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Review
. 2013 Mar;14(3):166-80.
doi: 10.1038/nrm3528. Epub 2013 Feb 13.

Exploring mechanisms of FGF signalling through the lens of structural biology

Regina Goetz  1 , Moosa Mohammadi
Affiliations
Review

Exploring mechanisms of FGF signalling through the lens of structural biology

Regina Goetz et al. Nat Rev Mol Cell Biol. 2013 Mar.

Abstract

Fibroblast growth factors (FGFs) mediate a broad range of functions in both the developing and adult organism. The accumulated wealth of structural information on the FGF signalling pathway has begun to unveil the underlying molecular mechanisms that modulate this system to generate a myriad of distinct biological outputs in development, tissue homeostasis and metabolism. At the ligand and receptor level, these mechanisms include alternative splicing of the ligand (FGF8 subfamily) and the receptor (FGFR1-FGFR3), ligand homodimerization (FGF9 subfamily), site-specific proteolytic cleavage of the ligand (FGF23), and interaction of the ligand and the receptor with heparan sulphate cofactor and Klotho co-receptor.

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Conflict of interest statement

Competing interests statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. The FGF signalling system
a | The 18 mammalian fibroblast growth factor (FGF) ligands are listed, grouped by subfamily and mode of action. The ligand which each subfamily is named after is boxed in orange. The crystal structures of FGF2, a prototypical paracrine FGF (Protein Databank identifier (PDB ID): 1FQ9), and FGF19, an endocrine FGF (PDB ID: 2P23) are shown. The conserved globular core domain consists of 12 β-strands in FGF2 and 11 β-strands in FGF19. Residues from the loop connecting β-strand 1 and β-strand 2 and from the region between β-strand 10 and β-strand 12 comprise the heparan sulphate-binding site (shown in blue). The conformation of the FGF19 heparan sulphate-binding site differs from the conserved conformation of the heparan sulphate-binding site in paracrine FGFs, which are represented here by FGF2. b | The domain architecture of a prototypical FGF receptor and Klotho co-receptor are shown. Alternative splicing in the D3 domain of FGFR1, FGFR2 and FGFR3 generates FGFRb and FGFRc isoforms with distinct ligand-binding specificity,,. FGFRs interact with cell surface heparan sulphate via their D2 domain. Heparan sulphate is the obligatory cofactor for paracrine FGF signalling. A schematic of an heparan sulphate proteoglycan and the crystal structure of an heparan sulphate octasaccharide (PDB ID: 1FQ9) are shown. c | The crystal structure of the 2:2:2 ternary complex of FGF2, FGFR1c and heparan sulphate (PDB ID: 1FQ9) as well as a schematic of the paracrine FGF signal transduction unit based on this structure are shown. In the ternary complex, each FGF ligand interacts extensively with one receptor through the primary ligand-binding site comprising D2, D3 and the D2 D3 linker of the receptor. Each ligand also binds to the adjoining receptor in the complex through a secondary ligand-binding site on the D2 domain, which is adjacent to the site mediating the receptor receptor interaction. Heparan sulphate promotes the formation of the 2:2 paracrine FGF FGFR complex through concomitant interaction with both ligand and receptor.d | A schematic of two working models for the endocrine FGF FGFR Klotho co-receptor signal transduction unit is shown. A recent study on the ternary complex formation between FGF21, FGFR1c and β-Klotho supports the 1:2:1 model rather than the 2:2:2 model. AB, acid box; TMD, transmembrane domain. The FGF19 structure in parta is reproduced, with permission, from REF. © (2007) American Society for Microbiology.
Figure 2
Figure 2. FGF signalling pathways
Binding of fibroblast growth factor (FGF) to the FGF receptor (FGFR) induces FGFR dimerization, which juxtaposes the intracellular Tyr kinase domains of the receptors so that kinase activation by transphos- phorylation can occur,. Activated FGFR kinase in turn activates its intracellular substrates by phosphorylation, setting in motion distinct but potentially interactive signalling pathways that generate diverse cellular responses. Major substrates of FGFR kinase are FGFR substrate 2α (FRS2α), which is constitutively associated with the receptor kinase, and phospholipase Cγ1 (PLCγ1),. Activated FRS2α binds the adaptor protein growth factor receptor-bound 2 (GRB2). GRB2 then recruits either the guanine nucleotide exchange factor son of sevenless (SOS) (a) or the adaptor protein GRB2-associated binding protein 1 (GAB1) (b) to