0 1992 Harwood Academic Publishers GmbH
Growth Factors, 1992, Vol. 7, pp. 289-296 Reprints available directly from the publisher Photocopying permitted by license only
Printed in the United Kingdom
Interaction of Heparin-Binding EGF-Like Growth Factor (HB-EGF) with the Epidermal Growth Factor Receptor: Modulation by Heparin, Heparinase, or Synthetic Heparin-Binding HB-EGF Fragments GAIL E. BESNER*, DIANE WHELTON, MELISSA A. CRISSMAN-COMBS, CHRISTY L. STEFFEN, GREGORY Y. KIM and DAVID R. BRIGSTOCK Growth Factors Downloaded from informahealthcare.com by Chulalongkorn University on 01/12/15 For personal use only.
Department of Surgery, Ohio State University, Division of Pediatric Surgery, Children’s Hospitd, 700 Children’s Drive, Columbus, OH 43205
(Received April 20 1992, Accepted June 12 1992) The binding of heparin-binding EGF-like growth factor (HB-EGF) to the epidermal growth factor (EGF) receptor of human endometrial carcinoma cells was compared to that of EGF using an ‘*%EGF radioreceptor assay. The inhibitory effect of HB-EGF on Iz5I-EGFbinding was reversed either in the presence of heparin (but not by chondroitin sulfate) or by pre-treating the cells with heparinase. These treatments did not affect the binding of EGF to its receptor. To map potential regions in the HB-EGF molecule that mediate its heparin-dependent interaction with the EGF receptor, HB-EGF peptides were synthesized that were non-homologous to EGF. Accordingly residues 20-25 and 36-41, but not residues 8-19, of HB-EGF were found to be (i) heparin-binding and (ii) modulators of HB-EGF (but not of EGF) binding to the EGF receptor. KEYWORDS: heparin, HB-EGF, EGF, peptide mapping, extracellular matrix ABBREVIATIONS EGF, epidermal growth factor; HB-EGF, heparin-binding EGF-like growth factor; AR, amphiregulin; TGF-a, transforming growth factor alpha; aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; HBGF, heparin-binding growth factor; ECM, extracellular matrix; FPLC, fast protein liquid chromatography.
INTRODUCTION
family such as EGF, transforming growth factoralpha (TGF-a), and amphiregulin (AR) Heparin-binding EGF-like growth factor (HB- (Higashiyama et al., 1991). This was recently proEGF), a novel heparin-binding mitogen for ven with the purification and amino acid sesmooth muscle cells and fibroblasts, was first quencing of native HB-EGF proteins (Higashirecognized as a secreted product of cultured yama et al., 1992). Complementary DNA analysis human macrophages (Besner and Klagsbrun, has suggested that in common with other EGF 1988; Besner et al., 1990). Recently sufficient family members, HB-EGF is initially synthesized quantities of HB-EGF were purified from con- as a large precursor (208 amino acids) which is ditioned medium of the U-937 macrophage-like then cleaved to yield a mature protein compriscell line, allowing for the determination of its ing at least 86 amino acids (Higashiyama et al., unique N-terminal amino acid sequence 1991, 1992). Full length mature HB-EGF is a (Higashiyama et al., 1991). From the cDNA of glycosylated protein with an apparent molecular HB-EGF it was predicted that the amino acid weight of 22,000 as assessed by sodium dodecyl sequence of HB-EGF is similar to those of other sulfate polyacrylamide gel electrophoresis members of the epidermal growth factor (EGF) (Higashiyama et al., 1992). The HB-EGF precursor contains a highly hydrophobic sequence similar to the transmembrane domain of the precur“To whom correspondence should be addressed at: sor for TGF-a (Luetteke and Lee, 1990; Department of Surgery, Children’s Hospital, 700 Children‘s Drive, Columbus OH 43205. Tel: (614) 460-2015 Fax: (614) 460- Higashiyama et al., 1991, 1992). This suggests that HB-EGF may exist as a high molecular 7088. 289
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weight membrane-anchored protein as well as a fully processed secretory protein. This notion is supported by a very recent demonstration that a diphtheria toxin receptor is identical to the HBEGF precursor (Naglich et al., 1992). HB-EGF competes with EGF for binding to the EGF receptor on A-431 cells or bovine vascular smooth muscle cells (Higashiyama et al., 1991) and it stimulates the proliferation of EGF-responsive cells (Besner et al., 1990; Higashiyama et al., 1991).In addition, HB-EGF stimulates EGF receptor tyrosine kinase activity and EGF receptor tyrosine autophosphorylation (Higashiyama et al., 1992). These observations are consistent with the structural relatedness of HB-EGF to EGF, and demonstrate that these two factors bind to the same signal-transducing receptor. However, despite their structural and biological similarities, a principal difference between EGF and HB-EGF lies in their affinity for heparin. HB-EGF binds strongly to heparin-affinity matrices, requiring 1 M NaCl for elution, whereas EGF and TGF-a do not bind to heparin resins at all (Besner and Klagsbrun, 1988; Besner et al., 1990; Higashiyama et al., 1991). In addition, it was recently reported that AR is a heparin-binding protein (Cook et al., 1991). Thus, the EGF family contains members that have a strong affinity for heparin (AR, HB-EGF) and others that do not (EGF, TGFa). This dichotomy points to the importance of understanding, firstly, the functign (if any) of heparin-affinity of certain EGF-like growth factors and, secondly, the molecular basis of heparin-binding. Among the best characterized heparin-binding growth factors (HBGFs) are acidic and basic fibroblast growth factors (aFGF, bFGF) which were isolated by purification regimens that utilized heparin-affinity chromatography (Shing et al., 1984; Lobb and Fett, 1984; Bohlen et al., 1985; Esch et al., 1985).It is well established that bFGF binds to immobilized heparin, is present in the extracellular matrix (ECM) and basement membrane in association with heparin sulfate proteoglycan (Baird and Ling, 1987; Vlodavsky et al., 1987; Folkman et al., 1988; Bashkin et al., 1989), and is protected by heparin from inactivation (Gospodarowicz and Cheng, 1986). However, only recently has a functional significance of this interaction become apparent-namely that cell surface heparan sulfate proteoglycan is required for bFGF binding to its high affinity receptor
__
(Yayon et al., 1991; Ornitz et al., 1992) and subsequent biological actions including fibroblast growth (Rapraeger et al., 19911, myoblast differentiation (Rapraeger et al., 1991), and DNA synthesis in a lymphoid cell line transfected with the FGF receptor (Ornitz et al., 1992). Accordingly, the role played by the ECM or cell surface proteoglycans in binding FGFs and regulating their availability to high affinity receptors is attracting considerable interest (Ruosiahti and Yamaguchi, 1991; Klagsbrun and Baird, 1991). Although most reports to date have addressed the issue of FGF-heparin interactions, recent studies suggest that proteoglycan regulation of growth factor activity may be a common phenomenon. Examples include the interaction of the core protein of decorin and betaglycan with TGF-P (Ruosiahti and Yamaguchi, 1991)and of SPARC (secreted protein, acidic and rich in cysteine) with platelet-derived growth factor -AB and -BB (Raines et al., 1992). Since the existence of heparin-binding members of the EGF family has been recognized for only a short time, little has been reported regarding the interaction of AR or HB-EGF with heparin. However in a recent report it was shown that heparin inhibited EGF receptor binding and stimulation of DNA synthesis by a heparin-binding human keratinocyte-derived autocrine growth factor similar or identical to AR (Cook et al., 1991). In the studies described here we investigated the effect of heparin on the interaction between the EGF receptor expressed by a cultured endometrial cell line and either HB-EGF or EGF. In addition, since synthetic peptides were useful in the mapping of possible heparin-binding and receptor-binding domains of bFGF (Baird et al., 1988), we adopted this approach to identify potential heparin-binding domains in the HBEGF protein.
