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1.
A focused library of novel, sulfated glycoconjugates was synthesized by utilizing carbohydrate-derived blocks in the four-component Ugi condensation. Library members comprise a sulfated monosaccharide linked by various spacers to either an aromatic or monosulfated moiety, or a second sulfated monosaccharide. The affinities of these heparan sulfate (HS) mimetics for the HS-binding fibroblast growth factors FGF-1 and FGF-2 were measured via a surface plasmon resonance solution affinity assay. 相似文献
2.
Binding of fibroblast growth factors (FGFs) to receptor tyrosine kinases (FGFRs) and signaling is facilitated by binding of FGF to heparan sulfate proteoglycans (HSPGs). There are multiple families of HSPGs, including extracellular and cell surface forms. An important and potentially controversial question is whether cell surface forms of HSPGs act as positive or negative regulators of FGF signaling. This study examines the ability of the cell surface HSPG syndecan-1 to regulate FGF binding and signaling. HSPG-deficient Raji lymphoma cells, expressing a transfected syndecan-1 cDNA (Raji S1 cells), were used as HSPG “donor” cells. BaF3 cells, expressing an FGFR1 cDNA (FR1C-11 cells), were used as FGFR “reporter” cells. Using Raji S1 cells preincubated with FGF, it was found that they formed heterotypic aggregates with FR1C-11 cells in the presence of FGF-2, but not FGF-1. In addition, the FR1C-11 cells demonstrated FGF-2, but not FGF-1, dependent survival when cultured on fixed Raji S1 cells. Thus, Raji syndecan-1 (1) differentially regulates the binding and signaling of FGFs 1 and 2 and (2) acts as a positive regulator of FGF-2 signaling. J. Cell. Physiol. 174:310–321, 1998. © 1998 Wiley-Liss, Inc. 相似文献
3.
Identification of common and specific growth factor binding sites in heparan sulfate proteoglycans 总被引:2,自引:0,他引:2
The structural complexity within heparan sulfate has suggested that it contains multiple protein-specific binding sites. To evaluate the selectivity of growth factor binding to heparan sulfate, we conducted a detailed study of the intercompetition of fibroblast growth factor-2 (FGF-2) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) binding to heparan sulfate (HS) on bovine aortic smooth muscle cells. Radioligand binding assays were conducted, and an analytical method was developed for determining the apparent binding constants and numbers of specific and shared binding sites within HS. These studies revealed the presence of two general classes of HS-binding sites for FGF-2 and HB-EGF. The major class (approximately 10(6) sites per cell) was able to bind to either growth factor with relatively low affinity (K(d) = 12 and 44 nM for FGF-2 and HB-EGF, respectively) and was termed "common" binding sites. However, both FGF-2 and HB-EGF also showed specific high affinity (0.6 and 6.1 nM for FGF-2 and HB-EGF, respectively) binding to a minor subset (118,000 and 28,000 sites per cell for FGF-2 and HB-EGF, respectively) of "unique" binding sites, which were unable to bind the other growth factor. These studies indicate that growth factor binding to HS involves multiple binding sites of variable affinity, density, and selectivity. The approach outlined in this study could be applied to aid in the evaluation of the relative biological roles of these selective and nonselective growth factor binding domains within HS. 相似文献
4.
