sulfate proteoglycan; PBS, phosphate-buffered saline; HUVEC, endothelial cells; AT, antithrombin. signal transduction system is ubiquitous and a local mediator of developmental processes in the embryo and homeostasis in the adult (1McKeehan W.L. Wang F. Kan M. Prog. Nucleic Acid Res. Mol. Biol. 1998; 59: 135-176Crossref PubMed Scopus (363) Google Scholar). Heparin or heparan sulfate interact independently with both activating FGF polypeptides, of which there are currently nineteen, and the ectodomain of the FGFR transmembrane kinases, which are encoded in four genes that give rise to multiple variants as a consequence of alternate splicing (1McKeehan W.L. Wang F. Kan M. Prog. Nucleic Acid Res. Mol. Biol. 1998; 59: 135-176Crossref PubMed Scopus (363) Google Scholar). Through the interactions, the FGF signal transduction system responds both negatively and positively to changes in the peri-cellular matrix. Heparan sulfate plays potentially multiple roles in FGF signaling in stability and proteolytic modification of FGF (2Luo Y. Gabriel J.L. Wang F. Zhan X. Maciag T. Kan M. McKeehan W.L. J. Biol. Chem. 1996; 271: 26876-26883Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), in control of access of FGF to the FGFR kinase complex (3Flaumenhaft R. Moscatelli D. Rifkin D.B. J. Cell Biol. 1990; 111: 1651-1659Crossref PubMed Scopus (200) Google Scholar, 4Kato M. Wang H. Kainulainen V. Fitzgerald M.L. Ledbetter S. Ornitz D.M. Bernfield M. Nat. Med. 1998; 4: 691-697Crossref PubMed Scopus (289) Google Scholar), oligomerization of FGF (5Moy F.J. Safran M. Seddon A.P. Kitchen D. Bohlen P. Aviezer D. Yayon A. Powers R. Biochemistry. 1997; 36: 4782-4791Crossref PubMed Scopus (104) Google Scholar, 6Herr A.B. Ornitz D.M. Sasisekharan R. Venkataraman G. Waksman G. J. Biol. Chem. 1997; 272: 16382-16389Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 7DiGabriele A.D. Lax I. Chen D.I. Svahn C.M. Jaye M. Schlessinger J. Hendrickson W.A. Nature. 1998; 393: 812-817Crossref PubMed Scopus (331) Google Scholar) and FGFR complexes (1McKeehan W.L. Wang F. Kan M. Prog. Nucleic Acid Res. Mol. Biol. 1998; 59: 135-176Crossref PubMed Scopus (363) Google Scholar, 5Moy F.J. Safran M. Seddon A.P. Kitchen D. Bohlen P. Aviezer D. Yayon A. Powers R. Biochemistry. 1997; 36: 4782-4791Crossref PubMed Scopus (104) Google Scholar, 6Herr A.B. Ornitz D.M. Sasisekharan R. Venkataraman G. Waksman G. J. Biol. Chem. 1997; 272: 16382-16389Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 7DiGabriele A.D. Lax I. Chen D.I. Svahn C.M. Jaye M. Schlessinger J. Hendrickson W.A. Nature. 1998; 393: 812-817Crossref PubMed Scopus (331) Google Scholar), and in conformational activation of oligomers of FGFR complexes (1McKeehan W.L. Wang F. Kan M. Prog. Nucleic Acid Res. Mol. Biol. 1998; 59: 135-176Crossref PubMed Scopus (363) Google Scholar, 9Kan M. Wang F. Kan M. To B. Gabriel J.L. McKeehan W.L. J. Biol. Chem. 1996; 271: 26143-26148Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Heparin-derived oligosaccharides ranging from simple unsulfated disaccharide or trisaccharide units to sulfated six to ten units have been co-crystallized with FGF (7DiGabriele A.D. Lax I. Chen D.I. Svahn C.M. Jaye M. Schlessinger J. Hendrickson W.A. Nature. 1998; 393: 812-817Crossref PubMed Scopus (331) Google Scholar, 8Ornitz D.M. Herr A.B. Nilsson M. Westman J. Svahn C.M. Waksman G. Science. 1995; 268: 432-436Crossref PubMed Scopus (270) Google Scholar, 10Faham S. Hileman R.E. Fromm J.R. Linhardt R.J. Rees D.C. Science. 