Regulation of FGF Signaling by Heparan Sulfate

The fibroblast growth factors (FGFs) are a family of 23 polypeptide growth factors that have roles in early development and in disease [1]. The prototypic members of the family are acidic FGF (FGF1) and basic FGF (FGF2). These were originally identified with roles in neuron survival and neurite outgrowth, and fibroblast and endothelial cell proliferation, respectively. These two FGFs are also known to have potent angiogenic activity. FGF2 is released by many tumors both as an autocrine proliferation and survival factor, and also as an angiogenic agent that fosters tumor cell survival by promoting the growth of new blood vessels. Many or all of the FGFs have roles in early development. For example, FGFs 4, 7, 8 and 10 have been identified as important signaling factors in the limb bud, the classical model of ectoderm (the apical ectodermal ridge of the limb bud) signaling to the underlying mesoderm (the progress zone) to direct the formation of proximal-distal limb structures. These and other FGFs also have roles in development of the heart, nervous system, bone, etc.

All of the FGFs bind to heparan sulfate (HS), a glycosaminoglycan found on proteoglycans of the cell surface (e.g., syndecans and glypicans) and the extracellular matrix (e.g., perlecan) [2, 3]. The FGFs also bind and signal through a family of receptor tyrosine kinases, composed of FGF receptors (FRs) 1, 2, 3, and 4. Importantly, the FRs also contain a heparan sulfate-binding domain [4]. The formation of the high affinity signaling complex that signals into the cell requires the participation of the HS, the FGF and the FR (Figure 1). We first demonstrated this using cells treated with chlorate, a competitive inhibitor of sulfation [5]. Chlorate-treated cells lost their ability to respond to FGF-2, but could be recovered by adding soluble heparin which substituted for the cell surface heparan sulfate and restored assembly of FGF with its receptor. In this complex, the HS chain is believed to interact simultaneously with the FGF and the FR, thus enhancing the affinity of the interaction. We also showed that heparin that had been selectively de-sulfated could still bind FGF2 via its remaining 2-O-sulfate groups, but could not promote signaling because it lacked the groups (6-O-sulfates) necessary to bind effectively to the FGF receptor tyrosine kinase [6]. This suggested that the binding to the HS chain is dependent on its sulfate residues and that specific (and different) sulfate residues are critical for recognition of the FGF and the receptor. This pattern of sulfation is the consequence of concerted enzymatic action during synthesis of the chain as the proteoglycan passed through the Golgi apparatus. In addition, our hypothesis, based on our current data and that of others, is that the pattern of sulfation may be cell- or tissue-type specific and changes during development to coincide with FGF signaling. Thus, the ability of the HS chain to simultaneously assemble with a specific FGF and FR may be highly regulated, dictated by the sulfation pattern(s) expressed by that cell.

The question of how HS structure may vary in vivo has been difficult to answer. However, we have devised a simple, but elegant method for this analysis which we call the “ligand and carbohydrate engagement assay” or LACE. LACE relies on using FGF and soluble receptor as HS-specific probes and using these probes on frozen sections of developing mouse embryos. AP-tagged receptor ectodomain will assemble on HS with high affinity only when it forms a complex with a specific FGF and the HS. Thus, we detect probe binding only when the receptor and the FGF form a high affinity complex on a specific domain of HS expressed within a developing organ [7,8]. Where possible, we confirm that the HS decorated by these probes is indeed active by isolating it and testing it on cells (BaF3 cells) expressing specific FGF receptors and in the presence of a specific FGF (see Figure 2). This work has also shown that the assembly of the FGF, FGF receptor and HS ternary complex is more complex that the binding dictated by the individual FGF and FGF receptor. Our model, which we call the “synergistic model” predicts that interactions between the FGF and the FGF receptor determine how they will be oriented when they contact the HS (see figure 3). Thus, the sites on the HS chain that may bind an individual FGF or receptor are not necessarily the same sites that will bind the FGF and receptor complex.

Our current work is aimed at identifying the structure of the HS domains that capture these probes and the enzymes that are necessary for their specific synthesis.