Characterization of growth factor-binding structures in heparin/heparan sulfate using an octasaccharide library 总被引:8,自引:0,他引:8
Ashikari-Hada S Habuchi H Kariya Y Itoh N Reddi AH Kimata K 《The Journal of biological chemistry》2004,279(13):12346-12354
Heparan sulfate (HS) chains interact with various growth and differentiation factors and morphogens, and the most interactions occur on the specific regions of the chains with certain monosaccharide sequences and sulfation patterns. Here we generated a library of octasaccharides by semienzymatic methods by using recombinant HS 2-O-sulfotransferase and HS 6-O-sulfotransferase, and we have made a systematic investigation of the specific binding structures for various heparin-binding growth factors. An octasaccharide (Octa-I, DeltaHexA-GlcNSO(3)-(HexA-GlcNSO(3))(3)) was prepared by partial heparitinase digestion from completely desulfated N-resulfated heparin. 2-O- and 6-O-sulfated Octa-I were prepared by enzymatically transferring one to three 2-O-sulfate groups and one to three 6-O-sulfate groups per molecule, respectively, to Octa-I. Another octasaccharide containing 3 units of HexA(2SO(4))-GlcNSO(3)(6SO(4)) was prepared also from heparin. This octasaccharide library was subjected to affinity chromatography for interactions with fibroblast growth factor (FGF)-2, -4, -7, -8, -10, and -18, hepatocyte growth factor, bone morphogenetic protein 6, and vascular endothelial growth factor, respectively. Based upon differences in the affinity to those octasaccharides, the growth factors could be classified roughly into five groups: group 1 needed 2-O-sulfate but not 6-O-sulfate (FGF-2); group 2 needed 6-O-sulfate but not 2-O-sulfate (FGF-10); group 3 had the affinity to both 2-O-sulfate and 6-O-sulfate but preferred 2-O-sulfate (FGF-18, hepatocyte growth factor); group 4 required both 2-O-sulfate and 6-O-sulfate (FGF-4, FGF-7); and group 5 hardly bound to any octasaccharides (FGF-8, bone morphogenetic protein 6, and vascular endothelial growth factor). The approach using the oligosaccharide library may be useful to define specific structures required for binding to various heparin-binding proteins. Octasaccharides with the high affinity to FGF-2 and FGF-10 had the activity to release them, respectively, from their complexes with HS. Thus, the library may provide new reagents to specifically regulate bindings of the growth factors to HS. 相似文献
5.
Fibroblast growth factors (FGFs) require heparan sulfate proteoglycans (HSPGs) as cofactors for signaling. The heparan sulfate chains (HS) mediate stable high affinity binding of FGFs to their receptor tyrosine kinases (FR) and may specifically regulate FGF activity. A novel in situ binding assay was developed to examine the ability of HSPGs to promote FGF/FR binding using a soluble FR fusion construct (FR1-AP). This fusion protein probe forms a dimer in solution, simulating the dimerization or oligomerization that is thought to occur at the cell surface physiologically. In frozen sections of human skin, FGF-2 binds to keratinocytes and basement membranes of epidermis and dermal blood vessels. In contrast, in skin preincubated with FGF-2, FR1-AP binds avidly to FGF-2 immobilized on keratinocyte cell surfaces, but fails to bind to basement membranes at the dermo-epidermal junction or dermal microvessels despite the fact that these structures bind large amounts of FGF-2. Apparently, basement membrane and cell surface HSPGs differ in their ability to mediate the assembly of a FGF/FR signaling complex presumably due to structural differences of the heparan sulfate chains. 相似文献
6.
A divalent cation-dependent association between heparin or heparan sulfate and the ectodomain of the FGF receptor kinase (FGFR) restricts FGF-independent trans-phosphorylation and supports the binding of activating FGF to self-associated FGFR. Here we show that in contrast to heparin, cellular heparan sulfate forms a binary complex with FGFR that discriminates between FGF-1 and FGF-2. FGFR type 4 (FGFR4) in liver parenchymal cells binds only FGF-1, whereas FGFR1 binds FGF-1 and FGF-2 equally. Cell-free complexes of heparin and recombinant FGFR4 bound FGF-1 and FGF-2 equally. However, in contrast to FGFR1, when recombinant FGFR4 was expressed back in epithelial cells by transfection, it failed to bind FGF-2 unless heparan sulfate was depressed by chlorate or heparinase treatment. Isolated heparan sulfate proteoglycan (HSPG) from liver cells in cell-free complexes with FGFR4 restored the specificity for FGF-1 and supported the binding of both FGF-1 and FGF-2 when complexed with FGFR1. In contrast, FGF-2 bound equally well to complexes of both FGFR1 and FGFR4 formed with endothelial cell-derived HSPG, but the endothelial HSPG was deficient for the binding of FGF-1 to both FGFR complexes. These data suggest that a heparan sulfate subunit is a cell type- and FGFR-specific determinant of the selectivity of the FGFR signaling complex for FGF. In a physiological context, the heparan sulfate subunit may limit the redundancy among the current 18 FGF polypeptides for the 4 known FGFR. 相似文献
7.