MATERIALS AND METHODS
Materials HEC-1-B endometrial carcinoma cells and U-937 monocyte cells were obtained from ATCC. Cell culture -reagents and recombinant human EGF were from Gibco BRL. Iodinated mouse EGF (Iz5IEGF) (80-135 pCi/pg) was from ICN. Heparin, chondroitin sulfate,-heparinase, chondroiiinase
RECEPTOR BINDING OF HB-EGF
HB-EGF AR
29 1
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8-19 20-25 36-41 YTLSSIZP--QALATP~KEEHGKRKKKGKGLG------~RDEZ;LRKYKD~-~~~PS€IUP~H~HGLSL SVIULEQWIZPPQNKTESE~TSDKPKRKKKGGKN~~RRNR~KNEZ;NAEFQN~-~IEHLEBVT€K€QQEXFEEK NSDSE€PLSHDGY.€LHDGV€MXIEALDKYA€N~W~I~QYRDLKWWELR
EGF TGF-a
WSHFND€PDSHTQKFH-GTCRFLVQEDKPACVUS~GA~E€IADLLA
FIGURE 1 Amino acid sequence of EGF-like growth factors. The amino acid sequences of human AR, EGF and TGF-a are compared to that of human HB-EGF (adapted from Higashiyama et al., 1991, 1992). Amino acids that are underlined represent residues that are homologous between HB-EGF and other family members when the cysteine residues in each protein are coaligned. The solid bars above the HB-EGF sequence depict the HB-EGF domains that were generated synthetically.
u Growth Factors Downloaded from informahealthcare.com by Chulalongkorn University on 01/12/15 For personal use only.
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ABC, and bovine serum albumin (BSA) were from Sigma. Human HB-EGF was purified from U-937 cell conditioned medium by cationexchange chromatography, copper-affinity chromatography, and TSK-heparin affinity fast protein liquid chromatography (FPLC) as described (Higashiyama et al., 1991). Three HB-EGF peptide sequences were generated by fmoc solid phase synthesis by BioSynthesis Inc., Texas. The amino acid sequences of two of these peptides were QALATPNKEEHG and LRKYKD, corresponding to residues 8-19 and 36-41 of HB-EGF respectively. These sequences are non-homologous to the corresponding regions of EGF, TGF-a, and AR (Higashiyama et al., 1991). The third HB-EGF sequence, KRKKKG, corresponds to residues 20-25 of HB-EGF. This region is nonhomologous to EGF or TGF-a but is identical to residues 25-30 of AR (Higashiyama et al., 1991). The peptides are depicted schematically in Fig. 1.
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HEC-1-B cells were seeded in 48-well tissue culture plates at a density of 20,000 cells/we11/250 FIGURE 2 Effect of HB-EGF on '251-EGFreceptor binding. (A) p1 in Minimum Essential Medium (MEM) conPurification of HB-EGF by heparin-affinity FPLC and its taining 10% fetal bovine serum, 1 mM sodium detection using a '251-EGF radioreceptor assay. HB-EGF purified from U-937 conditioned medium by a combination of pyruvate, 292 pg/ml glutamine, non-essential cation exchange chromatography and copper chelating amino acids and antibiotics, and allowed to grow chromatography was applied to a TSK-heparin-5PW column in a humidified atmosphere of 10% COz for 4-6 (8 mmx7.5 cm) in 10 mM Tris HCI (pH 7.4) containing 0.5 M NaCI, essentially as described (Higashiyama et al., 1991). After days. The medium in each well was then aspirsample application, the column was washed with 10ml of ated and the cells washed with 2 5 0 ~ 1of cold 10 mM Tris HCI (pH 7.4) containing 0.2 M NaCl and then binding medium (composed of MEM containing subjected to a 40 ml linear salt gradient of 0.2-2 M NaCl in 10 mM Tris HC1 (pH 7.4). Fractions (1 ml) collected during 50 mM BES, 0.1 pM KI and 0.1% BSA, pH 7.2). gradient formation were tested for their ability to inhibit 1251- Each well then received 150 pl of 0.5 ng/ml '"IEGF binding by addin 20 p1 of each fraction to HEC-I-B cells EGF in binding medium pIus test substances in 150 pl of 0.5 ng/ml' 5'1-EGF. (B) Competition curve showing dose-dependent inhibition of lZ5I-EGFbinding by HB-EGF. (glycosaminoglycans, HB-EGF peptides, etc.) and Fractions 19-21 (see Fig. 2A) were pooled and assayed in either 0.5-3 ng/ml of EGF (to reduce cellular '"1triplicate at the indicated doses on HEC-1-B cells in 150 p1 of EGF binding to 30-60% of its maximal value) or a binding medium containing 0.5 ng/ml lZ51-EGF.The figure shows the specific lZ51-EGFbinding in the presence of the comparable dose of HB-EGF. The cells were incubated at 4°C for 2 hours after which they were different amounts of HB-EGF. I-B-EGF (ul/well)
BESNER el al.
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washed three times with cold phosphatebuffered saline containing 0.1 pM KI and 0.1% BSA and then solubilized in 25Opl of 0.3N NaOH for counting of cell-associated lZ5I.Nonspecific binding as determined in the presence of a 1000-fold excess of unlabelled EGF (i.e. 500ng/ml) was typically 5-10% of maximal binding.
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RESULTS HEC-1-B cells express approximately 2x106 EGF receptors per cell to which binding of lZ5I-EGFis saturable, time- and temperature-dependent, and specific (i.e. non-displaceable by a 40-fold excess of bFGF, aFGF, PDGF, or IGF-I) (data not shown). Since HB-EGF was reported to bind to the EGF receptor of A-431 cells or smooth muscle cells (Higashiyama et al., 1991), column fractions of U-937 cell conditioned medium were moni: tored for the presence of HB-EGF by testing their
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ability to displace Iz5I-EGFbinding to HEC-I-B cells. As shown in Fig. 2A, a single peak of Iz5IEGF-competing activity was eluted from a heparin-affinity FPLC column by 1 M NaCl, characteristic of the elution position of HB-EGF (Besner et al., 1990; Higashiyama et al., 1991). These results further show that the lZ5I-EGFbinding assay is a very reliable and rapid means of screening column fractions for HB-EGF since other heparin-binding mitogens such as PDGF (which is also present in macrophage cell conditioned medium (Besner et al., 1990; Higashiyama et al., 1991, 1992)) and FGFs do not affect EGF binding under the conditions described (Figure 2A and unpublished results). As shown in Figure 2B, HB-EGF competed with Iz5I-EGFin a dose-dependent manner. To investigate the mechanism by which HBEGF interacts with the EGF receptor, cells were co-incubated with heparin or chondroitin sulfate (0-300 pglml) and unlabelled EGF or HB-EGF. Neither heparin nor chondroitin sulfate affected
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FIGURE 3 Heparin-dependence of HB-EGF-mediated reduction of "'I-EGF binding. HEC-I-B cells were incubated in 0.5 ng/ml "'I-EGF containing the indicated concentrations of heparin ( A X ) or chondroitin sulfate (D-F) plus either unlabelled EGF at 0.5 ng/ml (to reduce Iz5I-EGFbinding by about 40%) (B,E) or an approximately equivalent inhibitory dose of HB-EGF (C,F). The specific binding of '"I-EGF to the cells after 2 hours at 4°C is shown ( m e a n h d . of triplicate determinations).