the signalling complex. Recruited SOS activates RAS GTPase, which initiates activation of the MAPK cascade (a), whereas recruited GAB1 leads to PI3K-mediated activation of AKT kinase (also known as protein kinase B) (b). Activated MAPK translocates from the cytoplasm to the nucleus, where it phosphorylates and hence activates immediate early gene transcription factors, such as FOS to induce transcription of specific genes (a). The outcome of this pathway is primarily cell proliferation but can also lead to cell differentiation, cell migration or another cellular response. Activated AKT kinase, on the other hand, inactivates pro-apoptotic effectors such as the BCL-2 antagonist of cell death (BAD) and forkhead box class O (FOXO) transcription factors, thereby promoting cell survival, (b). Recruitment and phosphorylation of PLCγ1 by FGFR kinase initiates a distinct signalling pathway that is thought to have roles in cell migration and cell differentiation and that can influence the RAS–MAPK and PI3K–AKT pathways (c). Activated PLCγ1 catalyses the hydrolysis of the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)(P2) into diacylglycerol (DAG) and inositol-1,4,5,-trisphosphate (IP3). DAG activates protein kinase C (PKC), which in turn activates its substrates byphosphorylation, including the myristoylated Ala-rich C kinase substrate (MARCKS), a regulator of cell motility. IP3 stimulates the release of calcium ions from intracellular stores, and this triggers the activation of calcium-dependent proteins such as the phosphatase calcineurin. Activated calcineurin induces nuclear translocation of the transcription factor nuclear factor of activated T cells (NFAT), which stimulates the expression of proteins essential for cell motility. BAX, BCL-2-associated X protein; MAPKK, MAPK kinase; PDK, phosphoinositide-dependent protein kinase.
Figure 3
Figure 3. Heparan sulphate-binding affinity determines the morphogenetic activity of FGF7 subfamily ligands
a | Structural model of heparan sulphate binding to fibroblast growth factor 7 (FGF7) and FGF10. FGF7 (Protein Databank identifier (PDB ID): 1QQK) and FGF10 (PDB ID: 1NUN) were superimposed onto FGF2 in the FGF2–FGFR1c–heparan sulphate complex (PDB ID: 1FQ9). The heparan sulphate octasaccharide from this complex and the α-carbon traces of the heparan sulphate-binding regions of FGF7 (orange) and FGF10 (teal) are shown. Note that Arg187 of FGF10, a crucial residue for heparan sulphate-binding affinity, makes hydrogen bonds (dashed black lines) with the heparan sulphate octasaccharide, whereas Val174, the corresponding residue of FGF7, cannot interact with the cofactor.b | Diffusion profiles of FGF10, FGF7 and an FGF10 mutant with reduced heparan sulphate-binding affinity (FGF10R187V) in the extracellular matrix. Heparan sulphate beads loaded with fluorescently labelled FGF ligand were embedded in a laminin-rich hydrogel (also known as Matrigel), and ligand diffusion was monitored by measuring fluorescence intensity along a line passing through the centre of the bead. The dashed circles indicate the radii of ligand diffusion; FGF10 forms a short, steep gradient, FGF7 forms a long, shallow gradient and the FGF10 mutant (FGF10R187V) diffuses similarly to FGF7. c | Epithelial growth response of developing submandibular gland tissue to FGF10, FGF7 and FGF10R187V mutant. Explants of prebranched submandibular gland epithelial buds were embedded in a Matrigel matrix and exposed to FGF ligand. FGF10 causes elongation of the epithelial gland buds, whereas FGF7 and FGF10R187V induce bud branching. d | Model for the regulation of branching morphogenesis by paracrine FGFs with different heparan sulphate-binding affinity. An FGF ligand with high binding affinity for heparan sulphate has a restricted diffusion range in the extracellular matrix (red sector), and hence can only signal to cells (red circles) closest to the FGF source (black asterisk). This leads to elongation of an epithelial gland bud towards the FGF source. Conversely, an FGF with low heparan sulphate-binding affinity can diffuse over a greater distance in the extracellular matrix, reaching cells further from the FGF source. The activation of cells both close to and distant from the FGF source results in branching of an epithelial gland bud. Image in part a is modified and the images in parts b, c, and d are reproduced, with permission, from REF. © (2009) AAAS.
Figure 4
Figure 4. Autoinhibition of FGF9 by homodimerization