Ruoyan Xu Alessandro Ori Timothy R. Rudd Katarzyna A. Uniewicz Yassir A. Ahmed Scott E. Guimond Mark A. Skidmore Giuliano Siligardi Edwin A. Yates David G. Fernig 《The Journal of biological chemistry》2012,287(47):40061-40073
The functions of a large number (>435) of extracellular regulatory proteins are controlled by their interactions with heparan sulfate (HS). In the case of fibroblast growth factors (FGFs), HS binding determines their transport between cells and is required for the assembly of high affinity signaling complexes with their cognate FGF receptor. However, the specificity of the interaction of FGFs with HS is still debated. Here, we use a panel of FGFs (FGF-1, FGF-2, FGF-7, FGF-9, FGF-18, and FGF-21) spanning five FGF subfamilies to probe their specificities for HS at different levels as follows: binding parameters, identification of heparin-binding sites (HBSs) in the FGFs, changes in their secondary structure caused by heparin binding and structures in the sugar required for binding. For interaction with heparin, the FGFs exhibit KD values varying between 38 nm (FGF-18) and 620 nm (FGF-9) and association rate constants spanning over 20-fold (FGF-1, 2,900,000 m−1 s−1 and FGF-9, 130,000 m−1 s−1). The canonical HBS in FGF-1, FGF-2, FGF-7, FGF-9, and FGF-18 differs in its size, and these FGFs have a different complement of secondary HBS, ranging from none (FGF-9) to two (FGF-1). Differential scanning fluorimetry identified clear preferences in these FGFs for distinct structural features in the polysaccharide. These data suggest that the differences in heparin-binding sites in both the protein and the sugar are greatest between subfamilies and may be more restricted within a FGF subfamily in accord with the known conservation of function within FGF subfamilies. 相似文献
8.
Murphy PV Pitt N O'Brien A Enright PM Dunne A Wilson SJ Duane RM O'Boyle KM 《Bioorganic & medicinal chemistry letters》2002,12(22):3287-3290
Inhibitors of FGF-2 binding to a heparin-albumin conjugate were identified by ELISA from a library of glucuronic acid derivatives. These compounds were also inhibitors of endothelial cell survival that is dependant on FGF-2 and heparin or heparan sulfate proteoglycans. The results indicate that these bioactive compounds may prove useful as lead structures for the further development of pharmaceutical agents capable of modulating biological activity of FGF-2. 相似文献
9.
Harwood LJ Gerber H Sobrino F Summerfield A McCullough KC 《Journal of virology》2008,82(13):6379-6394
Dendritic cells (DC), which are essential for inducing and regulating immune defenses and responses, represent the critical target for vaccines against pathogens such as foot-and-mouth disease virus (FMDV). Although it is clear that FMDV enters epithelial cells via integrins, little is known about FMDV interaction with DC. Accordingly, DC internalization of FMDV antigen was analyzed by comparing vaccine virus dominated by heparan sulfate (HS)-binding variants with FMDV lacking HS-binding capacity. The internalization was most efficient with the HS-binding virus, employing diverse endocytic pathways. Moreover, internalization relied primarily on HS binding. Uptake of non-HS-binding virus by DC was considerably less efficient, so much so that it was often difficult to detect virus interacting with the DC. The HS-binding FMDV replicated in DC, albeit transiently, which was demonstrable by its sensitivity to cycloheximide treatment and the short duration of infectious virus production. There was no evidence that the non-HS-binding virus replicated in the DC. These observations on virus replication may be explained by the activities of viral RNA in the DC. When DC were transfected with infectious RNA, only 1% of the translated viral proteins were detected. Nevertheless, the transfected cells, and DC which had internalized live virus, did present antigen to lymphocytes, inducing an FMDV-specific immunoglobulin G response. These results demonstrate that DC internalization of FMDV is most efficient for vaccine virus with HS-binding capacity, but HS binding is not an exclusive requirement. Both non-HS-binding virus and infectious RNA interacting with DC induce specific immune responses, albeit less efficiently than HS-binding virus. 相似文献
10.