RECEPTOR BINDING OF HB-EGF
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EGF binding was antagonized in a dose-dependent manner by heparin. Reversal of the HB6o EGF-mediated inhibition of 1251-EGFbinding 0 50 C occurred at heparin concentrations between 1 p.and 30 ,ug/ml (Fig. 3C) whereas the same concen0 trations of chondroitin sulfate were ineffective 40 (Fig. 3F). A slight reduction in the ability of HBW EGF to inhibit 1251-EGFbinding was observed at I 30very high concentrations (>lo0 pg/ml) of chondroitin sulfate (Fig. 3F) which is consistent with a previous report in which similar concentrations .g 20 of this glycosaminoglycan were shown to also marginally reduce cellular binding of bFGF 10 (Bashkin et al., 1989). These results suggest that HB-EGF binding to the EGF receptor of HEC-I-B cells may be regu0 lated in a specific manner by soluble heparin. To determine whether heparan sulfate on the cell surface can exert a similar regulatory role, cells FIGURE 4 Effect of heparinase pre-treatment of HEC-1-8 were pre-incubated with heparinase to remove cells on the ability of HB-EGF to bind to the EGF receptor. these potential HB-EGF binding sites. As shown HEC-I-B cells were treated for 4 hours at 37°C with 30 U/ml of in Fig. 4, the ability of HB-EGF to bind to the EGF heparinase-I. The culture medium was then removed from both heparinase-treated and non-treated well of cells, and receptor on heparinase-treated cells was reduced replaced with binding medium containing Iz5I-EGF(0.5 ng/ml) as compared to control cell cultures. Heparinase and HB-EGF (I p1 of pooIed fractions 20 and 21 from Fig. 2A). treatment did not affect the ability of unlabelled The figure shows '"I-EGF binding for each treatment group expressed as a percentage of the maximal lZ51-EGFbinding in EGF to compete for I2'I-EGF binding sites (data the absence of any competing HB-EGF (meanfs.d. of triplicate not shown). These results suggest that cell-surdeterminations). face heparan sulfate acts to regulate receptor binding of HB-EGF. maximal binding of Iz5I-EGF(Fig. 3A, D) or the To identify putative heparin-binding domains decreased level of Iz5I-EGFbinding in the pres- of the HB-EGF molecule, synthetic HB-EGF pepence of 0.5 ng/ml of unlabelled EGF (Fig. 3B, El. tides that do not share sequence homology with However, the ability of HB-EGF to inhibit Iz5I- non-heparin-binding members of the EGF family
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FIGURE 5 Heparin-affinity FPLC of synthetic HB-EGF peptides. One milligram of each of the peptides corresponding to residues 8-19, 20-25 and 36-41 of HB-EGF was individually subjected to heparin-affinity FPLC essentially as described in the legend of Fig. 2, except that tht unbound fractions were also collected prior to NaCl gradient elution. The elution of each peptide from the heparin column was determined by monitoring the absorbance of each fraction at 214 nm.
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Peptide concentration (ug/mll FIGURE 6 Modulation of HB-EGF-mediated inhibition of lz5I-EGFbinding by heparin-bindingHB-EGF peptides. HEC-I-B cells were incubatd with '=I-EGF (approx. 0.5 ng/ml) containing the indicated concentrations of (A) residues 8-19, (B)residues 20-25 and ( C )residues 36-41 alone (W), in the presence of unlabelled EGF (O), or in the presence of unlabelled HB-EGF (A). EGF and HB-EGF were added at comparable concentrations so that maximal '"I-EGF binding was reduced by 60% by both competitors. Binding of '"I-EGF is expressed as a percentage of maximal IZ5I-EGFbinding that was determined in the absence of any added factor (meanhd. of triplicate determinations).
such as EGF and TGF-a (see Fig. 1) were generated. The heparin-binding property of each peptide was determined by individually subjecting them to TSK-heparin-affinity FPLC. As shown in Fig. 5A, HB-EGF[8-19] was eluted in the void volume showing that it was non-heparin-binding. In contrast, HB-EGF[20-25] and HBEGF[3641] each bound strongly to TSK-heparin and were eluted by 1.5M NaCl and 1 M NaCl respectively (Fig. 5B, C). These findings suggested that residues 20-25 and/or 36-41 of HBEGF may contribute, at least in part, to the binding of HB-EGF to cell surface heparin-like molecules-and hence the EGF receptor. To test this possibility, all three peptides were tested for their ability to modulate the inhibition of cellular Iz5I-EGFbinding that is caused by HB-EGF or EGF. None of the three peptides affected lz5I-EGF binding in the absence of any competitor or in the presence of unlabelled EGF (Fig. 6A-C). While the binding of HB-EGF to the EGF receptor was unaffected by the non-heparin-binding peptide (i.e. residues 8-19) (Fig. 6A), binding was influenced by the two heparin-binding peptides (Fig. 6B, C). In four separate experiments, residues 20-25 at 0.3-1 pg/ml or residues 36-41 at 3-10 ,ug/ml were found to potentiate the inhibitory effect of HB-EGF on '251-EGFbinding (Fig. 6B,C). Higher doses of each peptide reproducibly antagonized the ability of HB-EGF to bind to
the EGF receptor (Fig. 6B, C). However, the peptides were no more effective at 500 pg/ml than at 100 pglml (data not shown) and only partially reversed the HB-EGF-mediated inhibition of lz5I-EGFbinding (Fig. 6B, C).
