Heparan sulfate moieties of cell-surface proteoglycans modulate the biological responses to fibroblast growth factors (FGFs). Cell-associated heparan sulfates inhibit the binding of the keratinocyte growth factor (KGF), but enhance the binding of acidic FGF to the KGF receptor, both in keratinocytes, which naturally express this receptor, and in rat myoblasts, which ectopically express it. The proteoglycan bearing these modulatory heparan sulfates was purified to homogeneity from salt extracts of rat myoblasts by anion-exchange and FGF affinity chromatography and was identified as rat glypican. Affinity-purified glypican augments the binding of acidic FGF and basic FGF to human FGF receptor-1 in a cell-free system. This effect is abolished following digestion of glypican by heparinase. Addition of purified soluble glypican effectively replaces heparin in supporting basic FGF-induced cellular proliferation of heparan sulfate-negative cells expressing recombinant FGF receptor-1. In keratinocytes, glypican strongly inhibits the mitogenic response to KGF while enhancing the response to acidic FGF. Taken together, these findings demonstrate that glypican plays an important role in regulating the biological activity of fibroblast growth factors and that, for different growth factors, glypican can either enhance or suppress cellular responsiveness (Bonneh-Barkay, 1997).

Loss-of-function mutations in glypican-3 (GPC3), one of the six mammalian glypicans, causes the Simpson-Golabi-Behmel overgrowth syndrome (SGBS), and GPC3 null mice display developmental overgrowth. Because the Hedgehog signaling pathway positively regulates body size, it was hypothesized that GPC3 acts as an inhibitor of Hedgehog activity during development. This study show that GPC3 null embryos display increased Hedgehog signaling and that GPC3 inhibits Hedgehog activity in cultured mouse embryonic fibroblasts. In addition, it is reportd that GPC3 interacts with high affinity with Hedgehog but not with its receptor, Patched, and that GPC3 competes with Patched for Hedgehog binding. Furthermore, GPC3 induces Hedgehog endocytosis and degradation. Surprisingly, the heparan sulfate chains of GPC3 are not required for its interaction with Hedgehog. It is concluded that GPC3 acts as a negative regulator of Hedgehog signaling during mammalian development and that the overgrowth observed in SGBS patients is, at least in part, the consequence of hyperactivation of the Hedgehog signaling pathway (Capurro, 2008).

Heparan sulfate proteoglycans (HSPG) are obligatory for receptor binding and mitogenic activity of basic fibroblast growth factor (bFGF). The capacity of various species of heparin and heparan sulfate (HS) to promote bFGF receptor binding was investigated using both Chinese hamster ovary mutant cells deficient in cell surface HSPG and a soluble bFGF receptor-alkaline phosphatase fusion protein. Highly sulfated oligosaccharides are more effective than medium and low sulfate fractions of the same size oligosaccharide. O-Sulfation in heparin is critical for its capacity to promote binding of bFGF to its receptors. The highest level of bFGF-receptor binding is achieved in the presence of over-sulfated heparin fragments, regardless of whether the N-position is sulfated or acetylated. Unlike receptor binding of bFGF, which requires oligosaccharides containing at least 8-10 sugar units, displacement of heparin- or HS-bound bFGF is obtained by oligosaccharides containing as little as four sugar units and by an N-sulfated, O-desulfated heparin fragment. A preparation of total cell surface-derived HS induces bFGF receptor binding. A preliminary survey of several defined and affinity purified species of cell surface HSPG, including syndecan, fibroglycan, and glypican fail to identify natural HSPG that promotes high affinity receptor binding of bFGF. A similar lack of activity is observed with species of HS isolated from bovine arterial tissue and characterized for their effect on vascular smooth muscle cell proliferation. Most of these species of HS inhibit in a dose-dependent manner the restoration of bFGF-receptor binding induced by heparin or by total HSPG. These results suggest the involvement of defined heparin-like oligosaccharide sequences and unique species of cell surface and extracellular matrix HS in the regulation of bFGF receptor binding and biological activity (Aviezer, 1994).

The formation of distinctive basic FGF-heparan sulfate complexes is essential for the binding of bFGF to its cognate receptor. In previous experiments, cell-surface heparan sulfate proteoglycans extracted from human lung fibroblasts could not be shown to promote high affinity binding of bFGF when added to heparan sulfate-deficient cells that express FGF receptor-1 (FGFR1). In alternative tests to establish whether cell-surface proteoglycans can support the formation of the required complexes, K562 cells were first transfected with the IIIc splice variant of FGFR1 and then transfected with constructs coding for either syndecan-1, syndecan-2, syndecan-4 or glypican, or with an antisense syndecan-4 construct. Cells cotransfected with receptor and proteoglycan show a two- to three- fold increase in neutral salt-resistant specific 125I-bFGF binding in comparison to cells transfected with only receptor or cells cotransfected with receptor and anti-syndecan-4. Exogenous heparin enhances the specific binding and affinity cross-linking of 125I-bFGF to FGFR1 in receptor transfectants that are not cotransfected with proteoglycan, but has no effect on this binding and decreases the yield of bFGFR cross-links in cells that are cotransfected with proteoglycan. Receptor-transfectant cells show a decrease in glycophorin A expression when exposed to bFGF. This suppression is dose-dependent and obtained at significantly lower concentrations of bFGF in proteoglycan-cotransfected cells. Complementary cell-free binding assays indicate that the affinity of 125I-bFGF for an immobilized FGFR1 ectodomain is increased threefold when the syndecan-4 ectodomain is coimmobilized with receptor. Equimolar amounts of soluble syndecan-4 ectodomain, in contrast, have no effect on this binding. It is concluded that, at least in K562 cells, syndecans and glypican can support bFGF-FGFR1 interactions and signaling, and that cell-surface association may augment their effectiveness (Steinfeld, 1996).

