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cytoplasmic domain cross-phosphorylation, which initiates intracellular signaling cascades (reviewed in Ref. 58). HSPGs modulate this system by acting as low affinity “co-receptors” that facilitate ligand-driven assembly of a functional ternary complex59−61 (Fig. 3A).
Multiple endothelial HSPGs serve as co-receptors (including perlecan, glypican-1, and syndecans-1,-2, and-4), 62−64 which suggests the HS chain plays a critical role. One potential role of HS is to facilitate FGF dimerization into a configuration that is essential for receptor binding, as indicated by solution phase studies.65 Mechanistic insights have been gained from X-ray crystallography of ligand:receptor complexes containing short HS (heparin) fragments. However, two distinct HS-FGF-FGFR ternary complexes have been crystallized so the precise means of ternary complex formation is disputed (reviewed in Ref. 58) (Fig.3A). In one case, the complex is symmetric, with each single FGF2-FGFR1 dimer being stabilized by the ends of two separate HS fragments.66 In the other case, the complex is asymmetric, with the pair of FGF1-FGFR2 dimers being bridged by a single HS disaccharide.67 Each complex was formed with different isoforms of ligand and receptor, which provides one possible explanation for these structural differences. Perhaps there are multiple ways to generate ternary complexes, given the large number of combinations of FGF ligands and receptors. Regardless, both models show that similar contacts are formed between the HS fragment(s) and HS-binding residues of the ligand and receptor. Thus, both models confirm that HS serves to crosslink FGF to the FGFR (reviewed in Ref. 58).
6.2. HSPG co-receptors confer unique regulatory properties
The deployment of an HSPG co-receptor influences growth factor signaling kinetics, strength and specificity and engenders several novel properties. Such distinct characteristics in part reflect fundamental differences between growth factors and HSPGs. For example, growth factors have a rapid turnover; whereas HSPGs are long-lived, exhibiting half-lives in the range of hours to days.16 Consequently, HSPGs can drive sustained signaling events by stabilizing growth factors. For example, in the absence of HS, FGF2 binding to FGFRs produces
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Fig. 3. HSPGs regulate signaling by multiple mechanisms. Schematically depicted are FGFs and the cell membrane with FGFRs (as indicated) and usually a syndecan-4 type HSPG, which can bear variable ratios of HS (solid lines) and CS (dotted lines) chains. Formation of an HS:FGF:FGFR ternary complex produces cell signaling (lightening bolt). For simplicity, asymmetric signaling complexes are predominantly depicted.
(A)Potential forms of ternary complexes. The symmetric complex requires the free ends of two HS chains; whereas, the asymmetric complex requires only a single HS chain.
(B)Extremely high HS levels potentially inhibit signaling by partitioning receptors and ligands onto separate HS chains, thereby preventing ternary complex formation.
(C)Regulation of signaling by the extracellular matrix. Signaling can be inhibited by high levels of extracellular matrix HSPGs, such as perlecan (partially shown), which can sequester growth factors away from cell membrane receptors. Conditions such as tissue injury can liberate matrix-bound growth factors by cell-secreted proteases and heparanase, which initiate signaling by releasing HS:growth factor complexes that diffuse to the cell surface receptors. (D) Hypoxia enhances cellular responsiveness to FGF by increasing the HS content of heteroglycan-type HSPGs. (E) Certain FGFR splice variants can be non-glycanated or glycanated with either an HS or CS chain. The HS glycanated receptor can form a ternary complex in the absence of other HSPGs.
(F)HS can regulate signaling of specific ligand:receptor isoforms. Shown are two cells exposed to two FGF isoforms and expressing two FGFR isoforms. HS sequence motifs (circles versus triangles) can determine which FGF:FGFR isoform combinations are functionally operable.
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a transient phosphorylation of mitogen-activated protein kinase that does not lead to cell proliferation.68 In contrast, sustained signaling that leads to mitogenesis requires the presence of HS.69,70 Another difference is the nanomolar levels of growth factors can be greatly exceeded by the concentrations of pericellular HSPGs, which can achieve micromolar levels (reviewed in Ref. 16). HSPG concentration regulates signaling by dramatically influencing the stoichiometry of the ligand-receptor-HS ternary complex.