DISCUSSION There is increasing evidence that availability of bFGF to its high affinity receptor is regulated by cell surface heparan sulfate proteoglycan molecules (Klagsbrun and Baird, 1991; Rapraeger et al., 1991; Ruosiahti et al., 1991; Yayon et al., 1991; Ornitz et al., 1992). Since bFGF is but one HBGF, these findings raise the question of whether the interaction between other HBGFs and cell surface heparin-like molecules constitutes a common receptor-binding mechanism for this class of mitogen. Our results suggest that, in common with bFGF, HB-EGF (which is structurally unrelated to bFGF) binds to cell surface heparan sulfate and that this binding primarily mediates the interaction of HB-EGF with its high affinity receptor. Evidence in support of this notion is that exogenous heparin or pretreatment of HEC1 -B cells with heparinase perturbs the ability of HB-EGF to bind to the EGF receptor whereas condroitin sulfate is ineffective. Our findings are similar to those of Cook et al. (1991) who showed
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RECEPTOR BINDING OF HB-EGF
that 30 pglml of heparin sulfate completely abolished the ability of AR to bind to the EGF receptor and to stimulate DNA synthesis in AKR-2B cells. Thus it would appear that EGF receptor occupancy by HB-EGF and AR-the two heparinbinding members of the EGF family-is heparindependent. These observations are in marked contrast to the binding of EGF to the EGF receptor which does not appear to be heparin-dependent for either human HEC-1-B cells (these results) or mouse AKR-2B cells (Cook et al., 19911, consistent with the lack of heparin-affinity of EGF (Shing et al., 1984). Since saturating concentrations of antagonists such as heparin (see Fig. 3C) or heparin-binding HB-EGF peptides (see Fig. 6B,C) did not completely reverse the binding of HB-EGF to the EGF receptor, the possibility that a small proportion of HB-EGF binds directly to the EGF receptor cannot be excluded. Since cell-surface heparan sulfate was shown to potentiate HB-EGF receptor binding, it might be expected that exogenously added heparin would do the same. However, we found that added heparin actually inhibited the ability of HB-EGF to displace Iz5I-EGFbinding to its receptor. This apparent discrepancy may be accounted for if HB-EGF receptor binding is a function of the proximity and spatial relationship of cell-surface heparan sulfate with the EGF receptor. Therefore a complex of HB-EGF and soluble (i.e. exogenously added) heparin may result in inappropriate conformational and/or spatial presentation of HB-EGF to the EGF receptor. Given the pronounced difference between HBEGF and EGF in their affinities for heparin, we reasoned that the heparin-binding properties of HB-EGF might reside in the N-terminal non-EGFlike domain (i.e. between residues 1 and 29; see Fig. 1). This region of HB-EGF is highly hydrophilic (Higashiyama et al., 1991) and shows areas of homology with the corresponding region of AR (residues 1-34) which is also highly hydrophilic (Cook et al., 1991). Moreover the N-terminal hydrophilic domain of AR has been suggested as a heparin-regulatory site (Cook et al., 1991) although evidence to support this has not been presented. Since synthetic peptides have been used in structure-function mapping of EGF, TGF-a and bFGF (Nestor et al., 1985; Heath et al., 1986; Baird et al., 1988), we adopted this approach to map potential heparin-binding
295
regions of HB-EGF. The ability of residues 20-25 to bind to heparin and modulate HB-EGF receptor binding suggests that this region constitutes at least part of a putative heparin-binding domain. Interestingly, this domain is also present in the AR hydrophilic region (see Fig. 1) suggesting that, if involved in heparin-binding, its function may be conserved and common to both AR and HB-EGF. Similar EGF receptor modulatory effects were also exhibited by residues 36-41 that are unique to HB-EGF but are present in the EGFlike region of HB-EGF (see Fig. 1). This finding was somewhat unexpected considering that this domain is not located in the N-terminus, and suggests that multiple amino acid domains of HB-EGF may account for its heparin-binding properties. Although residues 20-25 and 36-41 are highly cationic, a previous study of bFGF peptides showed that a high positive charge does not necessarily correlate with heparin-affinity (Baird et al., 1988). Therefore the highly specific interaction between heparin and the heparin-binding HB-EGF peptides identified here may not be due solely to charge effects. Baird et al. (1988) also showed that the heparin-binding bFGF peptides were capable of inhibiting lZ5I-bFGFbinding to high affinity receptors. In our study this would also seem to be the case since the heparin-binding HB-EGF peptides were able to modulate receptor binding of HB-EGF but not that of EGF. An unexpected feature was that the heparinbinding peptides modulated EGF receptor binding by HB-EGF in a bi-phasic manner in that they reproducibly potentiated HB-EGF receptor binding at low doses but inhibited it at high doses. These findings suggest a complex interaction of the peptides or HB-EGF with their respective cellular binding sites. Further studies using control peptides of similar size and composition as the heparin-binding HB-EGF peptides will help to establish the underlying mechanism of the biphasic phenomenon. Future studies will also address the question of whether the various modulators of HB-EGF binding reported in this study are effective regulators of HB-EGF biological activity. Taken together, these results highlight the importance of ECM and cell surface heparin-like molecules in regulating the binding of HB-EGF to its signal-transducing receptor. Since this mechanism is shared with at least two other HBGFs
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(AR and bFGF), it is clear that local concentration changes in heparin and/or heparin-degrading enzymes act as pivotal control mechanisms whereby the accessibility of a variety of HBGFs to their respective high affinity receptors may be regulated.
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ACKNOWLEDGMENTS G.E.B. and D.R.B. were s u p p o r t e d by grants 020-871 and 020-872 a w a r d e d by Children’s Hospital Research Foundation, Columbus, OH. G.E.B. was also supported by a n Ohio State University Seed Grant a n d by a w a r d s from the Bremer Foundation and the O h i o State University Department of Surgery Medical Research a n d Development Fund. We thank Dr. D.R. Cooney for his generous support.
REFERENCES Baird, A. and Ling, N. (1987). Fibroblast growth factors are present in the extracellular matrix produced by endothelial cells in vitro: implications for a role of heparinase-like enzymes in the neovascular response. Biochem. Biophys. Res. Comrnun. 142,428435. Baird, A., Schubert, D., Ling, N. and Guillemin, R. (1988). Receptor- and heparin-binding domains of basic fibroblast growth factor. Proc. Natl. Acad. Sci. U S A 85,2324-232. Bashkin, P., Doctrow, S., Klagsbrun, M., Svahn, C-M., Folkman, J. and Vlodavsky, I. (1989). Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules. Biochemistry 28,1737-1743. Besner, G. E. and Klagsbrun, M. (1988). Characterization and purification of a heparin-binding growth factor from mononuclear cell conditioned medium. J. Cell. Biol. 107, (6, Pt.3) A2706. Besner, G. E., Higashiyama, S. and Klagsbrun, M. (1990). Isolation and characterization of a macrophage-derived heparin-binding growth factor. Cell R e p / . 1,811-819. Bohlen, P., Esch, F., Baird, A. and Gospodarowicz, D. (1985). Acidic fibroblast growth factor (FGF) from bovine brain: amino terminal sequence and comparison with basic FGF. EMBO J. 4,1951-1956. Cook, P. W., Mattox, P. A,, Keeble, W. W., Pittelkow, M. R., Plowman, G. D., Shoyab, M., Adelman, J. P. and Shipley, G. D. (1991). A heparin sulfate-regulated human keratinocyte autocrine factor is similar or identical to amphiregulin. Mol. Cel Bid. 11,2547-2557. Esch, F., Ueno, N., Baird, A,, Hill, F., Denoroy, L., Ling N., Gospodarowicz, D. and Guillemin, R. (1985). Primary structure of bovine brain acidic fibroblast growth factor (FGF). Biochem. Biophys. Res. Commun. 133,554-562. Folkman, J., Klagsbrun, M., Sasse, J., Wadsinski, M., Ingber, D. and Vlodavsky, I. (1988). A heparin-binding angiogenic