OCI-5 encodes the rat homolog of glypican-3, a membrane-bound heparan sulfate proteoglycan that is mutated in the Simpson-Golabi-Behmel overgrowth syndrome. OCI-5 and glypican-3 are 95% identical. It has been recently suggested that glypican-3 interacts with insulin-like growth factor-2 (IGF-2) and that this interaction regulates IGF-2 activity. OCI-5 was transfected into two different cell lines and an interaction between the OCI-5 proteoglycan produced by the transfected cells and IGF-2 could not be detected. In contrast to this, OCI-5 interacts with FGF-2, as has already been shown for glypican-1. This interaction is mediated by the heparan sulfate chains of OCI-5 because it can be inhibited by heparin or by heparitinase (Song, 1997).

A comparative study was undertaken of the interaction of the three mammalian transforming growth factor-betas (TGF-beta) with heparin and heparan sulfate. TGF-beta1 and -beta2, but not -beta3, bind to heparin and the highly sulfated liver heparan sulfate. These polysaccharides potentiate the biological activity of TGF-beta1 (but not the other isoforms), whereas a low sulfated mucosal heparan sulfate fails to do so. Potentiation is due to antagonism of the binding and inactivation of TGF-beta1 by alpha2-macroglobulin, rather than by modulation of growth factor-receptor interactions. TGF-beta2.alpha2-macroglobulin complexes are more refractory to heparin/heparan sulfate, and those involving TGF-beta3 cannot be affected. Comparison of the amino acid sequences of the TGF-beta isoforms strongly implicates the basic amino acid residue at position 26 of each monomer as being a vital binding determinant. A model is proposed in which polysaccharide binding occurs at two distinct sites on the TGF-beta dimer. Interaction with heparin and liver heparan sulfate may be most effective because of the ability of the dimer to co-operatively engage two specific sulfated binding sequences, separated by a distance of approximately seven disaccharides, within the same chain (Lyon, 1997).

The transforming growth factor-beta (TGF-beta) binding site in betaglycan, the type III TGF-beta receptor, has been variously assigned to the C-terminal half and N-terminal one-third of the extracellular domain. There are at least two TGF-beta-binding sites in betaglycan. Bacterially expressed fragments bg 1,2 and bg3, which represent the N-terminal two-thirds and C-terminal one-third of betaglycan extracellular domain, both compete for the binding of 125I-TGF-beta to mink lung epithelial cells. 125I-bg1,2 binds to immobilized TGF-beta with an affinity about 4-fold higher than does bg3. Both bg3 and bg1,2 enhance the bioactivity of TGF-beta. The whole ectodomain of betaglycan is more active than either bg3 or bg1,2 in the assays. The binding of 125I-bg3 to TGF-beta is inhibited by bg1,2 and vice versa. The binding of 125I-bg3 and 125I-bg1,2 to TGF-beta is also inhibited by the small decorin family of proteoglycans. These results indicate that there are at least two binding sites for TGF-beta in betaglycan and that these sites recognize the same or overlapping sites in TGF-beta (Kaname, 1996).

Although a number of growth factors bind cell-surface heparan sulphate proteoglycans, the role of this interaction is unclear except for fibroblast growth factor, which requires HSPG binding for signaling. Hepatocyte growth factor/scatter factor (HGF/SF) plays important roles in mammalian development and tissue regeneration and acts on target cells through a specific receptor tyrosine kinase encoded by the c-met proto-oncogene. This factor also binds HSPGs with high affinity, but conflicting data have been reported on the role of HSPG binding in HGF/SF signaling. To map the binding sites for HSPG and the Met receptor in HGF/SF, a number of HGF/SF mutants were engineered in which several clusters of solvent-accessible residues either in the hairpin structure of the amino-terminal domain or in kringle 2 were replaced. Two of the mutants (HP1 and HP2) show greatly decreased (more than 50-fold) affinity for heparin and HSPGs but retain full mitogenic and motogenic activities on target cells in culture. When compared with wild-type HGF/SF, the HP1 mutant exhibits a delayed clearance from the blood, higher tissue levels and a higher induction of DNA synthesis in normal, adult murine liver. These results establish the following: (1) the binding sites in HGF/SF for Met and for HSPGs can be dissociated by protein engineering; (2) high-affinity binding of HGF/SF to HSPGs is not essential for signalling; (3) one role of HSPG binding in the HGF/SF system appears to be sequestration and degradation of the growth factor, and (4) HGF/SF mutants with decreased affinity for HSPGs exhibit enhanced activity in vivo (Hartmann, 1998).

