INTRODUCTION

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The fibroblast growth factor (FGF) family consists of 22 polypeptides (FGF1 to-22) with diverse biological activities (16, 21, 41). FGFs modulate proliferation and differentiation of a variety of cells of mesenchymal and neuroectodermal origin (1). FGFs play critical roles during embryonic processes such as mesoderm induction, postimplantation blastocyst development, and limb and lung development (7, 40). Increased FGF signaling leads to a variety of human skeletal disorders, including dwarfism and craniosynostosis syndromes (15, 19, 39). In adult organisms, FGFs are thought to be involved in physiological angiogenesis and wound healing as well as in pathological angiogenesis, such as in tumor neovascularization and diabetic retinopathy (1).

The diverse effects of FGFs are mediated by four receptor tyrosine kinases, FGFR1 to -4, which are composed of an extracellular ligand binding portion consisting of three immunoglobulin (Ig)-like domains (D1 to -3), a single transmembrane helix, and a cytoplasmic portion with protein tyrosine kinase activity. Ligand binding and specificity reside in D2, D3, and the short D2-D3 linker (29, 30, 34).

Receptor dimerization is a prerequisite for FGF signaling and requires heparin or heparan sulfate proteoglycans (HSPGs) (22, 31). The recent crystal structure of a ternary FGF2-FGFR1-heparin complex has provided a mechanistic view of the process by which heparin aids FGFs to induce FGFR dimerization (32). According to the proposed "two-end" model, heparin interacts via its nonreducing end with the heparin binding sites of FGF and FGFR and promotes the formation of a ternary 1:1:1 FGF-FGFR-heparin complex. A second ternary 1:1:1 FGF-FGFR-heparin complex is then recruited to the first ternary complex via interactions of FGFR, FGF, and heparin in one ternary complex with the FGFR in the adjoining ternary complex. A fundamentally different model has emerged from the recent crystal structure of a dimeric FGF1-FGFR2-heparin ternary complex (27). In this structure, a single heparin molecule links two FGF ligands into a dimer that bridges between two receptor chains. The asymmetric heparin binding involves contacts with both FGF molecules, but only one receptor chain. There is essentially no protein-protein interface between the two 1:1 FGF-FGFR complexes in the dimer.

With the exception of FGF1, which is the universal ligand for all FGFRs, most FGFs exhibit specific, albeit promiscuous, patterns of receptor binding affinity (23). Comparison of the crystal structures of FGF1-FGFR1, FGF2-FGFR1, and FGF2-FGFR2 complexes defined a general binding interface for FGF-FGFR complexes that involves contacts made by FGF with D2 and with the D2-D3 linker (30). It was also shown that specificity is achieved through interactions of the FGF N-terminal (immediately preceding the FGF's -trefoil core domain) and central regions with FGFR D3. These structures have also provided a molecular basis for how alternative splicing in FGFR modulates specificity. In both FGF2-FGFR1 and FGF2-FGFR2 structures, FGF2 makes specific contacts with the C'-E loop in D3, which is subject to alternative splicing. Consequently, FGF2 discriminates between the IIIc and IIIb variants of FGFRs. In contrast, FGF1 does not interact with the C'-E loop and therefore can bind all FGFRs irrespective of alternative splicing in D3 (30).

FGF4 shares about 30% sequence identity with the prototypical members of the FGF family, FGF1 and FGF2 (4). FGF4, unlike FGF1 and FGF2, has a classical signal peptide and thus is efficiently secreted from cells (2). Most receptor binding studies indicate that FGF4 binds and activates the IIIc splice forms of FGFR1 to -3 to comparable levels, but it shows little activity towards the IIIb splice forms of FGFR1 to -3 as well as towards FGFR4 (23, 36). As for FGF1 and FGF2, heparin greatly augments the biological activity of FGF4 on cells lacking endogenous cell surface HSPG (14). However, employing selectively O-desulfated heparins, Guimond et al. (8) showed that both 2-O- and 6-O-desulfated heparin were able to support the mitogenic activity of FGF4, while neither of these heparins could support the biological activity of FGF1 and FGF2. It has been suggested the sulfation motifs in heparin required for FGF4 activity may differ from those required for the actions of FGF1 and FGF2 (8, 9).