N Raboudi J Julian L H Rohde D D Carson 《The Journal of biological chemistry》1992,267(17):11930-11939
The interaction of heparin (HP) with the cell-surface components of a human uterine epithelial carcinoma cell line (RL95) was studied. Binding of [3H]HP to cell surfaces was saturable in a dose- and time-dependent manner. HP and certain forms of heparan sulfate (HS) efficiently compete for [3H]HP binding. In contrast, other glycosaminoglycans, such as chondroitin sulfate, keratan sulfate, hyaluronic acid, and dermatan sulfate, do not compete for binding to these sites. Scatchard analysis revealed that [3H]HP bound to these sites with an apparent KD of 0.7-0.9 microM and a binding capacity of 9 x 10(6) sites/cell to attached cells. EDTA-detached cells displayed a similar apparent KD, but an approximately 2-fold increase in binding capacity. Protease digestion of cells on ice markedly reduced [3H]HP binding, indicating that these binding sites were associated with proteins. In contrast, heparinase treatment of cells stimulated binding by approximately 2-fold, indicating that a large fraction of these binding sites were occupied with endogenous ligand. We examined the structural features of HP/HS required for HP/HS binding. O-Sulfation, substitution of amino groups, and, to a lesser extent, the presence of carboxyl groups were important recognition features of HP/HS by cell-surface HP/HS-binding sites. N-Sulfation was not required. Photoaffinity labeling with 125I-sulfosuccinimidyl 2-(p-azidosalicylamido)-ethyl-1, 3-dithiopropionate-HP was used to identify HP/HS-binding proteins on RL95 cell surfaces. Proteins with M(r) values of 14,000-18,500 and 31,000 were photolabeled at the surfaces of attached cells. Photolabeling was blocked by the addition of excess HP, but not chondroitin sulfate. Additional proteins with M(r) values greater than 31,000 were photolabeled specifically on EDTA-detached cells. Moreover, the M(r) 14,000-18,500 and 31,000 proteins were retained on the EDTA-detached cells. These observations indicated that certain cell-surface HP/HS-binding proteins were not exposed when cells were attached to substrata. Proteins of similar M(r) values as the photolabeled components as well as many additional proteins were identified by heparin-agarose chromatographic selection of extracts of cells labeled metabolically with [35S]methionine or vectorially with Na125I at the cell surface. Fragments of cell-surface HP/HS-binding proteins were released from intact RL95 and mouse uterine epithelial cells by mild trypsinization and isolated by heparin-agarose affinity chromatography. Three peptides with M(r) values between 6000 and 14,000 required greater than 0.5 M salt for elution from heparin-agarose, retained HP binding activity in a 125I-HP gel overlay assay, and selectively bound [3H]HP in a solid-phase binding assay.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
11.