A novel cell surface HP/HS interacting protein (HIP) has been identified from human uterine epithelia and a variety of other human epithelial and endothelial cells and cell lines. HIP from HEC cells, a human uterine epithelial cell line, as well as recombinant HIP from a bacterial expression system were purified and characterized. HIP supports attachment of the human trophoblastic cell line, JAR, in a HS-dependent fashion. Predigestion of JAR cells with a mixture of heparitinases, but not chondroitinase AC, abolishes cell attachment to HIP. JAR cell attachment to HIP is highly sensitive to HP inhibition and much more selective for HP/HS than other glycosaminoglycans. Dermatan sulfate displays partial inhibitory activity as well, consistent with the observation that chondroitinase ABC digestion partially reduces JAR cell attachment to HIP. HIP binds labelled HP with high affinity, and HIP binds cell surface/extracellular matrix-associated HS, expressed by RL95 cells, a human uterine epithelial cell line. Anti-HIP antibody generated against a synthetic peptide derived from a putative HP/HS-binding motif resident within HIP inhibits about half of labelled HP binding to HIP, indicating that this domain is a functional HP-binding domain of HIP. Similarly, this same synthetic peptide motif of HIP can block about 50% of labelled HP binding to HIP; however, this peptide almost completely inhibits cell attachment to HIP, suggesting a critical role, in this regard. Collectively, these results suggest that HIP can function as a HP/HS-binding cell-cell/cell-matrix adhesion molecule (Liu, 1997).

Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by the formation of cartilage-capped tumors (exostoses) that develop from the growth plate of endochondral bone. This condition can lead to skeletal abnormalities, short stature and malignant transformation of exostoses to chondrosarcomas or osteosarcomas. Linkage analyses have identified three different genes for HME, EXT1 on 8q24.1, EXT2 on 11p11-13 and EXT3 on 19p. Most HME cases have been attributed to missense or frameshift mutations in these tumor-supressor genes, whose functions have remained obscure. EXT1 is shown to be an ER-resident type II transmembrane glycoprotein whose expression in cells results in the alteration of the synthesis and display of cell surface heparan sulfate glycosaminoglycans (GAGs). Two EXT1 variants containing etiologic missense mutations fail to alter cell-surface glycosaminoglycans, despite retaining their ER-localization (McCormick).

Dickkopf-1 (Dkk1) is a secreted protein that negatively modulates the Wnt/βcatenin pathway. Lack of Dkk1 function affects head formation in frog and mice, supporting the idea that Dkk1 acts as a 'head inducer' during gastrulation. Lack of Dkk1 function accelerates internalization and rostral progression of the mesendoderm and gain of function slows down both internalization and convergence extension, indicating a novel role for Dkk1 in modulating these movements. The motility phenotype found in the morphants is not observed in embryos in which the Wnt/βcatenin pathway is overactivated, and dominant-negative Wnt proteins are not able to rescue the gastrulation movement defect induced by absence of Dkk1. These data strongly suggest that Dkk1 is acting in a βcatenin independent fashion when modulating gastrulation movements. The glypican 4/6 homolog Knypek (Kny) binds to Dkk1, and they are able to functionally interact in vivo. Moreover, Dkk1 regulation of gastrulation movements is kny dependent. Kny is a component of the Wnt/planar cell polarity (PCP) pathway. Indeed Dkk1 is able to activate this pathway in both Xenopus and zebrafish. Furthermore, concomitant alteration of the βcatenin and PCP activities is able to mimic the morphant accelerated cell motility phenotype. These data therefore indicate that Dkk1 regulates gastrulation movement through interaction the Frizzled coreceptor transmembrane protein LRP5/6 and Kny and coordinated modulations of Wnt/βcatenin and Wnt/PCP pathways (Caneparo, 2007).

Skin fibroblasts treated with brefeldin A produce a recycling variant of glypican (a glycosylphosphatidylinositol anchored heparan-sulfate proteoglycan) that is resistant to inositol-specific phospholipase C and incorporate sulfate and glucosamine into heparan sulfate chains. Structural modifications of recycling glypican have been examined, such as fatty acylation from [3H]palmitate, and degradation and assembly of heparan sulfate side chains. Most of the 3H-radioactivity is recovered as lipid-like material after de-esterification. To distinguish between formation of heparan sulfate at vacant sites, elongation of existing chains or degradation followed by re-elongation of chain remnants, cells were pulse-labeled with [3H]glucosamine and then chase-labeled with [14C]glucosamine. Material isolated from the cells during the chase consisted of proteoglycan and mostly [3H]-labeled heparan-sulfate degradation products (molecular mass, 20-80 kDa) showing that the side chains are degraded during recycling. The degradation products are initially glucuronate-rich, but become more iduronate-rich with time. The glypican proteoglycan formed during the chase is degraded either with alkali to release intact side chains or with heparinase to generate distally located chain fragments that are separated from the core protein, containing the proximally located, covalently attached chain remnants. All of the [14C]-radioactivity incorporated during the pulse is found in peripheral chain fragments, and the chains formed are not significantly longer than the original ones. It is therefore concluded that newly made heparan-sulfate chains are neither made on vacant sites, nor by extension of existing chains but rather by re-elongation of degraded chain remnants. The remodeled chains made during recycling appear to be more extensively modified than the original ones (Edgren, 1997).