protein, basic fibroblast growth factor, is stored within basement membrane. Am. J. Pathol. 130,393400. Gospodarowicz, D. and Cheng, J. (1986). Heparin protects basic and acidic FGF from inactivation. J. Cell. Physiol. 128, 475484. Heath, W. F. and Merrifield, R. 8. (1986). A synthetic approach to structure-function relationships in the murine epidermal growth factor molecule. Proc. Natl. Acad. Sci. U S A 83,6367-6371. Higashiyama, S., Abraham, J. A,, Miller, J., Fiddes, J. C . and Klagsbrun, M. (1991). A heparin-binding growth factor secreted by macrophage-like cells that is related to EGF. Science 251,936-939. Higashiyama, S., Lau, K., Besner, G. E., Abraham, J. A. and Klagsbrun, M. (1992). Structure of heparin-binding EGFlike growth factor; multiple forms, primary structure and glycosylation of the mature protein. J. Biol. Chem. 267, 6205-6212. Klagsbrun, M. and Baird, A. (1991). A dual receptor system is required for basic fibroblast growth factor activity. Cell 67, 229-231. Lobb, R. R. and Fett, J. W. (1984). Purification of two distinct growth factors from bovine neural tissue by heparin-affinity chromatography. Biochemistry 23,6295-6299. Luetteke, N. C. and Lee, D. C. (1990). Transforming growth factor alpha: expression, regulation and biological action of its integral precursor. Sem. Cancer. Biol. 1,265-275. Naglich, J. G., Metherall, J. E., Russell, D. W. and Eidels, L. (1992). Expression cloning of a Diphtheria toxin receptor: Identity with heparin-binding EGF-like growth factor precursor. Cell 69,1051-1061. Nestor, J. J., Newman, S. R., DeLustro, B., Todaro, G. J. and Schreiber, A. B. (1985). A synthetic fragment of rat transforming growth factor a with receptor binding and antigenic properties. Biochem. Biophys. Res. Comm. 129,226-232. Ornitz, D. M., Yayon, A., Flanagan, J. G., Svahn, C. M., Levi, E. and Leder, P. (1992). Heparin is required for cell-free binding of basic fibroblast growth factor to a soluble receptor and for mitogenesis in whole cells. Mol. Cell. Bid. 12, 240-247. Raines, E. W., Lane, T. F., Iruela-Arispe, M. L., Ross, R. and Sage, E. H. (1992). The extracellular glycoprotein SPARC interacts with platelet-derived growth factor (PDGF)-AB and -BB and inhibits binding of PDGF to its receptors. Proc. Natl. Acad. Sci. U S A 89,1281-1285. Rapraeger, A. C., Krufka, A. and Olwin, B. B. (1991). Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation. Science 252, 1705-1 708. Ruosiahti, E. and Yamaguchi, Y. (1991). Proteoglycans as modulators of growth factor activities. Cell 64,867-869. Shing, Y., Folkman, J., Sullivan, R., Butterfield, C., Murray, J. and Klagsbrun, M. (1984).Heparin affinity: purification of a tumor-derived capillary endothelial cell growth factor. Science 223,1296-1299. Vlodavsky, I., Folkman, J., Sullivan, R., Fridman, R., IshaiMichaeli, R., Sasse, J. and Klagsbrun, M. (1987). Endothelial cell-derived basic fibroblast growth factor synthesis and deposition into subendothelial extracellular matrix. Proc. Natl. Acad. Sci. U S A 84,2292-2296. Yayon, A,, Klagsbrun, M., Esko, J. D., Leder, P. and Ornitz, D. M. (1991). Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 64,841-848.
Growth Factors, 1992, Vol. 7, pp. 289-296 Reprints available directly from the publisher Photocopying permitted by license only
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Interaction of Heparin-Binding EGF-Like Growth Factor (HB-EGF) with the Epidermal Growth Factor Receptor: Modulation by Heparin, Heparinase, or Synthetic Heparin-Binding HB-EGF Fragments GAIL E. BESNER*, DIANE WHELTON, MELISSA A. CRISSMAN-COMBS, CHRISTY L. STEFFEN, GREGORY Y. KIM and DAVID R. BRIGSTOCK Growth Factors Downloaded from informahealthcare.com by Chulalongkorn University on 01/12/15 For personal use only.
Department of Surgery, Ohio State University, Division of Pediatric Surgery, Children’s Hospitd, 700 Children’s Drive, Columbus, OH 43205
(Received April 20 1992, Accepted June 12 1992) The binding of heparin-binding EGF-like growth factor (HB-EGF) to the epidermal growth factor (EGF) receptor of human endometrial carcinoma cells was compared to that of EGF using an ‘*%EGF radioreceptor assay. The inhibitory effect of HB-EGF on Iz5I-EGFbinding was reversed either in the presence of heparin (but not by chondroitin sulfate) or by pre-treating the cells with heparinase. These treatments did not affect the binding of EGF to its receptor. To map potential regions in the HB-EGF molecule that mediate its heparin-dependent interaction with the EGF receptor, HB-EGF peptides were synthesized that were non-homologous to EGF. Accordingly residues 20-25 and 36-41, but not residues 8-19, of HB-EGF were found to be (i) heparin-binding and (ii) modulators of HB-EGF (but not of EGF) binding to the EGF receptor. KEYWORDS: heparin, HB-EGF, EGF, peptide mapping, extracellular matrix ABBREVIATIONS EGF, epidermal growth factor; HB-EGF, heparin-binding EGF-like growth factor; AR, amphiregulin; TGF-a, transforming growth factor alpha; aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; HBGF, heparin-binding growth factor; ECM, extracellular matrix; FPLC, fast protein liquid chromatography.
INTRODUCTION
family such as EGF, transforming growth factoralpha (TGF-a), and amphiregulin (AR) Heparin-binding EGF-like growth factor (HB- (Higashiyama et al., 1991). This was recently proEGF), a novel heparin-binding mitogen for ven with the purification and amino acid sesmooth muscle cells and fibroblasts, was first quencing of native HB-EGF proteins (Higashirecognized as a secreted product of cultured yama et al., 1992). Complementary DNA analysis human macrophages (Besner and Klagsbrun, has suggested that in common with other EGF 1988; Besner et al., 1990). Recently sufficient family members, HB-EGF is initially synthesized quantities of HB-EGF were purified from con- as a large precursor (208 amino acids) which is ditioned medium of the U-937 macrophage-like then cleaved to yield a mature protein compriscell line, allowing for the determination of its ing at least 86 amino acids (Higashiyama et al., unique N-terminal amino acid sequence 1991, 1992). Full length mature HB-EGF is a (Higashiyama et al., 1991). From the cDNA of glycosylated protein with an apparent molecular HB-EGF it was predicted that the amino acid weight of 22,000 as assessed by sodium dodecyl sequence of HB-EGF is similar to those of other sulfate polyacrylamide gel electrophoresis members of the epidermal growth factor (EGF) (Higashiyama et al., 1992). The HB-EGF precursor contains a highly hydrophobic sequence similar to the transmembrane domain of the precur“To whom correspondence should be addressed at: sor for TGF-a (Luetteke and Lee, 1990; Department of Surgery, Children’s Hospital, 700 Children‘s Drive, Columbus OH 43205. Tel: (614) 460-2015 Fax: (614) 460- Higashiyama et al., 1991, 1992). This suggests that HB-EGF may exist as a high molecular 7088. 289
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BESNER et a/.