Beer HD Vindevoghel L Gait MJ Revest JM Duan DR Mason I Dickson C Werner S 《The Journal of biological chemistry》2000,275(21):16091-16097
Fibroblast growth factors (FGFs) transmit their signals through four transmembrane receptors that are designated FGFR1-4. Alternative splicing in the extracellular region of FGFR1-3 generates receptor variants with different ligand binding affinities. Thus two types of transmembrane receptors (IIIb and IIIc isoforms) have been identified for FGFR2 and FGFR3, and the existence of analogous variants has been postulated for FGFR1 based on its genomic structure. However, only a single full-length transmembrane FGFR1 variant (FGFR1-IIIc) has been identified so far. Here we describe the cloning of a full-length cDNA encoding FGFR1-IIIb from a mouse skin wound cDNA library. This receptor isoform was expressed at the highest levels in a subset of sebaceous glands of the skin and in neurons of the hippocampus and the cerebellum. FGFR1-IIIb was expressed in L6 rat skeletal muscle myoblasts and used in cross-linking and receptor binding studies. FGF-1 was found to bind the receptor with high affinity, whereas FGF-2, -10, and -7 bound with significantly lower affinities. Despite their apparently similar but low affinities, FGF-10 but not FGF-7 induced the activation of p44/42 mitogen-activated protein kinase in FGFR1-IIIb-expressing L6 myoblasts and stimulated mitogenesis in these cells, demonstrating that this new receptor variant is a functional transmembrane receptor for FGF-10. 相似文献
12.
Herpes simplex virus types 1 and 2 differ in their interaction with heparan sulfate 总被引:7,自引:0,他引:7
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Cell surface heparan sulfate (HS) serves as an initial receptor for many different viruses, including herpes simplex virus types 1 and 2 (HSV-1 and 2, respectively). Glycoproteins C and B (gC and gB) are the major components of the viral envelope that mediate binding to HS. In this study, purified gB and gC homologous proteins as well as purified HSV-1 and HSV-2 virions were compared for the ability to bind isolated HS receptor molecules. HSV-1 gC and HSV-2 gC bound comparable amounts of HS. Similarly, HSV-1 gB and its HSV-2 counterpart showed no difference in the HS-binding capabilities. Despite the similar HS-binding potentials of gB and gC homologs, HSV-1 virions bound more HS than HSV-2 particles. Purified gC and gB proteins differed with respect to sensitivity of their interaction with HS to increased concentrations of sodium chloride in the order gB-2 > gB-1 > gC-1 > gC-2. The corresponding pattern for binding of whole HSV virions to cells in the presence of increased ionic strength of the medium was HSV-2 gC-neg1 > HSV-1 gC(-)39 > HSV-1 KOS 321 > HSV-2 333. These results relate the HS-binding activities of individual glycoproteins with the cell-binding abilities of whole virus particles. In addition, these data suggest a greater contribution of electrostatic forces for binding of gB proteins and gC-negative mutants compared with binding of gC homologs and wild-type HSV strains. Binding of wild-type HSV-2 virions was the least sensitive to increased ionic strength of the medium, suggesting that the less extensive binding of HS molecules by HSV-2 than by HSV-1 can be compensated for by a relatively weak contribution of electrostatic forces to the binding. Furthermore, gB and gC homologs exhibited different patterns of sensitivity of binding to cells to inhibition with selectively N-, 2-O-, and 6-O-desulfated heparin compounds. The O-sulfate groups of heparin were found to be more important for interaction with gB-1 than gB-2. These results indicate that HSV-1 and HSV-2 differ in their interaction with HS. 相似文献
13.
J Kovensky P Duchaussoy F Bono M Salmivirta P Sizun J M Herbert M Petitou P Sina? 《Bioorganic & medicinal chemistry》1999,7(8):1567-1580
It has been suggested that the FGF-2 binding site on heparan sulfate chains is a trisulfated pentasaccharide containing three hexuronic acid units. The configuration at C-5 of two of them being undetermined, we have synthesized the four possible pentasaccharides, and have evaluated their FGF-2 binding affinity through in vitro biological assays. The pentasaccharide containing L-iduronic acid as the sole hexuronic acid showed higher affinity for FGF-2 than the other pentasaccharides, where one hexuronic acid unit at least is D-glucuronic acid. 相似文献
14.