To explore the structural determinants of FGF4 involved in receptor and heparin binding, we determined the crystal structure of FGF4 at a 1.8-Å resolution. As anticipated, FGF4 adopts a -trefoil fold similar to those of other FGFs. Superimposition of FGF4 structure onto FGF2 bound to FGFR1 and heparin allowed us to identify potential receptor and heparin binding sites in FGF4. Mutation of several key FGF4 residues, observed to interact with FGFR1 in the model, produced ligands with reduced receptor binding and extremely low mitogenic potential. Significantly, the observed interactions between FGF4 and FGFR1 D3 provide a potential basis for preferential affinity of FGF4 towards IIIc splice variants of FGFR1 to -3. Moreover, the presented modeling studies along with mutational data suggest a two-step model for FGF-FGFR binding that involves initial formation of a crucial FGF-D2 interface stabilized by heparin binding followed by secondary FGF-D3 interactions.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Protein expression and purification of FGF4. DNA fragments generated by PCR of N-terminally truncated human FGF4 cDNA (encoding residues Gly-79 to Leu-206 [Gly⁷⁹-Leu²⁰⁶]) were subcloned into the pET-15b bacterial expression vector by using NcoI and XhoI cloning sites. The resulting construct (FGF4-pET15b) was transformed into Escherichia coli strain BL21 (DE3) bacteria, and FGF4 expression was induced with 1 mM isopropyl-1-thio--D-galactopyranoside for 5 h. The bacteria were then centrifuged and subsequently lysed in a 25 mM Na/K phosphate buffer (pH 7.5) containing 300 mM NaCl with a French cell press. The N-terminally truncated FGF4 (Gly⁷⁹-Leu²⁰⁶) was found primarily in the insoluble fraction and was extracted in 25 mM Na/K phosphate buffer (pH 7.5) containing 1 M NaCl at 4°C overnight. Following centrifugation, soluble FGF4 was diluted five times with 25 mM Na/K phosphate buffer (pH 7.5) and loaded onto a Source S column (Pharmacia). Bound FGF4 was eluted by a linear gradient of NaCl to 1 M in a 25 mM Na/K phosphate buffer (pH 7.5). Matrix-assisted laser desorption ionization mass spectrometry of the purified FGF4 gave a molecular mass of 14,244 Da (calculated mass, 14,409 Da). The mass difference was due to the cleavage of initiation methionine of FGF4 upon expression in E. coli and a point mutation (Ser-182Gly) that resulted from PCR. This mutation had no effect on FGF4 biological activity (data not shown).

Crystallization and data collection. Crystals of FGF4 were grown by vapor diffusion at 20°C by the hanging drop method. Two microliters of protein solution (2 mg/ml in 25 mM HEPES-NaOH buffer [pH 7.5] containing 150 mM NaCl) was mixed with an equal volume of the crystallization buffer (30% polyethylene glycol 8000, 0.2 M ammonium sulfate). FGF4 crystals belong to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 40.37 Å, b = 53.3 Å, and c = 56.23 Å. There is one molecule of FGF4 in the asymmetric unit with a solvent content of ~43%. Diffraction data were collected from a flash-frozen (in a dry nitrogen stream with mother liquor containing 10% glycerol as cryoprotectant) crystal on an R-Axis IV image plate detector at Beamline X4-A at the National Synchrotron Light Source, Brookhaven National Laboratory, Long Island, N.Y. The data were processed with DENZO and SCALEPACK (25).

Structure determination and refinement. A molecular replacement solution was found for one copy of FGF4 in the asymmetric unit by using the program AmoRe (20) and the structure of FGF2 (Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank, Rutgers, the State University of New Jersey, entry 2FGF) (42) as the search model. Simulated annealing and positional/B-factor refinement were performed with CNS (3). Bulk solvent and anisotropic B-factor corrections were applied. Model building into 2F_o-F_c and F_o-F_c electron density maps was performed with the program O (10). The atomic model contains residues 79 to 206 of FGF4, 3 sulfate ions, and 96 water molecules. The average B-factors are 10.5 Å² for the FGF4 molecule, 40.5 Å² for the sulfate ions, and 17.5 Å² for the water molecules.