weight membrane-anchored protein as well as a fully processed secretory protein. This notion is supported by a very recent demonstration that a diphtheria toxin receptor is identical to the HBEGF precursor (Naglich et al., 1992). HB-EGF competes with EGF for binding to the EGF receptor on A-431 cells or bovine vascular smooth muscle cells (Higashiyama et al., 1991) and it stimulates the proliferation of EGF-responsive cells (Besner et al., 1990; Higashiyama et al., 1991).In addition, HB-EGF stimulates EGF receptor tyrosine kinase activity and EGF receptor tyrosine autophosphorylation (Higashiyama et al., 1992). These observations are consistent with the structural relatedness of HB-EGF to EGF, and demonstrate that these two factors bind to the same signal-transducing receptor. However, despite their structural and biological similarities, a principal difference between EGF and HB-EGF lies in their affinity for heparin. HB-EGF binds strongly to heparin-affinity matrices, requiring 1 M NaCl for elution, whereas EGF and TGF-a do not bind to heparin resins at all (Besner and Klagsbrun, 1988; Besner et al., 1990; Higashiyama et al., 1991). In addition, it was recently reported that AR is a heparin-binding protein (Cook et al., 1991). Thus, the EGF family contains members that have a strong affinity for heparin (AR, HB-EGF) and others that do not (EGF, TGFa). This dichotomy points to the importance of understanding, firstly, the functign (if any) of heparin-affinity of certain EGF-like growth factors and, secondly, the molecular basis of heparin-binding. Among the best characterized heparin-binding growth factors (HBGFs) are acidic and basic fibroblast growth factors (aFGF, bFGF) which were isolated by purification regimens that utilized heparin-affinity chromatography (Shing et al., 1984; Lobb and Fett, 1984; Bohlen et al., 1985; Esch et al., 1985).It is well established that bFGF binds to immobilized heparin, is present in the extracellular matrix (ECM) and basement membrane in association with heparin sulfate proteoglycan (Baird and Ling, 1987; Vlodavsky et al., 1987; Folkman et al., 1988; Bashkin et al., 1989), and is protected by heparin from inactivation (Gospodarowicz and Cheng, 1986). However, only recently has a functional significance of this interaction become apparent-namely that cell surface heparan sulfate proteoglycan is required for bFGF binding to its high affinity receptor
__
(Yayon et al., 1991; Ornitz et al., 1992) and subsequent biological actions including fibroblast growth (Rapraeger et al., 19911, myoblast differentiation (Rapraeger et al., 1991), and DNA synthesis in a lymphoid cell line transfected with the FGF receptor (Ornitz et al., 1992). Accordingly, the role played by the ECM or cell surface proteoglycans in binding FGFs and regulating their availability to high affinity receptors is attracting considerable interest (Ruosiahti and Yamaguchi, 1991; Klagsbrun and Baird, 1991). Although most reports to date have addressed the issue of FGF-heparin interactions, recent studies suggest that proteoglycan regulation of growth factor activity may be a common phenomenon. Examples include the interaction of the core protein of decorin and betaglycan with TGF-P (Ruosiahti and Yamaguchi, 1991)and of SPARC (secreted protein, acidic and rich in cysteine) with platelet-derived growth factor -AB and -BB (Raines et al., 1992). Since the existence of heparin-binding members of the EGF family has been recognized for only a short time, little has been reported regarding the interaction of AR or HB-EGF with heparin. However in a recent report it was shown that heparin inhibited EGF receptor binding and stimulation of DNA synthesis by a heparin-binding human keratinocyte-derived autocrine growth factor similar or identical to AR (Cook et al., 1991). In the studies described here we investigated the effect of heparin on the interaction between the EGF receptor expressed by a cultured endometrial cell line and either HB-EGF or EGF. In addition, since synthetic peptides were useful in the mapping of possible heparin-binding and receptor-binding domains of bFGF (Baird et al., 1988), we adopted this approach to identify potential heparin-binding domains in the HBEGF protein.
MATERIALS AND METHODS
Materials HEC-1-B endometrial carcinoma cells and U-937 monocyte cells were obtained from ATCC. Cell culture -reagents and recombinant human EGF were from Gibco BRL. Iodinated mouse EGF (Iz5IEGF) (80-135 pCi/pg) was from ICN. Heparin, chondroitin sulfate,-heparinase, chondroiiinase
RECEPTOR BINDING OF HB-EGF
HB-EGF AR
29 1
-
8-19 20-25 36-41 YTLSSIZP--QALATP~KEEHGKRKKKGKGLG------~RDEZ;LRKYKD~-~~~PS€IUP~H~HGLSL SVIULEQWIZPPQNKTESE~TSDKPKRKKKGGKN~~RRNR~KNEZ;NAEFQN~-~IEHLEBVT€K€QQEXFEEK NSDSE€PLSHDGY.€LHDGV€MXIEALDKYA€N~W~I~QYRDLKWWELR
EGF TGF-a
WSHFND€PDSHTQKFH-GTCRFLVQEDKPACVUS~GA~E€IADLLA
FIGURE 1 Amino acid sequence of EGF-like growth factors. The amino acid sequences of human AR, EGF and TGF-a are compared to that of human HB-EGF (adapted from Higashiyama et al., 1991, 1992). Amino acids that are underlined represent residues that are homologous between HB-EGF and other family members when the cysteine residues in each protein are coaligned. The solid bars above the HB-EGF sequence depict the HB-EGF domains that were generated synthetically.
u Growth Factors Downloaded from informahealthcare.com by Chulalongkorn University on 01/12/15 For personal use only.
C
2000,
ABC, and bovine serum albumin (BSA) were from Sigma. Human HB-EGF was purified from U-937 cell conditioned medium by cationexchange chromatography, copper-affinity chromatography, and TSK-heparin affinity fast protein liquid chromatography (FPLC) as described (Higashiyama et al., 1991). Three HB-EGF peptide sequences were generated by fmoc solid phase synthesis by BioSynthesis Inc., Texas. The amino acid sequences of two of these peptides were QALATPNKEEHG and LRKYKD, corresponding to residues 8-19 and 36-41 of HB-EGF respectively. These sequences are non-homologous to the corresponding regions of EGF, TGF-a, and AR (Higashiyama et al., 1991). The third HB-EGF sequence, KRKKKG, corresponds to residues 20-25 of HB-EGF. This region is nonhomologous to EGF or TGF-a but is identical to residues 25-30 of AR (Higashiyama et al., 1991). The peptides are depicted schematically in Fig. 1.
A
0
20
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40
Fractlm N m b r
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EGF Radioreceptor Assay 0
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HEC-1-B cells were seeded in 48-well tissue culture plates at a density of 20,000 cells/we11/250 FIGURE 2 Effect of HB-EGF on '251-EGFreceptor binding. (A) p1 in Minimum Essential Medium (MEM) conPurification of HB-EGF by heparin-affinity FPLC and its taining 10% fetal bovine serum, 1 mM sodium detection using a '251-EGF radioreceptor assay. HB-EGF purified from U-937 conditioned medium by a combination of pyruvate, 292 pg/ml glutamine, non-essential cation exchange chromatography and copper chelating amino acids and antibiotics, and allowed to grow chromatography was applied to a TSK-heparin-5PW column in a humidified atmosphere of 10% COz for 4-6 (8 mmx7.5 cm) in 10 mM Tris HCI (pH 7.4) containing 0.5 M NaCI, essentially as described (Higashiyama et al., 1991). After days. The medium in each well was then aspirsample application, the column was washed with 10ml of ated and the cells washed with 2 5 0 ~ 1of cold 10 mM Tris HCI (pH 7.4) containing 0.2 M NaCl and then binding medium (composed of MEM containing subjected to a 40 ml linear salt gradient of 0.2-2 M NaCl in 10 mM Tris HC1 (pH 7.4). Fractions (1 ml) collected during 50 mM BES, 0.1 pM KI and 0.1% BSA, pH 7.2). gradient formation were tested for their ability to inhibit 1251- Each well then received 150 pl of 0.5 ng/ml '"IEGF binding by addin 20 p1 of each fraction to HEC-I-B cells EGF in binding medium pIus test substances in 150 pl of 0.5 ng/ml' 5'1-EGF. (B) Competition curve showing dose-dependent inhibition of lZ5I-EGFbinding by HB-EGF. (glycosaminoglycans, HB-EGF peptides, etc.) and Fractions 19-21 (see Fig. 2A) were pooled and assayed in either 0.5-3 ng/ml of EGF (to reduce cellular '"1triplicate at the indicated doses on HEC-1-B cells in 150 p1 of EGF binding to 30-60% of its maximal value) or a binding medium containing 0.5 ng/ml lZ51-EGF.The figure shows the specific lZ51-EGFbinding in the presence of the comparable dose of HB-EGF. The cells were incubated at 4°C for 2 hours after which they were different amounts of HB-EGF. I-B-EGF (ul/well)
BESNER el al.