The secreted serine protease xHtrA1, expressed in early Xenopus embryos and transcriptionally activated by FGF signals, promotes posterior development in mRNA-injected embryos. xHtrA1 mRNA leads to the induction of secondary tail-like structures, expansion of mesoderm, and formation of ectopic neurons in an FGF-dependent manner. An antisense morpholino oligonucleotide or a neutralizing antibody against xHtrA1 has the opposite effects. xHtrA1 activates FGF/ERK signaling and the transcription of FGF genes. Xenopus Biglycan, Syndecan-4, and Glypican-4 are proteolytic targets of xHtrA1 and heparan sulfate and dermatan sulfate trigger posteriorization, mesoderm induction, and neuronal differentiation via the FGF signaling pathway. The results are consistent with a mechanism by which xHtrA1, through cleaving proteoglycans, releases cell-surface-bound FGF ligands and stimulates long-range FGF signaling (Hou, 2007).

HtrA1 belongs to the HtrA (High temperature requirement-A) family of serine proteases that is well conserved from bacteria to humans. HtrA1 was originally isolated as a gene downregulated in SV40-transformed human fibroblasts. Overexpression of HtrA1 in cancer cells suppresses growth and proliferation in vivo, suggesting that HtrA1 is a candidate tumor suppressor. More recently, a single nucleotide polymorphism in the HtrA1 promoter has been presented as a major risk factor for age-related macular degeneration. HtrA1 binds to and inactivates members of the TGFβ family and modulates insulin-like growth factor (IGF) signals, but its biological function is not yet known (Hou, 2007).

The Xenopus homolog of HtrA1 (xHtrA1) was identified in a direct screen for secreted proteins. xHtrA1 is a modulator of FGF signaling that participates in axial development, mesoderm formation, and neuronal differentiation. xHtrA1 is activated by FGF signals and induces ectopic FGF4 and FGF8 transcription. Biglycan, Syndecan-4, and Glypican-4 are proteolytic targets of xHtrA1; pure heparan sulfate and dermatan sulfate phenocopy xHtrA1 and FGF activities in Xenopus embryos. The results suggest that xHtrA1 acts as a positive regulator of FGF signals and, through proteolytic cleavage of proteoglycans, allows long-range FGF signaling in the extracellular space (Hou, 2007).

High density lipoprotein (HDL) particles and HDL cholesteryl esters are taken up by both receptor-mediated and non-receptor-mediated pathways. Cell surface heparan sulfate proteoglycans (HSPG) participate in hepatic lipase (HL)- and apolipoprotein (apo) E-mediated binding and uptake of mouse and human HDL by cultured hepatocytes. The HL secreted by HL-transfected McA-RH7777 cells enhances both HDL binding at 4 degrees C (approximately 2-4-fold) and HDL uptake at 37 degrees C (approximately 2-5-fold). The enhanced binding and uptake of HDL are partially inhibited by the 39-kDa protein, an inhibitor of low density lipoprotein receptor-related protein (LRP), but are almost totally blocked by heparinase, which removes the sulfated glycosaminoglycan chains from HSPG. Therefore, HL may mediate the uptake of HDL by two pathways: an HSPG-dependent LRP pathway and an HSPG-dependent but LRP-independent pathway. The HL-mediated binding and uptake of HDL are only minimally reduced when catalytically inactive HL or LRP binding-defective HL is substituted for wild-type HL, indicating that much of the HDL uptake requires neither HL binding to the LRP nor lipolytic processing. To study the role of HL in facilitating the selective uptake of cholesteryl esters, HDL has been used into which radiolabeled cholesteryl ether had been incorporated.. HL increases the selective uptake of HDL cholesteryl ether; this enhanced uptake is reduced by more than 80% by heparinase but is unaffected by the 39-kDa protein. Like HL, apoE enhances the binding and uptake of HDL (approximately 2-fold) but has little effect on the selective uptake of HDL cholesteryl ether. In the presence of HL, apoE does not further increase the uptake of HDL, and at a high concentration apoE impairs or decreases the HL-mediated uptake of HDL. Therefore, HL and apoE may utilize similar (but not identical) binding sites to mediate HDL uptake. Although the relative importance of cell surface HSPG in the overall metabolism of HDL in vivo remains to be determined, cultured hepatocytes clearly display an HSPG-dependent pathway that mediates the binding and uptake of HDL. This study also demonstrates the importance of HL in enhancing the binding and uptake of remnant and low density lipoproteins via an HSPG-dependent pathway (Ji, 1997).

Potential mechanisms of non-low-density lipoprotein (LDL) receptor-mediated uptake of triglyceride-rich particles (TGRP) in the presence of apolipoprotein E (apo E) were explored. Human fibroblasts were incubated with model intermediate-density lipoprotein- (IDL-) sized TGRP (10-1000 microg of neutral lipid/mL) containing apo E. At low particle concentrations, almost all apo E-TGRP uptake is via the LDL receptor. At higher particle concentrations, within the physiologic range, most particle uptake and internalization is via HSPG-mediated pathways. This HSPG pathway does not involve classical lipoprotein receptors, such as LRP or the LDL receptor. These data suggest that in peripheral tissues, such as the arterial wall, apo E may act in TGRP as a ligand for uptake not only via the LDL receptor and LRP pathways but also via HSPG pathways that are receptor-independent. Thus, at physiologic particle concentrations apo E-TGRP can be bound and internalized in certain cells by relatively low affinity but high capacity HSPG-mediated pathways (Al-Haideri, 1997).