Production of the mutant FGF4 proteins. Alanine substitutions were introduced into the N-terminally truncated FGF4 (Gly⁷⁹-Leu²⁰⁶) by PCR site-directed mutagenesis (Quik Change; Stratagene) with the FGF4-pET15b expression plasmid (described above) as the template and the following mutant oligonucleotides as primers: Y87A (5'-AAGCGGCTGCGGCGGCTCGCATGCAACGTGGGCATCGGC-3'), F129A (5'-GCGTGGTGAGCATCGCCGGCGTGGCCAGCCGG-3'), F151A (5'-CTATGGCTCGCCCTTCGCGACCGATGAGTGCACGTTC-3'), E159A (5'-GATGAGTGCACGTTCAAGGCCATTCTCCTTCCCAAC-3'), Y166A (5'-CTCCTTCCCAACAACGCGAACGCGTACGAGTCC-3'), L203A (5'-CCATGAAGGTCACCCACTTCGCCCCTAGGCTGTGACCC-3'), R205A (5'-CCCACTTCCTCCCCGCGCTGTGACCTTCCAGAGG-3'), N89A, (5'-CGGCGGCTCTACTGCGCCGTGGGCATCGGCTTC-3'), K198A (5'-GTGTCGCCCACCATGGCGGTCACCCACTTCCTC-3'), K183A/K188A (5'-GCCCTGAGCGCGAATGGGAAGACCGCGAAGGGGAAC-3'), R103A (5'-GCGCTCCCCGACGGCGCCATCGGCGGCGCGCAC-3'), and K144A (5'-ATGAGCAGCAAGGGCGCGCTCTATGGCTCGCCC-3').

DNA synthesis assay. NIH 3T3 cells were seeded at a density of 3 × 10⁴/well in 24-well plates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum. The following day, the medium was replaced with DMEM containing only 0.5% calf serum, and the cells were allowed to reach quiescence for 48 h. Thereafter, serial dilutions of wild-type full-length FGF4 (Ala³¹-Leu²⁰⁶) or N-terminally truncated wild-type or mutant FGF4 (Gly⁷⁹-Leu²⁰⁶) were added for 18 h. Cells were then labeled with 1 µCi of [³H]thymidine for 6 h, washed with Tris-HCl-buffered saline (pH 7.5), and lysed with 0.5 M NaOH. The lysates were then neutralized with 0.5 M HCl, and the radioactivity incorporated into the acid-precipitable material was measured with a -counter (LKB, Pharmacia). Each assay was performed in triplicate.

Receptor binding assay. N-terminally truncated FGF4 (Gly⁷⁹-Leu²⁰⁶) was radioiodinated by the chloramine-T method by a previously described protocol (2). Labeled FGF4 was separated from free iodine over a Sephadex G-25 column, which was previously equilibrated in phosphate-buffered saline containing 1% bovine serum albumin. CHO cells overexpressing FGFR2 (14) were seeded at 10⁶ cells/well in six-well plates in DMEM containing 10% fetal calf serum. The following day, the medium was removed, and the cells were allowed to bind labeled FGF4 (specific activity, 2.5 × 10⁴ cpm/ng) in DMEM containing 25 mM HEPES-NaOH (pH 7.4), 15% gelatin, 10 µg of heparin per ml, and increasing concentrations of the wild-type or mutant FGF4 proteins for 2 h at 4°C. Cells were then washed several times with ice-cold Tris-HCl-buffered saline (pH 7.5), and the receptor-bound radiolabeled FGF4 was released by using a 50 mM sodium acetate buffer (pH 4.0) containing 2 M NaCl. Radioactivity was measured with a -counter (LKB-Pharmacia). Binding assays were done in duplicate.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Structure determination. The mature, secreted form of human FGF4 spans amino acids Ala-31 to Leu-206 (2). Based on the crystal structure of FGF2 (5, 42, 45), the -trefoil core of FGF4 is expected to start at Leu-83 (Pro-29 in FGF2). Recent crystal structures of three different FGF-FGFR complexes have revealed that the residues immediately preceding the -trefoil core in FGF1 and FGF2 are involved in FGFR binding (29, 30, 33). These residues correspond to amino acids Gly-79 to Arg-82 of FGF4 (Fig. 1B). Thus, to maximize the likelihood of obtaining diffracting crystals without jeopardizing the biological activity of FGF4, we decided to crystallize an N-terminally truncated FGF4 containing residues Gly-79 to Leu-206 (Gly⁷⁹-Leu²⁰⁶). Truncated FGF4 was expressed in E. coli and purified to homogeneity (see Materials and Methods). The mitogenic activity of the truncated FGF4 on NIH 3T3 cells was only slightly lower than that of the mature FGF4, indicating that the truncated FGF4 contains the majority of receptor binding sites (not shown). Crystallization trials with FGF4 produced orthorhombic crystals with 1 molecule per asymmetric unit. The crystal structure of FGF4 was solved by molecular replacement (see Materials and Methods) and refined to a 1.8-Å resolution with an R-value of 19.4% (free R-value of 20.7%). The atomic model for FGF4 consists of 1 FGF4 molecule (residues 79 to 206), 3 sulfate ions, and 96 water molecules. Data collection and refinement statistics are given in Table 1.