292
washed three times with cold phosphatebuffered saline containing 0.1 pM KI and 0.1% BSA and then solubilized in 25Opl of 0.3N NaOH for counting of cell-associated lZ5I.Nonspecific binding as determined in the presence of a 1000-fold excess of unlabelled EGF (i.e. 500ng/ml) was typically 5-10% of maximal binding.
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RESULTS HEC-1-B cells express approximately 2x106 EGF receptors per cell to which binding of lZ5I-EGFis saturable, time- and temperature-dependent, and specific (i.e. non-displaceable by a 40-fold excess of bFGF, aFGF, PDGF, or IGF-I) (data not shown). Since HB-EGF was reported to bind to the EGF receptor of A-431 cells or smooth muscle cells (Higashiyama et al., 1991), column fractions of U-937 cell conditioned medium were moni: tored for the presence of HB-EGF by testing their
A
..-
z
0
ability to displace Iz5I-EGFbinding to HEC-I-B cells. As shown in Fig. 2A, a single peak of Iz5IEGF-competing activity was eluted from a heparin-affinity FPLC column by 1 M NaCl, characteristic of the elution position of HB-EGF (Besner et al., 1990; Higashiyama et al., 1991). These results further show that the lZ5I-EGFbinding assay is a very reliable and rapid means of screening column fractions for HB-EGF since other heparin-binding mitogens such as PDGF (which is also present in macrophage cell conditioned medium (Besner et al., 1990; Higashiyama et al., 1991, 1992)) and FGFs do not affect EGF binding under the conditions described (Figure 2A and unpublished results). As shown in Figure 2B, HB-EGF competed with Iz5I-EGFin a dose-dependent manner. To investigate the mechanism by which HBEGF interacts with the EGF receptor, cells were co-incubated with heparin or chondroitin sulfate (0-300 pglml) and unlabelled EGF or HB-EGF. Neither heparin nor chondroitin sulfate affected
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FIGURE 3 Heparin-dependence of HB-EGF-mediated reduction of "'I-EGF binding. HEC-I-B cells were incubated in 0.5 ng/ml "'I-EGF containing the indicated concentrations of heparin ( A X ) or chondroitin sulfate (D-F) plus either unlabelled EGF at 0.5 ng/ml (to reduce Iz5I-EGFbinding by about 40%) (B,E) or an approximately equivalent inhibitory dose of HB-EGF (C,F). The specific binding of '"I-EGF to the cells after 2 hours at 4°C is shown ( m e a n h d . of triplicate determinations).
RECEPTOR BINDING OF HB-EGF
293
EGF binding was antagonized in a dose-dependent manner by heparin. Reversal of the HB6o EGF-mediated inhibition of 1251-EGFbinding 0 50 C occurred at heparin concentrations between 1 p.and 30 ,ug/ml (Fig. 3C) whereas the same concen0 trations of chondroitin sulfate were ineffective 40 (Fig. 3F). A slight reduction in the ability of HBW EGF to inhibit 1251-EGFbinding was observed at I 30very high concentrations (>lo0 pg/ml) of chondroitin sulfate (Fig. 3F) which is consistent with a previous report in which similar concentrations .g 20 of this glycosaminoglycan were shown to also marginally reduce cellular binding of bFGF 10 (Bashkin et al., 1989). These results suggest that HB-EGF binding to the EGF receptor of HEC-I-B cells may be regu0 lated in a specific manner by soluble heparin. To determine whether heparan sulfate on the cell surface can exert a similar regulatory role, cells FIGURE 4 Effect of heparinase pre-treatment of HEC-1-8 were pre-incubated with heparinase to remove cells on the ability of HB-EGF to bind to the EGF receptor. these potential HB-EGF binding sites. As shown HEC-I-B cells were treated for 4 hours at 37°C with 30 U/ml of in Fig. 4, the ability of HB-EGF to bind to the EGF heparinase-I. The culture medium was then removed from both heparinase-treated and non-treated well of cells, and receptor on heparinase-treated cells was reduced replaced with binding medium containing Iz5I-EGF(0.5 ng/ml) as compared to control cell cultures. Heparinase and HB-EGF (I p1 of pooIed fractions 20 and 21 from Fig. 2A). treatment did not affect the ability of unlabelled The figure shows '"I-EGF binding for each treatment group expressed as a percentage of the maximal lZ51-EGFbinding in EGF to compete for I2'I-EGF binding sites (data the absence of any competing HB-EGF (meanfs.d. of triplicate not shown). These results suggest that cell-surdeterminations). face heparan sulfate acts to regulate receptor binding of HB-EGF. maximal binding of Iz5I-EGF(Fig. 3A, D) or the To identify putative heparin-binding domains decreased level of Iz5I-EGFbinding in the pres- of the HB-EGF molecule, synthetic HB-EGF pepence of 0.5 ng/ml of unlabelled EGF (Fig. 3B, El. tides that do not share sequence homology with However, the ability of HB-EGF to inhibit Iz5I- non-heparin-binding members of the EGF family
I
8
Growth Factors Downloaded from informahealthcare.com by Chulalongkorn University on 01/12/15 For personal use only.
E E
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B RESIDES 8-19
C RESIDES 36-41
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FIGURE 5 Heparin-affinity FPLC of synthetic HB-EGF peptides. One milligram of each of the peptides corresponding to residues 8-19, 20-25 and 36-41 of HB-EGF was individually subjected to heparin-affinity FPLC essentially as described in the legend of Fig. 2, except that tht unbound fractions were also collected prior to NaCl gradient elution. The elution of each peptide from the heparin column was determined by monitoring the absorbance of each fraction at 214 nm.
BESNER er a / .
294
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RESIDUES 8-19
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Growth Factors Downloaded from informahealthcare.com by Chulalongkorn University on 01/12/15 For personal use only.