Glypicans are heparan sulfate proteoglycans that are attached to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. Glypicans regulate the activity of Wnts, Hedgehogs, bone morphogentic proteins, and fibroblast growth factors. In the particular case of Wnts, it has been proposed that GPI-anchored glypicans stimulate Wnt signaling by facilitating and/or stabilizing the interaction between Wnts and their cell surface receptors. In contrast, when glypicans are secreted to the extracellular environment they can act as competitive inhibitors of Wnt. Genetic screens in Drosophila have recently identified a novel inhibitor of Wnt signaling named Notum. The Wnt-inhibiting activity of Notum was associated with its ability to release dally-like protein (a Drosophila glypican) from the cell surface by cleaving the GPI anchor. Because these studies showed that the other Drosophila glypican Dally was not released from the cell surface by Notum, it remains unclear whether this enzyme is able to cleave glypicans from mammalian cells. Furthermore, whether Notum cleaves GPI-anchored proteins that are not members of the glypican family is also unknown. This study shows that mammalian Notum can cleave several mammalian glypicans. Moreover, Notum is able to release GPI-anchored proteins other than glypicans. Another important finding of this study is that, unlike GPI-Phospholipase D, the other mammalian enzyme that cleaves GPI-anchored proteins, Notum is active in the extracellular environment. Finally, by using a cellular system in which glypican-3 stimulates Wnt signaling it was shown that Notum can act as a negative regulator of this growth factor (Traister, 2008).

Dally-like (Dlp) is a glypican-type heparan sulfate proteoglycan (HSPG), containing a protein core and attached glycosaminoglycan (GAG) chains. In Drosophila wing discs, Dlp represses short-range Wingless (Wg) signaling, but activates long-range Wg signaling. This study shows that Dlp core protein has similar biphasic activity as wild-type Dlp. Dlp core protein can interact with Wg; the GAG chains enhance this interaction. Importantly, it was found that Dlp exhibits a biphasic response, regardless of whether its glycosylphosphatidylinositol linkage to the membrane can be cleaved. Rather, the transition from signaling activator to repressor is determined by the relative expression levels of Dlp and the Wg receptor, Frizzled (Fz) 2. Based on these data, it is proposed that the principal function of Dlp is to retain Wg on the cell surface. As such, it can either compete with the receptor or provide ligands to the receptor, depending on the ratios of Wg, Fz2, and Dlp (Yan, 2009).

The mechanisms controlling Wg signaling and its gradient formation are highly complex. This study provides two lines of findings for the mechanistic roles of Dlp in Wg signaling. First, it is shown that the core protein of Dlp has similar biphasic activity to wild-type Dlp in Wg signaling. Consistent with this, the Dlp core protein can interact with Wg, while the attached HS chains can enhance Dlp's affinity for Wg binding. Second, it is demonstrated that Dlp can get a biphasic response without Notum cleavage, and the ratio of Dlp:Fz2 determines its biphasic activity in cell culture and in the wing disc. While a low ratio of Dlp:Fz2 can help Fz2 obtain more Wg, a high ratio of Dlp:Fz2 prevents Fz2 from capturing Wg. It is proposed that the main activity of Dlp in Wg signaling is to retain Wg on the cell membrane rather than to act as a classic coreceptor. Dlp can mediate the exchange of Wg between receptors and itself; the net flow of the ligand depends on the ratios of the ligand, receptor, and Dlp. In support of this model, it was found that Fz2-GPI also has biphasic activity in Wg signaling (Yan, 2009).

Previous studies have demonstrated that Dlp acts as a biphasic modulator for Wg signaling in the wing disc; however, the mechanism underlying this biphasic response is not clear. One model suggests that Notum expressed at the D/V boundary can cleave Dlp and release it together with bound Wg, converting Dlp from a membrane coreceptor to a secreted antagonist. The current data suggest that this model needs to be revised. First, it was shown that expression of a GPI-deleted secreted form of Dlp (similar to the form cleaved by Notum) does not inhibit Wg signaling in the wing discs. Second, expression of CD2 forms of Dlp, which cannot be cleaved by Notum, can also inhibit sens expression similar to GPI versions of Dlp. An alternative model is that Dlp competes with Wg receptors on the cell surface, locally inhibiting signaling, but it also promotes long-range Wg gradient formation, and thus provides more Wg in the distal part of the wing disc. However, this model cannot explain how Dlp has biphasic effects in vitro, where Wg gradients do not form (Yan, 2009).

On the basis of these results, an exchange factor model is favored to explain the biphasic activity of Dlp in Wg signaling. The model is very similar to a recently published mathematical model for biphasic activity of CV-2 in BMP signaling (Serpe, 2008). In this model, Dlp might either compete with the receptor or provide ligands for the receptor, its role changing depending on the relative levels of ligand, receptor, and exchange factor. This study has shown that, in the wing discs, raising the levels of Fz2 can convert Dlp from a repressor to an activator. In S2 cells, the biphasic activity of Dlp also depends on the Dlp:Fz2 ratio, with a low level of Dlp increasing Wg signaling reporter activity and a high level of Dlp reducing its activity. Using Co-IP experiments, it was directly shown that a small amount of Dlp provides Wg for Fz2 receptor, while a large amount of Dlp sequesters the Wg ligand. Moreover, it was found that, for a constant amount of Dlp, it is more likely to repress Wg signaling at high Wg concentration, but to promote signaling at low Wg concentration. In contrast, Dlp is more likely to promote Wg signaling at high Fz2 concentration, but to repress signaling at low Fz2 concentration. Thus, this model could explain the situation in wing disc, where Dlp inhibits Wg signaling in regions close to the D/V border (high Wg and low Fz2), and promotes signaling in regions far from the D/V border (low Wg and high Fz2). These data are consistent with a previous reports showing that in vitro Dlp promotes Wg signaling when the Wg level is low, but reduces signaling when the Wg level is high. This result also fits well with the theoretical modeling data of Serpe (2008) for different ligand levels, suggesting Dlp acts similarly to CV-2 in different systems. In order to work, their model contains a tripartite complex between CV-2, BMP, and the receptor. No Dlp coprecipitated with Fz2 is detected; however, as the Serpe study proposed, the intermediate is a transient complex with very rapid on-off kinetics, and it is difficult to demonstrate the tripartite intermediate directly. Finally, in further support of the current model, it was found that Fz2-GPI, which can stabilize Wg on the cell surface and compete with Fz2 for Wg binding, also has biphasic activity in Wg signaling (Yan, 2009).

