Heparin or heparin-like heparan sulfate proteoglycans are obligatory for activity of the heparin-binding fibroblast growth factor (FGF) family. Heparin interacts independently of FGF ligand with a specific sequence (K18K) in one of the immunoglobulin-like loops in the extracellular domain of the FGF receptor tyrosine kinase transmembrane glycoprotein. A synthetic peptide corresponding to K18K inhibited heparin and heparin-dependent FGF binding to the receptor. K18K and an antibody to K18K were antagonists of FGF-stimulated cell growth. Point mutations of lysine residues in the K18K sequence abrogated both heparin- and ligand-binding activities of the receptor kinase. The results indicate that the FGF receptor is a ternary complex of heparan sulfate proteoglycan, tyrosine kinase transmembrane glycoprotein, and ligand.

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Recent experiments have established that Sox9 is required for chondrocyte differentiation. Here, we show that fibroblast growth factors (FGFs) markedly enhance Sox9 expression in mouse primary chondrocytes as well as in C3H10T1͞2 cells that express low levels of Sox9. FGFs also strongly increase the activity of a Sox9-dependent chondrocyte-specific enhancer in the gene for collagen type II. Transient transfection experiments using constructs encoding FGF receptors strongly suggested that all FGF receptors, FGFR1-R4, can transduce signals that lead to the increase in Sox9 expression. The increase in Sox9 levels induced by FGF2 was inhibited by a specific mitogen-activated protein kinase kinase (MAPKK)͞mitogen-activated protein kinase͞ERK kinase (MEK) inhibitor U0126 in primary chondrocytes. In addition, coexpression of a dual-specificity phosphatase, CL100͞MKP-1, that is able to dephosphorylate and inactivate mitogen-activated protein kinases (MAPKs) inhibited the FGF2-induced increase in activity of the Sox9-dependent enhancer. Furthermore, coexpression of a constitutively active mutant of MEK1 increased the activity of the Sox9-dependent enhancer in primary chondrocytes and C3H10T1͞2 cells, mimicking the effects of FGFs. These results indicate that expression of the gene for the master chondrogenic factor Sox9 is stimulated by FGFs in chondrocytes as well as in undifferentiated mesenchymal cells and strongly suggest that this regulation is mediated by the MAPK pathway. Because Sox9 is essential for chondrocyte differentiation, we propose that FGFs and the MAPK pathway play an important role in chondrogenesis.

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.