Deepa SS Umehara Y Higashiyama S Itoh N Sugahara K 《The Journal of biological chemistry》2002,277(46):43707-43716
We previously observed that the cortical neuronal cell adhesion mediated by midkine (MK), a heparin (Hep)-binding growth factor, is specifically inhibited by oversulfated chondroitin sulfate-E (CS-E) (Ueoka, C., Kaneda, N., Okazaki, I., Nadanaka, S., Muramatsu, T., and Sugahara, K. (2000) J. Biol. Chem. 275, 37407-37413) and that CS-E exhibits neurite outgrowth promoting activities toward embryonic rat hippocampal neurons. We have also shown oversulfated CS chains in embryonic chick and rat brains and demonstrated that the CS disaccharide composition changes during brain development. In view of these findings, here we tested the possibility of CS-E interacting with Hep-binding growth factors during development, using squid cartilage CS-E. The binding ability of Hep-binding growth factors (MK, pleiotrophin (PTN), fibroblast growth factor-1 (FGF-1), FGF-2, Hep-binding epidermal growth factor-like growth factor (HB-EGF), FGF-10, FGF-16, and FGF-18) toward [(3)H]CS-E was first tested by a filter binding assay, which demonstrated direct binding of all growth factors, except FGF-1, to CS-E. The bindings were characterized further in an Interaction Analysis system, where all of the growth factors, except FGF-1, gave concentration-dependent and specific bindings. The kinetic constants k(a), k(d), and K(d) suggested that MK, PTN, FGF-16, FGF-18, and HB-EGF bound strongly to CS-E, in comparable degrees to the binding to Hep, whereas the intensity of binding of FGF-2 and FGF-10 toward CS-E was lower than that for Hep. These findings suggest the possibility of CS-E being a binding partner, a coreceptor, or a genuine receptor for various Hep-binding growth factors in the brain and possibly also in other tissues. 相似文献
15.
Berman B Ostrovsky O Shlissel M Lang T Regan D Vlodavsky I Ishai-Michaeli R Ron D 《The Journal of biological chemistry》1999,274(51):36132-36138
The keratinocyte growth factor (KGF or FGF-7) is unique among its family members both in its target cell specificity and its inhibition by the addition of heparin and the native heparan-sulfate proteoglycan (HSPG), glypican-1 in cells expressing endogenous HSPGs. FGF-1, which binds the FGF-7 receptor with a similar affinity as FGF-7, is stimulated by both molecules. In the present study, we investigated the modulation of FGF-7 activities by heparin and glypican-1 in HS-free background utilizing either HS-deficient cells expressing the FGF-7 receptor (designated BaF/KGFR cells) or soluble extracellular domain of the receptor. At physiological concentrations of FGF-7, heparin was required for high affinity receptor binding and for signaling in BaF/KGFR cells. In contrast, binding of FGF-7 to the soluble form of the receptor did not require heparin. However, high concentrations of heparin inhibited the binding of FGF-7 to both the cell surface and the soluble receptor, similar to the reported effect of heparin in cells expressing endogenous HSPGs. The difference in heparin dependence for high affinity interaction between the cell surface and soluble receptor may be due to other molecule(s) present on cell surfaces. Glypican-1 differed from heparin in that it stimulated FGF-1 but not FGF-7 activities in BaF/KGFR cells. Glypican-1 abrogated the stimulatory effect of heparin, and heparin reversed the inhibitory effect of glypican-1, indicating that this HSPG inhibits FGF-7 activities by acting, most likely, as a competitive inhibitor of stimulatory HSPG species for FGF-7. The regulatory effect of glypican-1 is mediated at the level of interaction with the growth factor as glypican-1 did not bind the KGFR. The effect of heparin and glypican-1 on FGF-1 and FGF-7 oligomerization was studied employing high and physiological concentrations of growth factors. We did not find a correlation between the effects of these glycosaminoglycans on FGFs biological activity and oligomerization. Altogether, our findings argue against the heparin-linked dimer presentation model as key in FGFR activation, and support the notion that HSPGs primarily affect high affinity interaction of FGFs with their receptors. 相似文献
16.