References:
1. Szebenyi, G. and J.F. Fallon, Fibroblast growth factors as multifunctional signaling factors. Int Rev Cytol, 1999. 185: p. 45-106.
2. Rapraeger, A.C., In the clutches of proteoglycans: how does heparan sulfate regulate FGF binding? Chem Biol, 1995. 2(10): p. 645-9.
3. Turnbull, J., A. Powell, and S. Guimond, Heparan sulfate: decoding a dynamic multifunctional cell regulator. Trends Cell Biol, 2001. 11(2): p. 75-82.
4. Kan, M., F. Wang, J. Xu, J.W. Crabb, J. Hou, and W.L. McKeehan, An essential heparin-binding domain in the fibroblast growth factor receptor kinase. Science, 1993. 259(5103): p. 1918-21.
5. Rapraeger, A., Krufka, A. and Olwin, B. (1991). Heparan sulfate is required for the action of bFGF on fibroblast growth and myoblast differentiation, Science, 252:1705-1708.
6. Guimond, S., Maccarana, M., Olwin, B., Lindahl, U. and Rapraeger, A. (1993). Activating and inhibitory heparin sequences for FGF-2 (basic FGF): Distinct requirements for FGF-1, FGF-2 and FGF-4. J. Biol. Chem., 268: 23906-23914.
7. Allen, B.L, Filla, M. and Rapraeger, A.C. (2001). Role of heparan sulfate as a tissue specific regulator of FGF4 and FGF receptor recognition. J. Cell Biol., 155: 845-857.
8. Allen, B.L. and Rapraeger, A.C. (2003). Spatial and temporal expression of distinct heparan sulfate domains in mouse development regulate FGF and FGF receptor complex assembly. J. Cell Biol., 163: 637-648.

Other relevant publications:
• Rapraeger, A. and Epel, D. (1981). The appearance of an extracellular arylsulfatase during morphogenesis of the sea urchin S. purpuratus. Dev. Biol., 88: 269-278.
• Rapraeger, A.C., Guimond, S., Krufka, A. and Olwin, B.B. (1994). Regulation by heparan sulfate in FGF signaling. Met. Enzymol., 245: 219-240.
• Krufka, A., Guimond, S. and Rapraeger, A.C. (1996). Two hierarchies of FGF-2 signaling in heparin: mitogenic stimulation and high affinity binding/receptor transphosphorylation. Biochemistry, 35:11131-11141.
• Chang, Z., Meyer, C., Rapraeger, A.C. and A. Friedl (2000). Differential ability of heparan sulfate proteoglycans to assemble the fibroblast growth factor receptor complex in situ. FASEB J., 14: 137-144.
• Cornelison, D.D.W., Filla, M., Stanley, H., Rapraeger, A.C. and B.B. Olwin (2001). Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell maintenance and muscle regeneration. Dev. Biol. 239: 79-94.
• Rapraeger, A.C. (2002). Heparan sulfate-growth factor interactions. Meth. Cell Biol. 69: 83-109.
• Cornelison, D.D.W., Wilcox-Adelman, S.A., Goetinck, P.F., Rauvala, H., Rapraeger, A.C. and Olwin, B.B. (2004). Essential and separable roles for Syndecan-3 and Syndecan-4 in skeletal muscle development and regeneration. Genes & Dev. 18: 2231-2236.
• Jasuja, R., Papanno, W. N., Allen, B.L., Rapraeger, A.C. and Greenspan, D. S. (2004). Cell surface heparan sulfate proteoglycans potentiate chordin’s antagonism of BMP signaling and are necessary for cellular uptake of chordin. J. Biol. Chem., 279: 51289-97.
• Dai, Y., Yang, Y., MacLeod, V., Yue, X., Rapraeger, A.C., Shriver, Z., Venkataraman, G.,Sasisekharan, R., and Sanderson, R.D. (2005). HSulf-1 and HSulf-2 are potent inhibitors of myeloma tumor growth in vivo. J. Biol. Chem., 280: 40066-40073.
•Ma, P., Beck, S., Raab, R., Robert McKown, R., George Coffman, G., William J. Chirico, W.J., Rapraeger, A.C. and Laurie, G.W. (2006). Deglycosylation of coreceptor syndecan-1 is required for core protein binding of epithelial-restricted mitogen ‘lacritin’. J. Cell Biol., 174: 1097-1106.