View larger version (41K): [in this window] [in a new window]   FIG. 1.   Structure and sequence alignment of FGF4. (A) Ribbon diagram of FGF4. Secondary structure assignments were obtained with the program PROCHECK (12). The -strands of FGF4 are labeled according to the conventional strand nomenclature for FGF1 and FGF2 (6). NT and CT denote the amino and carboxy termini, respectively. This figure was created with the programs Molscript (11) and Raster3D (17). (B) Structure-based sequence alignment of FGFs. Sequence alignment was performed with CLUSTALW (34). All of the FGFs used in this alignment are human. The locations and lengths of the -strands and -helices are shown on the top. The signal sequences of FGF4 and FGF6 are indicated by italics and underlining. The box demarcates the boundaries of the -trefoil core. A period indicates sequence identity to FGF4. A dash represents a gap introduced to optimize the alignment. FGF4 residues are colored with respect to the region on FGFR1 with which they interact: residues that interact with D2 are green, residues that interact with the linker region are gray, and residues that interact with D3 are cyan. FGF4 residues that interact with the C'-E loop in D3 of FGFR1 are purple. In red are FGF4 residues that constitute the conventional low- and high-affinity heparin binding sites. In addition, FGF4 residues that localize to the periphery of the high-affinity heparin-binding site and could potentially interact with heparin are yellow. A star indicates FGFR and heparin binding residues that were tested by site-directed mutagenesis.

Description of the structure. As anticipated by sequence similarity (36), FGF4 adopts a -trefoil fold (Fig. 1A). Superimposition of the C traces located within the -trefoil core of FGF4 with those of FGF1 (45) and FGF2 (42) gives root-mean-square (rms) deviations of only 0.86 Å (122 common C atoms) and 0.76 Å (123 common C atoms), respectively. Within the -trefoil core, the major differences between the FGF4 structure and the structures of FGF1 and FGF2 are the conformations of the 1-2, 3-4, and 9-10 loops. These loops vary in both length and sequence among the various members of the FGF family (Fig. 1B). In FGF4, the 1-2 loop is one residue longer than the corresponding loops of FGF1 and FGF2. In contrast, the 3-4 loop in FGF4 is shorter by one residue than the corresponding loops in FGF1 and FGF2. Like FGF2, the 9-10 loop in FGF4 is shorter by two residues than in FGF1 (Fig. 1B).