Fibroblast growth factors FGF-1 and FGF-2 mediate their biological effects via heparan sulfate-dependent interactions with cell surface FGF receptors. While the specific heparan sulfate domain binding to FGF-2 has been elucidated in some detail, limited information has been available concerning heparan sulfate structures involved in the recognition of FGF-1. In the current study we present evidence that the minimal FGF-1 binding heparan sulfate sequence comprises 5-7 monosaccharide units and contains a critical trisulfated IdoA(2-OSO3)-GlcNSO3(6-OSO3) disaccharide unit. N-Sulfated heparan sulfate decasaccharides depleted of FGF-1 binding domains showed dose-dependent and saturable binding to FGF-2. These data indicate that the FGF-1 binding domain is distinct from the minimal FGF-2 binding site, previously shown to contain an IdoA(2-OSO3) residue but no 6-O-sulfate groups. We further show that the FGF-1 binding heparan sulfate domain is expressed in human aorta heparan sulfate in an age-related manner in contrast to the constitutively expressed FGF-2 binding domain. Reduction of heparan sulfate O-sulfation by chlorate treatment of cells selectively impedes binding to FGF-1. The present data implicate the 6-O-sulfation of IdoA(2-OSO3)-GlcNSO3 units in cellular heparan sulfate in the control of the biological activity of FGF-1. 相似文献
17.
Sher I Lang T Lubinsky-Mink S Kuhn J Adir N Chatterjee S Schomburg D Ron D 《The Journal of biological chemistry》2000,275(45):34881-34886
Fibroblast growth factors (FGFs) mediate a multitude of physiological and pathological processes by activating a family of tyrosine kinase receptors (FGFRs). Each FGFR binds to a unique subset of FGFs and ligand binding specificity is essential in regulating FGF activity. FGF-7 recognizes one FGFR isoform known as the FGFR2 IIIb isoform or keratinocyte growth factor receptor (KGFR), whereas FGF-2 binds well to FGFR1, FGFR2, and FGFR4 but interacts poorly with KGFR. Previously, mutations in FGF-2 identified a set of residues that are important for high affinity receptor binding, known as the primary receptor-binding site. FGF-7 contains this primary site as well as a region that restricts interaction with FGFR1. The sequences that confer on FGF-7 its specific binding to KGFR have not been identified. By utilizing domain swapping and site-directed mutagenesis we have found that the loop connecting the beta4-beta5 strands of FGF-7 contributes to high affinity receptor binding and is critical for KGFR recognition. Replacement of this loop with the homologous loop from FGF-2 dramatically reduced both the affinity of FGF-7 for KGFR and its biological potency but did not result in the ability to bind FGFR1. Point mutations in residues comprising this loop of FGF-7 reduced both binding affinity and biological potency. The reciprocal loop replacement mutant (FGF2-L4/7) retained FGF-2 like affinity for FGFR1 and for KGFR. Our results show that topologically similar regions in these two FGFs have different roles in regulating receptor binding specificity and suggest that specificity may require the concerted action of distinct regions of an FGF. 相似文献
18.