6.2.1. Co-receptors engender stoichiometric control of signaling
Since HS interacts with both FGFs and FGFRs, the levels of HSPG present on the cell surface tightly control the levels of functional HS:FGF:FGFR ternary complex. Quantitative modulation of signaling can be achieved; this occurs in developmental morphogen gradients, where the regional level of HSPG expression defines the local strength of growth factor signaling.71 Much more extreme concentration effects can be demonstrated with HSPG-deficient cells, where the addition of exogenous HS/heparin restores FGF signaling. High concentrations of GAG inhibit FGF signaling but low concentrations are stimulatory.62,72,73 It is thought that low HS/heparin concentrations promote ternary complex formation at the cell surface. In contrast, high GAG concentrations that exceed levels of receptors and ligands can prevent ternary complex formation by partitioning each component onto separate HS chains (Fig. 3B). Indeed, mitogenic signaling at high HS/heparin concentrations can be re-established by simply increasing the concentration of FGF.74
In vivo, high HS levels can clearly prevent ternary complex formation by a mechanism involving spatial segregation (Fig.3C). High levels of growth factors are bound to HSPGs of the endothelial basement membrane; however, the ligands are physically separated from their cell surface-bound receptors so signaling does not occur. Thus, growth factors are sequestered by the basement membrane. During vessel injury or remodeling, this reservoir of growth factors can be recruited. The actions of heparinase or proteases can release HS-growth factor complexes that diffuse to activate their cell surface receptors (reviewed in Refs. 12, 16 and 58).
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6.2.2. The effects of glycanation
One means of regulating HS levels is to change glycanation, the type and number of GAG chains synthesized on a core protein. Glycanation control is germane to “heteroglycans” — where multiple GAG types occur on single core proteins. ECs express several heteroglycans (Table 2). For example, syndecan-4 exhibits three GAG attachment sites that can bear either HS or CS chains. Consequently, a variety of isoforms can be produced by a single cell type: pure CS-syndecan-4, pure HS-syndecan-4 and all possible permutations of heteroglycans.75 The ratio of these forms is biosynthetically controlled in response to external stimuli. For example, exposure of ECs to hypoxia enhances cellular responsiveness to FGF2 by increasing the HS to CS ratio.76 Core protein expression is not altered by hypoxia. Instead, hypoxia enhances the expression of EXTL2, which commits GAG attachment sites to synthesis of HS chains (Fig. 2). Major elevations in cell surface levels of HS occur because such biosynthetic control should increase the HS content on all heteroglycans expressed by a cell (Fig. 3D).
Glycanation of certain receptors results in additional signaling mechanisms (Fig. 3E). A single GAG chain occurs on a specific FGFR2 splice variant.77 An alternate exon of the variant encodes a GAG attachment site that can bear either HS or CS chains. The HS-bearing FGFR2, compared to non-modified or CS modified receptors, exhibits high affinity for FGF1. In the absence of exogenous HS/heparin, only the HS modified receptor responds to FGF1 stimulation with sustained and enhanced signaling events that lead to mitogenesis.77 Glycanation of FGFR3 may also be possible, as this receptor also has a similarly positioned potential GAG attachment site. Thus, the need for an HSPG co-receptor may be abrogated for select FGFRs that bear an HS chain. It follows that when cell surface levels of HSPGs are low, such receptors may play a dominant role in FGF signaling.
6.2.3. HS sequence motifs regulate signaling
The existence of distinct HS motifs provides multiple mechanisms for regulating signaling specificity. Specific HS motifs are critical
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for FGF signaling. For example, FGF2 binding requires an HS sequence with 2-O-sulfated iduronic acids.78−80 A cell mutant deficient in HS2ST activity, which produces HS lacking 2-O-sulfated iduronic acid, confirms this necessity. Mutant cells can neither bind to nor respond to FGF2.81 Successful signaling also requires 6-O- sulfates that form direct contacts with the FGFR.66,67,82 As described above, this requirement is exemplified by a HS6ST-deficient Drosophila mutant, which consequently has defective FGF-driven tracheal morphogenesis.
HS motifs may also allow for temporal and cell type control of the specificity of ligand-receptor signaling. Across both the respective families of FGFs and FGFRs, the residues of the HS-binding regions are non-conserved (reviewed in Ref. 58). Consequently, distinct HS motifs are recognized by distinct isoforms of FGFs and FGFRs.83 It follows that the structural composition of cellular HS will govern which ligand:receptor combinations are functionally operable and how efficiently they will signal.84 Since HS structure varies between cell types, cell type-specific signaling can result. Two different cell types bearing the same FGFRs, but different HS motifs, can exhibit differential responsiveness to a given FGF isoform (Fig. 3F). Indeed, distinct FGF:FGFR combinations are activated by HS isolated from different EC phenotypes.85 Physiological cues can also change the array of HS motifs on the cell surface, by altering the cellular expression of various HS sulfotransferases.76 Thus, the growth factor responsiveness of a single cell type is modulated in a temporal fashion by regulation of HS synthesis.
The array of HS motifs expressed by a cell can also be modulated subsequent to HS synthesis. For example, endosulfatases (Sulf1 and Sulf2) secreted from cells can function in the extracellular environment to remove 6-O-sulfates from intact HS chains. These enzymes can influence FGF signaling, since 6-O-sulfates are required for HS interaction with certain FGFRs. Indeed, forced Sulf1 expression can inhibit formation of HS:FGF:FGFR ternary complexes and thereby prevents mesoderm induction and angiogenesis stimulated by FGF.40 Thus, HS structure can be modulated to superimpose specificity control onto the myriad of FGFs and FGFRs.