1996; 271: 1116-1120Crossref PubMed Scopus (746) Google Scholar) which enhance oligomerization and affect FGF activity at the cellular level. Others have argued that FGF-2 associates more specifically with a pentasaccharide containing glucosamine-N-sulfate and a single iduronic acid-2-O-sulfate (11Turnbull J.E. Fering D.G. Ke Y.Q. Wilkinson M.C. Gallagher J.T. J. Biol. Chem. 1992; 267: 10337-10341Abstract Full Text PDF PubMed Google Scholar, 12Maccarana M. Casu B. Lindahl U. J. Biol. Chem. 1993; 268: 23898-23905Abstract Full Text PDF PubMed Google Scholar). The 6-O-sulfate of glucosamine-N-sulfate residues may contribute to the interaction with other FGFs (7DiGabriele A.D. Lax I. Chen D.I. Svahn C.M. Jaye M. Schlessinger J. Hendrickson W.A. Nature. 1998; 393: 812-817Crossref PubMed Scopus (331) Google Scholar, 14Guimond S. Maccarana M. Olwin B.B. Lindahl U. Rapraeger A.C. J. Biol. Chem. 1993; 268: 23906-23914Abstract Full Text PDF PubMed Google Scholar). A longer oligosaccharide that contains glucosamine-N-sulfate (6-O-sulfate) is more active in enhancing the interaction of FGF with FGFR and the activities elicited by FGF in various bioassays (12Maccarana M. Casu B. Lindahl U. J. Biol. Chem. 1993; 268: 23898-23905Abstract Full Text PDF PubMed Google Scholar, 13Ishihara M. Glycobiology. 1994; 4: 817-824Crossref PubMed Scopus (105) Google Scholar, 14Guimond S. Maccarana M. Olwin B.B. Lindahl U. Rapraeger A.C. J. Biol. Chem. 1993; 268: 23906-23914Abstract Full Text PDF PubMed Google Scholar, 15Ishihara M. Tyrrell D.J. Stauber G.B. Brown S. Cousens L.S. Stack R.J. J. Biol. Chem. 1993; 268: 4675-4683Abstract Full Text PDF PubMed Google Scholar, 16Ishihara M. Takano R. Kanda T. Hayashi K. Hara S. Kikuchi H. Yoshida K. J. Biochem. 1995; 118: 1255-1260Crossref PubMed Scopus (65) Google Scholar, 17Pye D.A. Vives R.R. Turnbull J.E. Hyde P. Gallagher J.T. J. Biol. Chem. 1998; 273: 22936-22942Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). The additional length has been proposed to reflect the requirement for spanning FGF dimers that bind an FGFR monomer or for spanning two FGFs that bind adjacent FGFR kinases (5Moy F.J. Safran M. Seddon A.P. Kitchen D. Bohlen P. Aviezer D. Yayon A. Powers R. Biochemistry. 1997; 36: 4782-4791Crossref PubMed Scopus (104) Google Scholar, 6Herr A.B. Ornitz D.M. Sasisekharan R. Venkataraman G. Waksman G. J. Biol. Chem. 1997; 272: 16382-16389Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 7DiGabriele A.D. Lax I. Chen D.I. Svahn C.M. Jaye M. Schlessinger J. Hendrickson W.A. Nature. 1998; 393: 812-817Crossref PubMed Scopus (331) Google Scholar, 8Ornitz D.M. Herr A.B. Nilsson M. Westman J. Svahn C.M. Waksman G. Science. 1995; 268: 432-436Crossref PubMed Scopus (270) Google Scholar). The length and 6-O-sulfate requirement may also reflect requirement for a bivalent interaction with FGF and FGFR to form a ternary unit (1McKeehan W.L. Wang F. Kan M. Prog. Nucleic Acid Res. Mol. Biol. 1998; 59: 135-176Crossref PubMed Scopus (363) Google Scholar, 9Kan M. Wang F. Kan M. To B. Gabriel J.L. McKeehan W.L. J. Biol. Chem. 1996; 271: 26143-26148Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar,18Kan M. Wang F. Xu J. Crabb J.W. Hou J. McKeehan W.L. Science. 