a | The structure of the crystallographic fibroblast growth factor 9 (FGF9) homodimer (Protein Databank identifier (PDB ID): 1IHK). The dimer interface can be divided into two regions (black circles), one containing residues of the β-trefoil core domain and the other containing the amino-terminal and carboxy-terminal regions flanking the core domain. Extensive hydrophobic and hydrogen-bonding contacts between the N-terminal and C-terminal regions of the two monomers in the dimer drive homodimerization. Note that Asn143, which is spontaneously mutated in mice with Elbow knee synostosis (Eks), is located in the core region of the dimer interface. b | The molecular surface of an FGF9 monomer (dark yellow; PDB ID: 1IHK) is shown with residues that exclusively mediate ligand dimerization depicted in light blue, and residues that mediate dimerization and are also predicted to bind FGF receptors shown in dark blue. Note that the bifunctional residues account for about half of the residues that form the ligand dimer interface (red boundary).c | Joint synostosis in developing limb tissue caused by the N143T mutation leading to Eks. Forelimbs of a newborn mouse with Eks and a newborn wild-type mouse are shown. The elbow joints (marked by arrows) are also shown at greater magnification. In the forelimb of the mouse with Eks, humerus (h) and radius (r) bones are fused together at the prospective elbow joint, and humerus and ulna (u) bones are barely separated by cartilaginous joint tissue. d | Hyperdiffusion of the FGF9 mutant that causes Eks in developing limb tissue in mice. Beads loaded with FGF9 or the FGF9N143T mutant were implanted into forelimb buds of an Fgf9−/− embryo around the initiation stage of joint synostosis, and ligand diffusion was detected immunochemically. As FGF9N143T exhibits reduced binding affinity for heparan sulphate, it diffuses further than wild-type FGF9. s, scapula. The image in partc is reproduced, with permission, from REF. © (2002) Springer, and the image in partd is reproduced, with permission, from REF. © (2009) Macmillan Publishers Limited. All Rights Reserved.
Figure 5
Figure 5. N-terminal alternative splicing regulates the biological activity of FGF8
a | Sequence alignment of the amino-terminal regions of the mature human fibroblast growth factor 8 (FGF8) splice isoforms. Residues that make up the secondary structure elements known for FGF8b (the N-terminal G helix and the β1 strand) are indicated. Phe32 and Val36 of the FGF8b G helix (shaded orange) are key residues that interact with the D3 domain of FGF receptor (FGFR) c isoforms. Mutations at Pro26 and Phe40 of FGF8f (shaded grey) have been associated with idiopathic hypogonadotropic hypogonadism in humans. b | FGF8 splice isoforms possess distinct abilities to transform midbrain (Mid) into cerebellum (Cb) in chick embryos. Shown are dorsal views of developing chick brains transfected on the right side (represented by green fluorescence in the inset in the first image) with plasmids encoding FGF8a, FGF8b or FGF8bF32A or empty vector (control). The asterisk to the right of the midline of the brain (dashed red line) marks transformation of midbrain into cerebellum, and the arrows point to the expansion of midbrain tissue. FGF8b, but not FGF8a, is able to transform midbrain into cerebellum, and mutation of Phe32 to Ala in FGF8b abrogates this isoform-specific patterning ability.c | Crystal structure of the FGF8b–FGFR2c complex (Protein Databank identifier: 2FDB). A view of the whole structure and a close-up view of the ligand receptor D3 domain interface are shown. Phe32 and Val36 from the G helix and Phe93 from the β4–β5 loop of FGF8b engage a hydrophobic groove in the D3 domain of FGFR2c (coloured yellow). Note that Phe32 is the sole isoform-specific residue of FGF8b that interacts with the receptor groove. Residues that are specific to the FGFRc splice isoforms of FGFR1, FGFR2 and FGFR3 mainly form this hydrophobic groove, thus the groove is unique to the FGFRc isoforms. Note that Klotho co-receptors also engage this receptor groove. Di, diencephalon; Tel, telencephalon. The image in part b is reproduced, with permission, from REF. © (2006) Cold Spring Harbor Laboratory Press.
Figure 6
Figure 6. Alternative splicing confers ligand-binding specificity on FGFR