Peptide concentration (ug/mll FIGURE 6 Modulation of HB-EGF-mediated inhibition of lz5I-EGFbinding by heparin-bindingHB-EGF peptides. HEC-I-B cells were incubatd with '=I-EGF (approx. 0.5 ng/ml) containing the indicated concentrations of (A) residues 8-19, (B)residues 20-25 and ( C )residues 36-41 alone (W), in the presence of unlabelled EGF (O), or in the presence of unlabelled HB-EGF (A). EGF and HB-EGF were added at comparable concentrations so that maximal '"I-EGF binding was reduced by 60% by both competitors. Binding of '"I-EGF is expressed as a percentage of maximal IZ5I-EGFbinding that was determined in the absence of any added factor (meanhd. of triplicate determinations).
such as EGF and TGF-a (see Fig. 1) were generated. The heparin-binding property of each peptide was determined by individually subjecting them to TSK-heparin-affinity FPLC. As shown in Fig. 5A, HB-EGF[8-19] was eluted in the void volume showing that it was non-heparin-binding. In contrast, HB-EGF[20-25] and HBEGF[3641] each bound strongly to TSK-heparin and were eluted by 1.5M NaCl and 1 M NaCl respectively (Fig. 5B, C). These findings suggested that residues 20-25 and/or 36-41 of HBEGF may contribute, at least in part, to the binding of HB-EGF to cell surface heparin-like molecules-and hence the EGF receptor. To test this possibility, all three peptides were tested for their ability to modulate the inhibition of cellular Iz5I-EGFbinding that is caused by HB-EGF or EGF. None of the three peptides affected lz5I-EGF binding in the absence of any competitor or in the presence of unlabelled EGF (Fig. 6A-C). While the binding of HB-EGF to the EGF receptor was unaffected by the non-heparin-binding peptide (i.e. residues 8-19) (Fig. 6A), binding was influenced by the two heparin-binding peptides (Fig. 6B, C). In four separate experiments, residues 20-25 at 0.3-1 pg/ml or residues 36-41 at 3-10 ,ug/ml were found to potentiate the inhibitory effect of HB-EGF on '251-EGFbinding (Fig. 6B,C). Higher doses of each peptide reproducibly antagonized the ability of HB-EGF to bind to
the EGF receptor (Fig. 6B, C). However, the peptides were no more effective at 500 pg/ml than at 100 pglml (data not shown) and only partially reversed the HB-EGF-mediated inhibition of lz5I-EGFbinding (Fig. 6B, C).
DISCUSSION There is increasing evidence that availability of bFGF to its high affinity receptor is regulated by cell surface heparan sulfate proteoglycan molecules (Klagsbrun and Baird, 1991; Rapraeger et al., 1991; Ruosiahti et al., 1991; Yayon et al., 1991; Ornitz et al., 1992). Since bFGF is but one HBGF, these findings raise the question of whether the interaction between other HBGFs and cell surface heparin-like molecules constitutes a common receptor-binding mechanism for this class of mitogen. Our results suggest that, in common with bFGF, HB-EGF (which is structurally unrelated to bFGF) binds to cell surface heparan sulfate and that this binding primarily mediates the interaction of HB-EGF with its high affinity receptor. Evidence in support of this notion is that exogenous heparin or pretreatment of HEC1 -B cells with heparinase perturbs the ability of HB-EGF to bind to the EGF receptor whereas condroitin sulfate is ineffective. Our findings are similar to those of Cook et al. (1991) who showed
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RECEPTOR BINDING OF HB-EGF
that 30 pglml of heparin sulfate completely abolished the ability of AR to bind to the EGF receptor and to stimulate DNA synthesis in AKR-2B cells. Thus it would appear that EGF receptor occupancy by HB-EGF and AR-the two heparinbinding members of the EGF family-is heparindependent. These observations are in marked contrast to the binding of EGF to the EGF receptor which does not appear to be heparin-dependent for either human HEC-1-B cells (these results) or mouse AKR-2B cells (Cook et al., 19911, consistent with the lack of heparin-affinity of EGF (Shing et al., 1984). Since saturating concentrations of antagonists such as heparin (see Fig. 3C) or heparin-binding HB-EGF peptides (see Fig. 6B,C) did not completely reverse the binding of HB-EGF to the EGF receptor, the possibility that a small proportion of HB-EGF binds directly to the EGF receptor cannot be excluded. Since cell-surface heparan sulfate was shown to potentiate HB-EGF receptor binding, it might be expected that exogenously added heparin would do the same. However, we found that added heparin actually inhibited the ability of HB-EGF to displace Iz5I-EGFbinding to its receptor. This apparent discrepancy may be accounted for if HB-EGF receptor binding is a function of the proximity and spatial relationship of cell-surface heparan sulfate with the EGF receptor. Therefore a complex of HB-EGF and soluble (i.e. exogenously added) heparin may result in inappropriate conformational and/or spatial presentation of HB-EGF to the EGF receptor. Given the pronounced difference between HBEGF and EGF in their affinities for heparin, we reasoned that the heparin-binding properties of HB-EGF might reside in the N-terminal non-EGFlike domain (i.e. between residues 1 and 29; see Fig. 1). This region of HB-EGF is highly hydrophilic (Higashiyama et al., 1991) and shows areas of homology with the corresponding region of AR (residues 1-34) which is also highly hydrophilic (Cook et al., 1991). Moreover the N-terminal hydrophilic domain of AR has been suggested as a heparin-regulatory site (Cook et al., 1991) although evidence to support this has not been presented. Since synthetic peptides have been used in structure-function mapping of EGF, TGF-a and bFGF (Nestor et al., 1985; Heath et al., 1986; Baird et al., 1988), we adopted this approach to map potential heparin-binding
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regions of HB-EGF. The ability of residues 20-25 to bind to heparin and modulate HB-EGF receptor binding suggests that this region constitutes at least part of a putative heparin-binding domain. Interestingly, this domain is also present in the AR hydrophilic region (see Fig. 1) suggesting that, if involved in heparin-binding, its function may be conserved and common to both AR and HB-EGF. Similar EGF receptor modulatory effects were also exhibited by residues 36-41 that are unique to HB-EGF but are present in the EGFlike region of HB-EGF (see Fig. 1). This finding was somewhat unexpected considering that this domain is not located in the N-terminus, and suggests that multiple amino acid domains of HB-EGF may account for its heparin-binding properties. Although residues 20-25 and 36-41 are highly cationic, a previous study of bFGF peptides showed that a high positive charge does not necessarily correlate with heparin-affinity (Baird et al., 1988). Therefore the highly specific interaction between heparin and the heparin-binding HB-EGF peptides identified here may not be due solely to charge effects. Baird et al. (1988) also showed that the heparin-binding bFGF peptides were capable of inhibiting lZ5I-bFGFbinding to high affinity receptors. In our study this would also seem to be the case since the heparin-binding HB-EGF peptides were able to modulate receptor binding of HB-EGF but not that of EGF. An unexpected feature was that the heparinbinding peptides modulated EGF receptor binding by HB-EGF in a bi-phasic manner in that they reproducibly potentiated HB-EGF receptor binding at low doses but inhibited it at high doses. These findings suggest a complex interaction of the peptides or HB-EGF with their respective cellular binding sites. Further studies using control peptides of similar size and composition as the heparin-binding HB-EGF peptides will help to establish the underlying mechanism of the biphasic phenomenon. Future studies will also address the question of whether the various modulators of HB-EGF binding reported in this study are effective regulators of HB-EGF biological activity. Taken together, these results highlight the importance of ECM and cell surface heparin-like molecules in regulating the binding of HB-EGF to its signal-transducing receptor. Since this mechanism is shared with at least two other HBGFs
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(AR and bFGF), it is clear that local concentration changes in heparin and/or heparin-degrading enzymes act as pivotal control mechanisms whereby the accessibility of a variety of HBGFs to their respective high affinity receptors may be regulated.
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ACKNOWLEDGMENTS G.E.B. and D.R.B. were s u p p o r t e d by grants 020-871 and 020-872 a w a r d e d by Children’s Hospital Research Foundation, Columbus, OH. G.E.B. was also supported by a n Ohio State University Seed Grant a n d by a w a r d s from the Bremer Foundation and the O h i o State University Department of Surgery Medical Research a n d Development Fund. We thank Dr. D.R. Cooney for his generous support.
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