1993; 259: 1918-1921Crossref PubMed Scopus (479) Google Scholar). The structural restrictions within heparan sulfate required for formation of an FGF-binding binary complex with the FGFR kinase or the ternary complex with both FGF and FGFR are less clear than the independent interaction with FGF. Characterization of the structural requirements in heparan sulfate for association with the FGFR kinase has been hampered in vivo by the interference with cellular heparan sulfates and in vitro by structural instability of isolated FGFR and the variability in the dependence on heparin/heparan sulfate for FGF binding (9Kan M. Wang F. Kan M. To B. Gabriel J.L. McKeehan W.L. J. Biol. Chem. 1996; 271: 26143-26148Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Recently we showed that divalent cations stabilize the FGFR ectodomain, squelch the heparin/heparan sulfate-independent FGF binding and mediate the high affinity interaction of heparin/heparan sulfate to FGFR (9Kan M. Wang F. Kan M. To B. Gabriel J.L. McKeehan W.L. J. Biol. Chem. 1996; 271: 26143-26148Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). This interaction restricts activating trans-phosphorylation between self-associated FGFR in absence of FGF (9Kan M. Wang F. Kan M. To B. Gabriel J.L. McKeehan W.L. J. Biol. Chem. 1996; 271: 26143-26148Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) but is required for and can mediate selectivity of binding of the activating FGF (19Kan M. Wu X. Wang F. McKeehan W.L. J. Biol. Chem. 1999; 19: 15947-15952Abstract Full Text Full Text PDF Scopus (128) Google Scholar). Using these improvements, we report here that, in contrast to the interaction of heparin/heparan sulfate with FGF, the functional complex with the FGFR kinase ectodomain requires all or a part of the structural motif that binds to antithrombin. Purified FGFR1β-GST was immobilized on GSH-agarose beads (Glutathione-Sepharose 4B, Amersham Pharmacia Biotech, Uppsala, Sweden) and incubated with heparin (No. H-3393, molecular weight 6,000–20,000, 195.2 USP units/mg from porcine intestinal mucosa) or rat liver HSPG in PBS containing 1% Triton X-100 and 10 mm MgCl2 for 1 h at room temperature (9Kan M. Wang F. Kan M. To B. Gabriel J.L. McKeehan W.L. J. Biol. Chem. 1996; 271: 26143-26148Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 19Kan M. Wu X. Wang F. McKeehan W.L. J. Biol. Chem. 1999; 19: 15947-15952Abstract Full Text Full Text PDF Scopus (128) Google Scholar). After washing extensively, 250 μl of125I-FGF-1 or 125I-FGF-2 (4 ng/ml at specific activity of 3.2 and 1.6 × 105, respectively) was added for 1 h at room temperature, the beads were washed with PBS three times, and the radioactivity was determined by γ-counter. The complex of 125I-FGF and FGFR was then chemically cross-linked by disuccinimidyl suberate and detected by autoradiography after SDS-polyacrylamide gel electrophoresis. Recombinant human FGF-2 was from Upstate Biotechnology, Inc. (Lake Placid, NY). FGF-1 was purified from bovine brain and the FGFR1β ectodomain fused to glutathione S-transferase (FGFR1β-GST) was expressed in Sf9 cells as described (9Kan M. Wang F. Kan M. To B. Gabriel J.L. McKeehan W.L. J. Biol. Chem. 1996; 271: 26143-26148Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Iodinated FGF-1 and FGF-2 were prepared as described (20Kan M. Shi E. McKeehan W.L. Methods Enzymol. 1991; 198: 158-171Crossref PubMed Scopus (49) Google Scholar). Unless otherwise indicated, data points in text illustrations were the mean of duplicates both for radiolabeled FGF and heparin binding. The experiments were representative of at least three reproductions using independent preparations of fractionated heparin or HSPG. At least two experiments were performed with different preparations of radiolabeled FGF. Heparin (1 μg) and 0.1 μg of 3H-heparin (0.41 mCi/mg; molecular weight 6,000–20,000, 142.5 units/mg from porcine intestinal mucosa from NEN Life Science Products, Boston, MA) was mixed with antithrombin III-agarose beads (Sigma) (0.2 