Receptor binding sites and specificity. To identify potential receptor binding sites in FGF4, we constructed an FGF4-FGFR1 model by superimposing the FGF4 structure onto the FGF2 structure bound to the ligand binding portion of FGFR1 consisting of Ig-like domains 2 (D2) and 3 (D3) (Fig. 2A). Careful inspection of the FGF4-FGFR1 interface showed that the majority of the interactions between FGF4 and FGFR1 in the FGF4-FGFR1 model could be accommodated by minor adjustments of FGF4 side-chain rotamers. Three loop regions, the 1-2 and 8-9 loops (within the -trefoil core) and the N terminus (outside the -trefoil core), sterically clash with the receptor (Fig. 2A). In the present crystal structure, these loop regions are involved in crystal lattice contacts, implying that the present conformations of these loops are dictated by the lattice contacts. However, upon FGFR binding, these regions are expected to undergo changes in backbone conformation to allow an engagement with FGFR1 to occur.

View larger version (43K): [in this window] [in a new window]   FIG. 2.   Mapping of receptor binding sites in FGF4. (A) A model of the FGF4-FGFR1 structure was generated by superimposition of the C traces within the -trefoil of the FGF4 structure onto the corresponding C traces of FGF2 in the FGF2-FGFR1 structure. Color coding is as follows: FGF4 is orange, D2 is green, D3 is cyan, and the linker region is gray. The FGF4 loop regions that clash with FGFR1 are red. NT and CT denote the amino and carboxy termini, respectively. (B) Stereo view of the receptor binding sites on FGF4. FGF4 residues are considered to be in the FGF4-FGFR1 interface if their side-chain or main-chain interatomic distance to FGFR1 is less than or equal to 3.8 Å. FGF4 residues are colored with respect to the FGFR1 regions with which they interact. FGF4 residues that interact with D2 are green, residues that interact with the linker region are gray, and residues that interact with D3 are cyan. FGF4 residues that interact with the C'-E loop in D3 of FGFR are purple. Oxygen and nitrogen atoms are red and blue, respectively. This figure was created by using Molscript and Raster3D.

Binding of the FGF4 mutants to FGFR2. We tested the mutant FGF4 proteins in a receptor binding assay to confirm that the diminished capacity of the mutant FGF4 proteins to induce DNA synthesis is a result of the reduced ability of the mutant FGF4 to interact with FGFR. CHO cells overexpressing FGFR2 (14) were allowed to bind radiolabeled N-terminally truncated FGF4 (Gly⁷⁹-Leu²⁰⁶) in the presence of increasing concentrations of unlabeled full-length (Ala³¹-Leu²⁰⁶), N-terminally truncated wild-type, or various N-terminally truncated mutant FGF4 proteins (Fig. 3). The N-terminally truncated wild-type FGF4 bound FGFR2 with only a slightly lower affinity than full-length FGF4 (Ala³¹-Leu²⁰⁶), indicating that the majority of receptor binding sites are contained within the Gly⁷⁹-Leu²⁰⁶ construct (Fig. 3). Substitutions of Tyr-87, Tyr-166, and Leu-203 with alanine severely reduced the affinity of FGF4 towards FGFR2 (Fig. 3 and Table 2), emphasizing the importance of the hydrophobic FGF4-D2 interface in providing FGF4-FGFR affinity. In contrast, the R205A mutant FGF4 showed only a slight reduction in FGFR2 binding affinity. The relative decrease in binding affinity of these mutants towards FGFR2 is consistent with the results of the DNA synthesis assay, thus implying that the impaired ability of these mutants to induce DNA synthesis is a consequence of loss of binding affinity to FGFR2.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Comparison of the binding affinities of various FGF4 mutants towards FGFR2. The capacity of the various FGF4 mutants to compete with binding of the N-terminally truncated wild-type FGF4 to FGFR2 binding was measured as described in Materials and Methods. The data are expressed as percent inhibition of wild-type FGF4 binding to FGFR2 by the indicated amount of unlabeled mutant FGF4. The results presented in this figure are also summarized in Table 2, where we have calculated for each mutant a 50% inhibitory concentration, or the concentration of mutant FGF4 necessary to compete off 50% of wild-type FGF4.