Previous studies reported that secreted Fz-related protein (sFRP), another family of Wnt-interacting proteins, can also exhibit biphasic activity in Wnt signaling, enhancing Wnt signaling at low concentration, but inhibiting it at high concentration. As mentioned above, the BMP-binding protein, CV-2, can act as a concentration-dependent, biphasic regulator for BMP signaling in the wing disc (Serpe, 2008). It is interesting to note that both sFRP and CV-2 can interact with HSPGs, and are likely to exert their function on the cell surface. In addition, another HSPG member, Xenopus Syndecan-1, shows a level-dependent activation or inhibition of BMP signaling during dorsoventral patterning of the embryonic ectoderm (Olivares, 2009). Moreover, it was found that Ihog, a recently identified Hh coreceptor (Yao, 2006), has biphasic activity in Hh morphogen signaling. Overexpression of Ihog represses high-threshold Hh target, and extends low-threshold Hh target gene expression. Together, other cell surface ligand-interacting proteins might regulate signaling by a similar mechanism. Traditionally, all cell surface ligand-binding receptors that cannot signal independently are equivocally called coreceptors, despite their diverse functions. On the basis of current results, it is proposed that some of the coreceptors may function as the exchange factors rather than the classical coreceptors, which only enhance signaling by providing ligand to the receptor (Yan, 2009).

Another important finding of this work is the demonstration that Dlp's major activity in Wg signaling depends on its core protein. Previous studies have shown that different HSPG proteins play very distinct roles in Wg signaling and distribution. However, the mechanism underlying this specificity is unknown. This study presents evidence that the specificity of Dlp in Wg signaling results from its core protein. First, the Dlp core protein has biphasic activity for short- and long-range signaling similar to that of wild-type Dlp. Second, the Dlp core protein interacts with Wg in co-IP experiment, cell-binding assay, as well as in the wing discs. Third, it is shown that the N-terminal domain of Dlp is essential for Wg binding, and that fusion of the N-terminal domain of Dlp to the Fz2 membrane and cytoplasmic domain can recapitulate Fz2 activity. These data are consistent with previous results indicating that Xenopus glypican-4 interacts with Wnt11 through its N-terminal domain. It is interesting to note that, similar to Fz2 CRD domain, the N-terminal domain of Dlp protein has 14 highly conserved cysteines, a shared feature of all glypican members (Yan, 2009).

Previously, Capurro (2005) has shown that vertebrate glypican-3 core protein is directly involved in Wnt signaling, whereas the GAG chains of glypican-3 are not required for the stimulatory effect in Wnt signaling . Moreover, recent data has shown that the glypican-3 core protein also binds to Sonic Hedgehog (Shh), but inhibits its signaling by competing with the receptor, Patched (Capurro, 2008). The opposite effects of the same glypican core protein on Wnt and Hh signaling are intriguing. Interestingly, it has also been observed that the Dlp core protein positively regulates Hh signaling in both Drosophila embryos and wing discs. Thus, the core proteins of glypican-3 and Dlp appear to have opposite roles in Wnt and Hh signaling. It is likely that different glypican cores may bear distinctive motifs to interact with Wnt and Hh proteins (Yan, 2009).

Although the Dlp core protein is able to bind Wg, it was found that the attached HS GAG chains are also important for the binding affinity between Dlp and Wg. Wild-type Dlp shows significantly stronger binding for Wg than the core protein alone. This result is consistent with previous genetic experiments showing that Wg signaling is compromised in HS-deficient mutants. In addition, biochemical studies also suggest that Wg is a heparin-binding protein. One possibility is that the Dlp core protein might have different membrane distribution than wild-type Dlp. However, this study did not observe obvious difference in the subcellular localizations between Dlp-GFP and Dlp(−HS)-GFP. It remains to be determined how the presence of HS GAG chains can enhance Dlp's ability to bind Wg (Yan, 2009).