ml) in 1 ml of PBS containing 1% Triton X-100 and 10 mmMgCl2 for 2 h at room temperature under constant shaking. The beads were then washed with the buffer extensively, and the bound heparin was eluted with 1 m NaCl in the buffer. The eluted fraction was dialyzed against the PBS buffer for assay. The bound heparin was about 14% of total heparin applied. Rat liver HSPG (10 μg) was prepared and partially purified as described below and then similarly fractionated by AT affinity chromatography. About 8% of the partially purified HSPG was retained on the column. 3H-Heparin (0.1 μg) was added to 5 μg unlabeled heparin in 1 ml of 1% Triton X-100 containing 10 mmMgCl2 in PBS and applied to an FGFR1β-GST affinity column with packed bead volume of 0.4 ml (2–4 μg FGFR) prepared as described (19Kan M. Wu X. Wang F. McKeehan W.L. J. Biol. Chem. 1999; 19: 15947-15952Abstract Full Text Full Text PDF Scopus (128) Google Scholar). The bound heparin was eluted with 1 m NaCl in PBS. Unretained material was desalted and repeatedly run on the column until bound heparin was negligible. FGFR extracted about 3% of the heparin applied. The FGFR-bound heparin was desalted and used for subsequent analysis. Competitive binding assays were performed by incubation of immobilized FGFR or FGF with 0.5 ml of 3H-heparin (0.1 μg/ml) in PBS containing 1% Triton X-100 and 10 mm MgCl2 in the presence or absence of different concentrations of unlabeled heparin or fractions from heparin for 1 h at room temperature. After washing the beads with buffer three times, the bound radioactivity was extracted by 0.5 ml of 1.5 NaCl in PBS and counted by liquid scintillation. FGFR1β-GST was immobilized on GST-Sepharose beads, and FGF was immobilized on copper-chelating beads (Chelating Sepharose Fast Flow, Amersham Pharmacia Biotech) as described (19Kan M. Wu X. Wang F. McKeehan W.L. J. Biol. Chem. 1999; 19: 15947-15952Abstract Full Text Full Text PDF Scopus (128) Google Scholar). Male rat livers (F-344) were perfused with 100 ml of 10 μg/ml trypsin and 0.02% EDTA in PBS for 10 min at room temperature after similar treatment without trypsin. The perfusate was clarified by centrifugation, dialyzed against water, and freeze-dried. The solid was then reconstituted with 1 ml of PBS and fractionated by gel-permeation (Bio-sil SEC-400, Bio-Rad, Richmond, CA) and ion exchange (Bio Gel TSK-DEAE-5-PW BIO-RAD, Richmond, CA) high performance liquid chromatography as described (19Kan M. Wu X. Wang F. McKeehan W.L. J. Biol. Chem. 1999; 19: 15947-15952Abstract Full Text Full Text PDF Scopus (128) Google Scholar). The activity of fractions was determined in the FGFR assembly assay, which measures both ability to form an FGF-independent binary complex with immobilized FGFR1β and the subsequent binding of radiolabeled FGF-1 and FGF-2 to the complex. Active fractions were pooled, dialyzed against water, lyophilized, and reconstituted in PBS. The carbohydrate concentration was determined by the carbazol method (21Bitter T. Muir H.M. Anal. Biochem. 1962; 4: 330-334Crossref PubMed Scopus (5314) Google Scholar). Fractions indicated in the text and 1 μg/ml antithrombin (Calbiochem-Novabiochem International, San Diego, CA) were added to assays containing 1 ml of 50 mm Tris-HCl (pH 7.4), 0.15 m NaCl, 10 mm calcium chloride, 1.0 μl of Factor Xa (10 μg/ml, New England BioLabs, Beverly, MA) and 25 μl of chromozym X (11.65 mg/ml, Roche Molecular Biochemicals, Mannheim, Germany). Incubation was carried out for 2 h at room temperature, and the absorption at 405 nm was measured. About 1% of size- and