a | Fibroblast growth factor (FGF) ligands secreted from mesenchymal tissue exhibit binding specificity for epithelial FGF receptor (FGFR) b isoforms, whereas ligands secreted from epithelial tissue specifically bind to mesenchymal FGFRc isoforms. These paracrine FGF signalling loops are essential for controlling developmental processes. b | D3 domain sequence alignment of FGFR2b and FGFR2c. The location of the β-strands in the D3 domain of FGFR2c are indicated by brackets, and the dashed line across the alignment marks the junction between the common amino-terminal portion and the spliced carboxy-terminal portion of the D3 domain. Isoform-specific residues of the receptor βC′–βE loop that are crucial in determining ligand-binding specificity are shaded orange. Boxes indicate the positions of mutations in FGFR2c that cause skeletal disorders in humans. c | The ligand–receptor D3 domain interface in the FGF10–FGFR2b structure (Protein Databank identifier (PDB ID): 1NUN) is shown, along with a view of the whole structure. The ligand is coloured orange, the receptor D3 domain is coloured green (common N-terminal portion) and teal (alternatively spliced C-terminal portion). A network of hydrogen bonds (dashed lines) is formed between FGF10 and the isoform-specific βC′–βE loop of FGFR2b. Within this network, the hydrogen bonds between Asp76 of FGF10 (a residue unique to FGF10 and other FGF7 subfamily ligands) and Ser315 of FGFR2b (an isoform-specific residue of the βC′–βE loop) determine binding specificity. The hydrogen-bonding network is buttressed by hydrophobic contacts between Phe146 of FGF10 and Ile317 of FGFR2b.d | The ligand–receptor D3 domain interface in the FGF2–FGFR2c structure (PDB ID: 1EV2) is shown. Ligand and receptor are coloured as in partc. Among the hydrogen bonds (dashed lines) that are formed at the ligand–receptor βC′–βE loop interface, unique bonds between Gln65 of FGF2 and Asp321 of FGFR2c dictate binding specificity. Mutation of Asp321 to Ala, which causes Pfeiffer syndrome in humans, overrides the binding specificity of FGFR2c by eliminating steric clashes and electrostatic repulsion between Asp76 of FGF10 and Asp321 of FGFR2c. This enables FGF10 to bind to the mutant receptor and signal illegitimately through FGFR2c. Hydrophobic contacts between Tyr82, Val97 and Phe102 of FGF2 and Val317 of FGFR2c strengthen the hydrogen bonding interactions at the ligand receptor βC′–βE loop interface.
Figure 7
Figure 7. Ligand-dependent differences at the FGF–FGFR interface differentially regulate FGFR dimerization
a | Superimposition of a 2:2 fibroblast growth factor 8b–FGF receptor 2c (FGF8b–FGFR2c) complex onto a 2:2 FGF2 – FGFR2c complex. The α-carbon traces of the complexes are shown. For the FGF8b–FGFR2c complex model, FGF8b from the FGF8b–FGFR2c structure (Protein Databank identifier (PDB ID): 2FDB) was superimposed onto each of the two FGF2 ligands in the 2:2:2 FGF2–FGFR1c–heparan sulphate complex (PDB ID: 1FQ9). The FGF2–FGFR2c model was created in a similar manner using FGF2 from the FGF2–FGFR2c structure (PDB ID: 1EV2). Note the differences in the orientation of the receptor D3 domain and D2–D3 linker between the two dimers of ligand-bound FGFR2c, which translate into differences in the spatial distance between the carboxy-terminal membrane insertion points of the D3 domains.b | The interface between the ligand and the D3 domain and D2–D3 linker of the receptor in the FGF2–FGFR2c structure (PDB ID: 1EV2) is shown. The ligand is coloured orange, the receptor is coloured green (D2–D3 linker and common amino-terminal portion of the D3 domain) and teal (alternatively spliced C-terminal portion of the D3 domain). The network of hydrogen bonds (dashed lines) formed at this interface is highly conserved among complexes of FGFR with FGF1 subfamily ligands or FGF10. Its key constituents comprise Glu105 and Asn113 of FGF2 and Arg251, Asp283 and Gln285 of FGFR2c. Most of these residues also form entropically favourable intramolecular hydrogen bonds with similarly conserved residues, such as Glu105 and Asn113 of FGF2 with Tyr115 of FGF2.c | The interface between the ligand and the D3 domain and D2–D3 linker of the receptor in the FGF8b–FGFR2c structure (PDB ID: 2FDB) is shown. Ligand and receptor are coloured as in partb. The composition and geometry of the network of hydrogen bonds (dashed lines) formed at this interface differ from that observed in the FGF2–FGFR2c complex (see, partb). For example, Asn113 and Tyr115 of FGF2 are replaced with Thr and Leu, respectively, in FGF8b. The side chain of Glu131 in FGF8b, the corresponding residue to Glu105 in FGF2, adopts a different rotamer conformation compared to that in the FGF2–FGFR2c complex as it makes an intramolecular hydrogen bond with Lys176, a residue specific to FGF8 subfamily ligands. A total of four hydrogen bonds unique to the FGF8b–FGFR2c complex (dashed red lines) are formed at this interface between FGF8b and FGFR2c. Images in partsb and c are modified, with permission, from REF. © (2006) Cold Spring Harbor Laboratory Press.
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