All glypicans anchor to the cell membrane via a GPI anchor. GPI proteins are enriched in specific membrane subdomains called lipid rafts, which are suggested to promote the signaling activities of GPI-anchored proteins. Thus, one important issue is whether the GPI anchor is required for Dlp's activity in Wg signaling. The results suggest that the GPI anchor of Dlp is not essential for its activity in Wg signaling. Several lines of evidence support this view. First, Dlp(−HS)-CD2, a transmembrane form of Dlp core protein, has similar biphasic activity to that of Dlp(−HS). Second, the subcellular localizations of different forms of Dlp was analyzed, and it was found that Dlp's major activity is to bind Wg at the cell surface. Dlp-GFP, which has the strongest binding affinity for Wg, accumulates more Wg on the cell surface. In Dlp(−HS)-GFP and Dlp(−HS)-CD2-GFP-expressing discs, less Wg was found accumulated on the cell membrane and more internalized Wg vesicles were found. These results are consistent with a recent work showing that accumulating Wg on Dlp-expressing cells is less accessible to internalization. Although Dlp-GFP and Dlp(−HS)-GFP, but not Dlp(−HS)-CD2-GFP, form many endocytic vesicles due to a role of the GPI anchor in trafficking, based on these functional data, it is suggested that the GPI anchor of Dlp is not essential for Wg signaling (Yan, 2009).

Recently, it has been proposed that the GPI anchor of Dlp is required for Wg internalization and long-range signaling (Gallet, 2008). This conclusion is mainly based on the evidence that expression of GFP-Dlp-CD2 can reduce the expression of Wg long-range target gene dll. This result is apparently different from the current data showing that expression of Dlp(−HS)-CD2-GFP construct leads to expanded dll expression. To resolve this issue, the GFP-Dlp-CD2 transgenic flies used by Gallet were obtained, and the activity of GFP-Dlp-CD2 in the wing discs was examined. Different results were observed from the previous study. It was found that their GFP-Dlp-CD2 has very similar biphasic activity to Dlp-GFP when it is expressed by en-Gal4 or ap-Gal4, and the effects on dll expression were examined. One possibility for the difference is that the previous study used only ap-Gal4, which will cause expression of Dlp to reduce the size of the compartment; this may complicate comparisons of the effect of GFP-Dlp-CD2 in long-range signaling. Therefore en-Gal4, which allows the use of the A compartment as an internal control, was used. Furthermore, while the previous showed that GFP-Dlp-CD2 induces a more severe wing defect than the GFP-Dlp construct, this study found that GFP-Dlp-CD2 does not generate a more severe wing defect than than the Dlp-GFP construct. In this regard, it is important to note that the GFP-Dlp-CD2 and GFP-Dlp constructs used by Gallet employed GFP inserted at two different sites in Dlp, and that the insertion in the GFP-Dlp construct leads to reduced activity. In conclusion, the current data demonstrate that CD2 forms of Dlp have similar activity to the GPI forms of Dlp, suggesting that the GPI anchor of Dlp is not essential for its activity in Wg signaling (Yan, 2009).

The kidney of the Gpc3-/- mouse, a novel model of human renal dysplasia, is characterized by selective degeneration of medullary collecting ducts preceded by enhanced cell proliferation and overgrowth during branching morphogenesis. Cellular and molecular mechanisms underlying this renal dysplasia have been identified. Glypican-3 (GPC3) deficiency is associated with abnormal and contrasting rates of proliferation and apoptosis in cortical (CCD) and medullary collecting duct (MCD) cells. In CCD, cell proliferation is increased threefold. In MCD, apoptosis was increased 16-fold. Expression of Gpc3 mRNA in ureteric bud and collecting duct cells suggests that GPC3 can exert direct effects in these cells. Indeed, GPC3 deficiency abrogates the inhibitory activity of BMP2 on branch formation in embryonic kidney explants, converts BMP7-dependent inhibition to stimulation, and enhances the stimulatory effects of KGF. Similar comparative differences are found in collecting duct cell lines derived from GPC3-deficient and wild type mice and induced to form tubular progenitors in vitro, suggesting that GPC3 directly controls collecting duct cell responses. It is proposed that GPC3 modulates the actions of stimulatory and inhibitory growth factors during branching morphogenesis (Grisaru, 2001).

The molecular basis for observations in ureteric bud and collecting duct cells remains to be determined. The demonstration that bFGF forms a molecular complex with cell surface heparan sulfate and the FGF cell surface receptor suggests that GPC3 may physically interact with receptors that bind BMP2, BMP7, and KGF. The opposite response of collecting duct cells to GPC3 deficiency, that is, inhibition of BMP2 activity and enhancement of KGF activity, suggests that the consequences of these interactions may differ. A second possibility is that GPC3 may function via independent signaling pathways that physically interact at the postreceptor level with BMP and KGF signaling intermediates. Alternatively, the GPC3 and growth factor-signaling pathways may interact indirectly by regulating competing or complementary gene products. Increasing evidence regarding the nature of inhibitory and stimulatory BMP-dependent signaling pathways in collecting duct cells provides a basis to determine the nature of GPC3 interactions with BMP2 and BMP7 (Grisaru, 2001).

Coordinated morphogenetic cell movements during gastrulation are crucial for establishing embryonic axes in animals. The non-canonical Wnt signaling cascade (PCP pathway) has been shown to regulate convergent extension movements in Xenopus and zebrafish. Heparan sulfate proteoglycans (HSPGs) are known as modulators of intercellular signaling, and are required for gastrulation movements in vertebrates. However, the function of HSPGs is poorly understood. The function of Xenopus glypican 4 (Xgly4), which is a member of membrane-associated HSPG family, has been analyzed. In situ hybridization revealed that Xgly4 is expressed in the dorsal mesoderm and ectoderm during gastrulation. Reducing the levels of Xgly4 inhibits cell-membrane accumulation of Dishevelled (Dsh), which is a transducer of the Wnt signaling cascade, and thereby disturbs cell movements during gastrulation. Rescue analyses with different Dsh mutants and Wnt11 demonstrate that Xgly4 functions in the non-canonical Wnt/PCP pathway, but not in the canonical Wnt/ß-catenin pathway, to regulate gastrulation movements. Evidence that the Xgly4 protein physically binds Wnt ligands. Therefore, the results suggest that Xgly4 functions is a positive regulator in non-canonical Wnt/PCP signaling during gastrulation (Ohkawara, 2003).