charge-enriched liver cell heparan sulfate proteoglycan with FGF complementation activity in cell growth and binding assays binds to immobilized FGFR1 or FGFR4 under optimized conditions (9Kan M. Wang F. Kan M. To B. Gabriel J.L. McKeehan W.L. J. Biol. Chem. 1996; 271: 26143-26148Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 19Kan M. Wu X. Wang F. McKeehan W.L. J. Biol. Chem. 1999; 19: 15947-15952Abstract Full Text Full Text PDF Scopus (128) Google Scholar). A similar analysis revealed that about 3% of a commercial heparin preparation bound to immobilized FGFR1 under the same conditions. Thus about 97% of heparin added into binding assays in soluble form may be incapable of participating in formation of a ternary FGFR complex. However, 5 and 15% of the heparin that is unretained by immobilized FGFR still binds FGF-1 or FGF-2, respectively, at 0.5 msalt (not shown). An even higher proportion binds at the 0.15m salt employed in binding assays (50–70% to FGF-2 and about 30% to FGF-1). The binding of 125I-FGF-1 or125I-FGF-2 to purified and immobilized FGFR1β-GST was employed to determine the structural requirements within the minority fraction of heparin and heparan sulfate that formed a divalent cation-dependent binary complex with FGFR (9Kan M. Wang F. Kan M. To B. Gabriel J.L. McKeehan W.L. J. Biol. Chem. 1996; 271: 26143-26148Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 18Kan M. Wang F. Xu J. Crabb J.W. Hou J. McKeehan W.L. Science. 1993; 259: 1918-1921Crossref PubMed Scopus (479) Google Scholar, 19Kan M. Wu X. Wang F. McKeehan W.L. J. Biol. Chem. 1999; 19: 15947-15952Abstract Full Text Full Text PDF Scopus (128) Google Scholar). Because the portion of commercial heparin that exhibits anticoagulant activity represents a distinct structural subset, porcine intestinal heparin was fractionated by AT-affinity chromatography. Activity of the bound and unbound fractions was analyzed for ability to bind to immobilized FGFR1β and the support of the binding of FGF to the immobilized binary complex (Fig. 1,A and B). Surprisingly, only the fraction of heparin that was retained by the AT column exhibited activity. Specific activity was increased by 8–10-fold. Activity of the fraction of heparin that failed to bind to the AT column was below detection limits, even when the immobilized FGFR1β was incubated with up to 0.4 μg per ml of heparin. Similar results were observed in separate experiments using immobilized FGFR2βIIIb-GST and FGF-1 and FGF-7 as radiolabel, and for FGFR4 with both FGF-1 and FGF-2 (results not shown). To determine whether the immobilized FGFR selected the anticoagulant fraction of heparin, the heparin that was captured by immobilized FGFR was recovered and assayed for ability to inhibit Factor Xa in the presence of antithrombin. Only the fraction of heparin (B) extracted by the immobilized FGFR exhibited anticoagulant activity as assessed by inhibition of Factor Xa activity in the presence of AT (Fig. 1C). Separate experiments confirmed that AT-bound heparin was 5 to 7 times more potent than crude heparin and that 1 and 10 ng/ml AT-bound heparin completely inhibited Factor Xa activity under the conditions indicated. To confirm that the selective activity of the AT-bound fraction of heparin for formation of an FGF-binding complex with FGFR1β reflected the FGF-independent interaction with FGFR1β, the AT-bound and unretained fractions were tested for ability to compete with receptor-bound radiolabeled heparin (Fig. 2A). Similar to the results from the FGF binding assays, only the AT-bound fraction of heparin competed