View larger version (23K): [in this window] [in a new window]   FIG. 4.   Differential effect of heparin on stimulation of DNA synthesis in NIH 3T3 cells by some FGF4 mutants. Thymidine uptake in NIH 3T3 cells in response to increasing concentrations of wild-type FGF4 and FGF4 mutants in the presence and absence of exogenous heparin was determined as described in Materials and Methods. (A) A representative experiment with the E159A and L203A mutants. (B) A representative experiment with the K183A/K188A and N89A/K198A double mutants. These results and those obtained with other mutants are summarized in Table 2, where we calculate the 50% effective dosethe concentration of mutant necessary to achieve 50% of maximum DNA synthesis produced by the wild-type FGF4.

Heparin binding sites. Recent biochemical and structural data demonstrate that FGF in the absence of heparin can form an initial low-affinity complex with FGFR (26). In the presence of heparin, the low-affinity complexes become stabilized, which in turn leads to stable 2:2 FGF-FGFR signaling complexes. The recent crystal structure of a dimeric 2:2:2 FGF2-FGFR1-heparin complex provides a molecular basis for how heparin enhances FGF-FGFR affinity and promotes dimerization (32). Within each ternary 1:1:1 FGF-FGFR-heparin complex, heparin makes numerous contacts with the heparin binding residues of FGF and FGFR, thereby increasing the affinity of FGF towards FGFRs. In addition, heparin also interacts with the heparin binding residues in D2 of the adjoining FGFR, thereby augmenting the weak interactions of FGF and FGFR in one ternary complex with the FGFR in the adjoining ternary complex. Since FGFs differ in the primary sequences of heparin binding sites, each FGF may require different heparin motifs (sulfation pattern and/or length) in order to exert its optimal biological activities (6, 32).

View larger version (45K): [in this window] [in a new window]   FIG. 5.   Heparin binding sites in FGF4. (A) A dimeric FGF4-FGFR1-heparin model was created by superimposition of the C traces of two FGF4 structures onto the C traces of the two FGF2 molecules in the FGF2-FGFR1-heparin ternary structure. NT and CT denote the amino and carboxy termini, respectively. The coloring for FGF4 and FGFR1 is as presented in Fig. 2. The heparin oligosaccharides are rendered in the ball-and-stick format. The atom coloring for oxygens and nitrogens is as presented in Fig. 2. In addition, sulfur atoms are yellow, and the carbon atoms of oligosaccharides are gray. (B) FGF4 residues that localize to the heparin binding surface of FGF4 in the context of the ternary FGF4-FGFR1-heparin structure are mapped onto the ribbon diagram of FGF4. FGF4 residues that localize to the peripheries of the high-affinity heparin binding site and could potentially bind heparin are labeled in purple letters. A sulfate ion is bound to the conventional high-affinity heparin binding site. Another sulfate ion is bound in the additional potential heparin binding site. The atom coloring is as presented in panel A. Dotted lines represent hydrogen bonds. The sugar rings of heparin are labeled A through H, starting at the nonreducing end of the oligosaccharide. This figure was created with Molscript and Raster3D.

Implications for the general mode of FGF-FGFR binding. It is important to note that some of the data presented in this report are not consistent with the model of FGF1-FGFR2 binding described in the recently published crystal structure of a ternary FGF1-FGFR2-heparin complex (27). In this structure, the FGFR-invariant Pro-253 located in the D2-D3 linker is found in a cis configuration, while in all previously reported binary FGF-FGFR structures, Pro-253 is found only in a trans configuration (29, 30, 34). Consequently, relative to its position in all binary FGF-FGFR structures, the receptor D3 in the ternary FGF1-FGFR2-heparin structure is swiveled around the linker region by more than 160°. This creates a completely different set of interactions at the FGF-D3 interface. Pellegrini et al. (27) propose that this D3 rotation is caused by a heparin-mediated trans-to-cis isomerization of Pro-253 in the D2-D3 linker region, but our mutagenesis data do not support this hypothesis. Based on this ternary FGF1-FGFR2-heparin structure (27), neither F129 nor F151 in FGF4 is predicted to make any contacts with D3. Thus, the drastically reduced mitogenic capacity of the F129A and F151A mutants is in disagreement with the mode of FGF-FGFR binding described by these authors. We believe that the cis isomerization of Pro-253 observed in the FGF1-FGFR2-heparin structure (27) is probably the result of partial refolding of FGFR2.