Heparan sulphate proteoglycans such as glypicans are essential modulators of intercellular communication during embryogenesis. In Xenopus laevis embryos, the temporal and spatial distribution of Glypican 4 (Gpc4) transcripts during gastrulation and neurulation suggests functions in early development of the central nervous system. The role of Xenopus Gpc4 has been functionally analyzed by using antisense morpholino oligonucleotides; Gpc4 is shown to be part of the signalling network that patterns the forebrain. Depletion of GPC4 protein results in a pleiotropic phenotype affecting both primary axis formation and early patterning of the anterior central nervous system. Molecular analysis shows that posterior axis elongation during gastrulation is affected in GPC4-depleted embryos, whereas head and neural induction are apparently normal. During neurulation, loss of GPC4 disrupts expression of dorsal forebrain genes, such as Emx2, whereas genes marking the ventral forebrain and posterior central nervous system continue to be expressed. This loss of GPC4 activity also causes apoptosis of forebrain progenitors during neural tube closure. Biochemical studies establish that GPC4 binds FGF2 and modulates FGF signal transduction. Inhibition of FGF signal transduction, by adding the chemical SU5402 to embryos from neural plate stages onward, phenocopies the loss of gene expression and apoptosis in the forebrain. It is proposed that GPC4 regulates dorsoventral forebrain patterning by positive modulation of FGF signalling (Gall, 2003).

The human parvovirus adeno-associated virus (AAV) infects a broad range of cell types, including human, nonhuman primate, canine, murine, and avian. Although little is known about the initial events of virus infection, AAV is currently being developed as a vector for human gene therapy. Using defined mutant CHO cell lines and standard biochemical assays, it has been demonstrated that heparan sulfate proteoglycans mediate both AAV attachment to and infection of target cells. Competition experiments using heparin, a soluble receptor analog, demonstrate dose-dependent inhibition of AAV attachment and infection. Enzymatic removal of heparan but not chondroitin sulfate moieties from the cell surface greatly reduce AAV attachment and infectivity. Mutant cell lines that do not produce heparan sulfate proteoglycans are significantly impaired for both AAV binding and infection. This is the first reported role for proteoglycan in the cellular attachment of a parvovirus. Together, these results demonstrate that membrane-associated heparan sulfate proteoglycan serves as the viral receptor for AAV type 2, and provide an explanation for the broad host range of AAV. Identification of heparan sulfate proteoglycan as a viral receptor should facilitate development of new reagents for virus purification and provide critical information on the use of AAV as a gene therapy vector (Summerford, 1998).

The amyloid precursor protein (APP) of Alzheimer's disease has been shown to stimulate neurite outgrowth in vitro. The effect of APP on neurite outgrowth can be enhanced if APP is presented to neurons in substrate-bound form, in the presence of heparan sulfate proteoglycans. To identify specific heparan sulfate proteoglycans that bind to APP, conditioned medium from neonatal mouse brain cells was subjected to affinity chromatography with recombinant APP695 as a ligand. Glypican binds strongly to the APP affinity column. Purified glypican binds to APP with an equilibrium dissociation constant of 2.8 nM and inhibits APP-induced neurite outgrowth from chick sympathetic neurons. The effect of glypican is specific for APP, as glypican do not inhibit laminin-induced neurite outgrowth. Treatment of cultures with 4-methylumbelliferyl-beta-D-xyloside, a competitive inhibitor of proteoglycan glycanation, inhibits APP-induced neurite outgrowth but does not inhibit laminin-induced neurite outgrowth. This result suggests that endogenous proteoglycans are required for substrate-bound APP to stimulate neurite outgrowth. Secreted glypican may act to inhibit APP-induced neurite outgrowth in vivo by competing with endogenous proteoglycans for binding to APP (Williamson, 1996).

Heparan sulfate proteoglycan (HSPG) has been found to be associated with amyloid deposits in a number of diseases including the cerebral amyloid plaques of Alzheimer's disease and the transmissible spongiform encephalopathies (TSEs). The role of HSPG in amyloid formation and the neurodegenerative pathology of these diseases have not been established. These questions were addressed using a scrapie mouse model, which exhibits both amyloid and nonamyloid deposition of abnormal PrP protein, the protein marker of TSE infection. The distribution of HSPG was examined throughout the course of the disease in the brains of experimentally infected mice and compared with the distribution of abnormal PrP. Abnormally high levels of HSPG are associated with most types of PrP pathology including all plaque types and diffuse neuroanatomically targeted forms. Scrapie-associated HSPG is present from 70 days after infection, the earliest time-point examined, in the same target areas as abnormal PrP. The association with amyloid plaques may indicate that HSPG is involved in amyloid plaque formation and/or persistence, but involvement with early diffuse forms of PrP suggests a more fundamental role in scrapie pathogenesis (McBride, 1998).