with FGFR-bound heparin. To determine the competition of the two heparin fractions to3H-heparin bound to FGF in absence of FGFR, FGF-1 and FGF-2 were immobilized on copper-chelating Sepharose beads, which then bound3H-heparin. Fig. 2B shows that, although the AT-bound fraction of heparin was more efficient, both AT-bound and unbound fractions competed with heparin bound to FGF. Native HSPG from rat liver was collected by perfusion, partially purified by gel filtration and ion exchange chromatography, and then fractionated by AT affinity chromatography. Similar to heparin, only the fraction that was retained on the AT column exhibited the ability to form an HSPG-FGFR1β complex that bound either FGF-1 or FGF-2 (Fig. 3). The stimulation of DNA synthesis of HUVEC by FGF-1 exhibits a stringent requirement for added heparin, whereas the stimulation by FGF-2 is relatively independent (22Kan M. Yan G. Xu J. Nakahara M. Hou J. In Vitro Cell. Dev. Biol. 1992; 28A: 515-520Crossref PubMed Scopus (23) Google Scholar, 23Hoshi H. Kan M. Chen J.K. McKeehan W.L. In Vitro Cell. Dev. Biol. 1988; 24: 309-320Crossref PubMed Scopus (46) Google Scholar). This appears to be because of a deficiency of a cellular HSPG that will form a binary complex with FGFR1 that is competent to bind FGF-1 (19Kan M. Wu X. Wang F. McKeehan W.L. J. Biol. Chem. 1999; 19: 15947-15952Abstract Full Text Full Text PDF Scopus (128) Google Scholar). The AT-bound fraction of heparin enhanced FGF-1-induced DNA synthesis, whereas the unretained fraction exhibited no activity (Fig. 4). In separate experiments not shown here, we have demonstrated that soluble antithrombin inhibited both basal and FGF-1- and FGF-2-stimulated DNA synthesis of the endothelial cells in a dose-dependent fashion. Moreover, antithrombin at 10–20 μg/ml inhibited 125I-FGF binding to the cells by 60%. These observations are consistent with the possibility that antithrombin competes with endogenous heparan sulfate that forms an obligatory binary complex with FGFR, although alternative activities of antithrombin through other mechanisms cannot be eliminated. Oligosaccharides with the structural motif associated with the anticoagulant activities of heparin or heparan sulfate, which requires 3-O-sulfation, appear to be unnecessary for the FGFR-independent interaction with FGF-1 and FGF-2 (7DiGabriele A.D. Lax I. Chen D.I. Svahn C.M. Jaye M. Schlessinger J. Hendrickson W.A. Nature. 1998; 393: 812-817Crossref PubMed Scopus (331) Google Scholar, 8Ornitz D.M. Herr A.B. Nilsson M. Westman J. Svahn C.M. Waksman G. Science. 1995; 268: 432-436Crossref PubMed Scopus (270) Google Scholar, 10Faham S. Hileman R.E. Fromm J.R. Linhardt R.J. Rees D.C. Science. 1996; 271: 1116-1120Crossref PubMed Scopus (746) Google Scholar, 11Turnbull J.E. Fering D.G. Ke Y.Q. Wilkinson M.C. Gallagher J.T. J. Biol. Chem. 1992; 267: 10337-10341Abstract Full Text PDF PubMed Google Scholar, 12Maccarana M. Casu B. Lindahl U. J. Biol. Chem. 1993; 268: 23898-23905Abstract Full Text PDF PubMed Google Scholar, 13Ishihara M. Glycobiology. 1994; 4: 817-824Crossref PubMed Scopus (105) Google Scholar, 14Guimond S. Maccarana M. Olwin B.B. Lindahl U. Rapraeger A.C. J. Biol. Chem. 1993; 268: 23906-23914Abstract Full Text PDF PubMed Google Scholar, 15Ishihara M. Tyrrell D.J. Stauber G.B. Brown S. Cousens L.S. Stack R.J. J. Biol. Chem. 1993; 268: 4675-4683Abstract Full Text PDF PubMed Google Scholar, 16Ishihara M. Takano R. Kanda T. Hayashi K. Hara S. Kikuchi H. Yoshida K. J. Biochem. 