Fernig DG Chen HL Rahmoune H Descamps S Boilly B Hondermarck H 《Biochemical and biophysical research communications》2000,267(3):770-776
The growth of the malignant human mammary MDA-MB-231 cells is stimulated by fibroblast growth factor-1 (FGF-1) but not by FGF-2. When these cells are cultured in the presence of chlorate, an inhibitor of heparan sulfate (HS) sulfation, their proliferation is stimulated by both FGF-1 and FGF-2. We analyzed the interactions of FGF-1 and FGF-2 with HS purified from the cell layer and the culture medium of control and chlorate-treated MDA-MB-231 cells. The HS from the cell layer bound FGF-1 with faster association kinetics than the HS from the culture medium, and so had a higher affinity for FGF-1. Chlorate treatment had no significant effect on the FGF-1 binding kinetics of the HS. In contrast to FGF-1, chlorate treatment of the cells significantly altered the FGF-2 binding kinetics. The HS from untreated cells possessed two binding sites for FGF-2, one with fast association kinetics (k(ass) 470,000 to 610,000 M(-1) s(-1)) and a high affinity (K(d) 46 to 70 nM) and one with slower association kinetics (k(ass) 74,000 to 100,000 M(-1) s(-1)) and a lower affinity (K(d) 290 to 400 nM). HS from chlorate-treated cells possessed just a single binding site for FGF-2 with fast association kinetics (k(ass) 270,000 to 290,000 M(-1) s(-1)) and a high affinity (K(d) 41 to 57 nM). These results show that there is a relationship between the binding kinetics of FGFs and their ability to stimulate cell growth. 相似文献
19.
Differential effects of heparin saccharides on the formation of specific fibroblast growth factor (FGF) and FGF receptor complexes. 总被引:8,自引:0,他引:8
Olga Ostrovsky Bluma Berman John Gallagher Barbara Mulloy David G Fernig Maryse Delehedde Dina Ron 《The Journal of biological chemistry》2002,277(4):2444-2453
Heparan sulfates (HS) play an important role in the control of cell growth and differentiation by virtue of their ability to modulate the activities of heparin-binding growth factors, an issue that is particularly well studied for fibroblast growth factors (FGFs). HS/heparin co-ordinate the interaction of FGFs with their receptors (FGFRs) and are thought to play a critical role in receptor dimerization. Biochemical and crystallographic studies, conducted mainly with FGF-2 or FGF-1 and FGF receptors 1 and 2, suggests that an octasaccharide is the minimal length required for FGF- and FGFR-induced dimerization and subsequent activation. In addition, 6-O-sulfate groups are thought to be essential for binding of HS to FGFR and for receptor dimerization. We show here that oligosaccharides shorter than 8 sugar units support activation of FGFR2 IIIb by FGF-1 and interaction of FGFR4 with FGF-1. In contrast, only relatively long oligosaccharides supported receptor binding and activation in the FGF-1.FGFR1 or FGF-7.FGFR2 IIIb setting. In addition, both 6-O- and 2-O-desulfated heparin activated FGF-1 signaling via FGFR2 IIIb, whereas neither one stimulated FGF-1 signaling via FGFR1 or FGF-7 via FGFR2 IIIb. These findings indicate that the structure of HS required for activating FGFs is dictated by the specific FGF and FGFR combination. These different requirements may reflect the differences in the mode by which a given FGFR interacts with the various FGFs. 相似文献
20.
Fibroblast growth factor-1 (FGF-1) has both extra- and intracellular functions. To identify intracellular binding partners for FGF-1, we isolated proteins from U2OS human osteosarcoma cells interacting specifically with FGF-1. One of the isolated proteins was identified as protein kinase CK2 (CK2). We here provide evidence that FGF-1 binds to both the catalytic alpha-subunit and to the regulatory beta-subunit of CK2. The interaction between FGF-1 and CK2 alpha and beta was characterized by surface plasmon resonance, giving K(D) values of 0.4 +/- 0.3 and 1.2 +/- 0.2 microM, respectively. By using a novel assay for intracellular protein interaction, FGF-1 and CK2 alpha are shown to interact in vivo. In vitro, FGF-1 and FGF-2 are phosphorylated by CK2, and the presence of FGF-1 or FGF-2 was found to enhance the autophosphorylation of CK2 beta. A correlation between the mitogenic potential of FGF-1 mutants and their ability to bind to CK2 alpha was observed. The possible involvement of CK2 in the FGF-induced stimulation of DNA synthesis is discussed. 相似文献