1995; 118: 1255-1260Crossref PubMed Scopus (65) Google Scholar, 17Pye D.A. Vives R.R. Turnbull J.E. Hyde P. Gallagher J.T. J. Biol. Chem. 1998; 273: 22936-22942Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). In this report we examined the direct association of heparin and heparan sulfate with the purified recombinant ectodomain of the FGFR kinase. This enabled study of the structural requirements required to form a binary complex that is competent to bind FGF in the absence of interfering cellular heparan sulfates or soluble heparin, or heparan sulfate that binds FGF, but is incapable of interaction with FGFR. The results revealed that only the fraction of heparin or cell-derived heparan sulfate that was competent to bind AT and to inhibit factor Xa in presence of AT was capable of forming a competent binary complex with FGFR. The results suggested that the FGFR kinase ectodomain specifically selects, from unfractionated heparin and heparan sulfate, the fraction that exhibits anticoagulant activity. The structural motif within heparin that is required for AT binding and anticoagulant activity is a penta- or hexasaccharide sequence, which can be up to 30% of unfractionated heparin, but is less than 10% of cellular heparan sulfates (24Rosenberg R.D. Shworak N.W. Liu J. Schwarz J.J. Zhang L. J. Clin. Invest. 1998; 99: 2062-2070Crossref Scopus (260) Google Scholar). A glucosamine-N-acetyl orN-sulfate-6-O-sulfate and a glucosamine-N-sulfate-3-O-sulfate (± 6-O-sulfate), with a residue in between, cooperate with an adjacent disaccharide comprised of iduronic acid-2-O-sulfate and glucosamine-N-sulfate-6-sulfate in AT binding (24Rosenberg R.D. Shworak N.W. Liu J. Schwarz J.J. Zhang L. J. Clin. Invest. 1998; 99: 2062-2070Crossref Scopus (260) Google Scholar). It is likely that all or a part of this structure within heparin or heparan sulfate participate in the specific divalent cation-dependent interaction with the FGFR ectodomain. However, it is noteworthy that less than 50% of AT-bound heparin will subsequently bind to FGFR1. This suggests a structural requirement in addition to the AT-binding motif for formation of the binary FGFR complex. Recently, it has become clear that tissue-specific and hormonally regulated isozymes of glucosaminyl-3-O-sulfotransferases (3-OST) are the final and rate-limiting step in heparan sulfate synthesis which generates 3-O-sulfate sites in positions dictated by the oligosaccharide sequence in precursors (24Rosenberg R.D. Shworak N.W. Liu J. Schwarz J.J. Zhang L. J. Clin. Invest. 1998; 99: 2062-2070Crossref Scopus (260) Google Scholar, 25Shworak N.W. Liu J. Fritze L.M. Schwartz J.J. Zhang L. Logeart D. Rosenberg R.D. J. Biol. Chem. 1997; 272: 28008-28019Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 26Zhang L. Yoshida K. Liu J. Rosenberg R.D. J. Biol. Chem. 1999; 274: 5681-5691Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Activity of these enzymes may be intimately involved in both the negative and positive regulation of FGF signaling through modification of the composition of heparan sulfate chains of the proteoglycan subunits of the oligomeric FGFR complex. The requirement for divalent cations and the AT-binding motif within heparin or heparan sulfate for formation of competent FGFR glycosaminoglycan-kinase complexes distinguish the FGFR complex from other indirect actions of heparin or heparan sulfate. This property should aid in characterization of the responsible proteoglycan based on the properties of its glycosaminoglycan chains. We thank Maki Kan, Kerstin McKeehan, and Thanh Tran for technical assistance.
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