Structural basis by which alternative splicing confers specificity in fibroblast growth factor receptors

Abstract

Binding specificity between fibroblast growth factors (FGFs) and their receptors (FGFRs) is essential for mammalian development and is regulated primarily by two alternatively spliced exons, IIIb ("b") and IIIc ("c"), that encode the second half of Ig-like domain 3 (D3) of FGFRs. FGF7 and FGF10 activate only the b isoform of FGFR2 (FGFR2b). Here, we report the crystal structure of the ligand-binding portion of FGFR2b bound to FGF10. Unique contacts between divergent regions in FGF10 and two b-specific loops in D3 reveal the structural basis by which alternative splicing provides FGF10-FGFR2b specificity. Structure-based mutagenesis of FGF10 confirms the importance of the observed contacts for FGF10 biological activity. Interestingly, FGF10 binding induces a previously unobserved rotation of receptor Ig domain 2 (D2) to introduce specific contacts with FGF10. Hence, both D2 and D3 of FGFR2b contribute to the exceptional specificity between FGF10 and FGFR2b. We propose that ligand-induced conformational change in FGFRs may also play an important role in determining specificity for other FGF-FGFR complexes.

Figure 1

Overall structure of the FGF10-FGFR2b complex. (a) Ribbon diagram of the FGF10-FGFR2b complex. D2 of FGFR2b is colored green. The first half of D3 is shown in blue. The alternatively spliced second half of D3 is colored purple. The linker connecting D2 and D3 is colored gray. FGF10 is shown in orange. The β-strands of FGF10 and FGFR2b are labeled according to published nomenclature (44). The N and C termini are labeled NT and CT, respectively. An arrow indicates the beginning of the alternatively spliced region of D3. (b) Molecular surface representation of FGFR2b. FGF10 is displayed as a Cα coil. αN, β1, and β4 of FGF10 are labeled accordingly. FGF10 regions that interact with D3 in the FGF10-FGFR2b structure are colored red. The ligand-binding cleft on D3 of FGFR2b, comprising the βB′-βC and βC′-βE loops, is outlined. Coloring is as in a.

Figure 2

Comparison of the FGF10 structure with other FGF structures. (a) Structure-based sequence alignment of human FGFs. Sequence alignment was performed by using the gcg wisconsin package. β-strand assignment is according to published nomenclature (44). The locations and lengths of the secondary structural elements are shown above the sequence alignment. The different lengths of β1 in the FGF1, FGF2, and FGF7 structures are indicated by boxes within the alignment. Both FGF1 and FGF2 have a proline N-terminal to β1 that restricts its length. The N-terminal 3/10 helix is labeled gN. FGF10 residues are colored with respect to the region of FGFR2b with which they interact: green for D2, gray for the linker region, blue for the first half D3, and purple for the alternatively spliced half of D3. Residues that interact with two regions are colored with both colors. A period indicates sequence identity with FGF10. A dash indicates a gap introduced to optimize the alignment. Asterisks denote residues in FGF10 that were mutated. The full sequences of the secreted ligands are shown. The numberings for FGF7, FGF10, and FGF22 start from the initiation methionine. (b) Superimposition of the FGF7 structure onto the FGF10 structure. FGF7 and FGF10 are colored purple and orange, respectively. The N and C termini are labeled NT and CT, respectively. Colored arrows mark the start of β1 in each ligand.

Figure 3

Detailed interactions between FGF10 and FGFR2b at the FGF-D3 interface. (a) Superimposition of the D3 domains of the FGFR2b and FGFR2c isoforms. The βC-βC′ and βC′-βE loops were excluded from the superimposition. D3 of FGFR2b is colored blue, with the alternatively spliced second half colored purple. An arrow indicates the beginning of the alternatively spliced region of FGFR2b-D3. D3 of FGFR2c is colored gray. The difference in conformation of the βC-βC′ loops is a result of crystal lattice contacts (25, 44). (b) Stereo view of the interactions made by Arg-78 of FGF10 with the βB′-βC loop of D3. (c) Stereo view of the interactions made by Asp-76 of FGF10 with the βC′-βE loop of D3. (d) Stereo view of the interface between FGF10 and the βF-βG loop of D3. The side chains of interacting residues are displayed. (Right) A view of the FGF10-FGFR2b complex, with the region of interest indicated by a square. FGF10 and FGFR2b are colored as in Fig. 1. Oxygen atoms are colored red, nitrogen blue, and carbon atoms the same color as the molecules to which they belong.

Figure 4

Structure-based mutagenesis of FGF10 confirms the importance of observed FGF10-FGFR2b contacts for FGF10 biological activity. Serum-starved Balb/MK cells were stimulated with increasing concentrations of WT or mutant FGF10. Sixteen hours later, [³H]thymidine was added for 6 h, and incorporation was determined as described (18). Each data point was performed in triplicate. WT FGF10 induced an 85-fold increase in DNA synthesis.

Figure 5

FGF10-induced D2 rotation contributes to FGF10-FGFR2b specificity. (a) Relationship between the D2 domains after superimposition of FGF10 and FGF2 from the FGF10-FGFR2b and FGF2-FGFR2c structures. For the sake of clarity, D3 and the linker region are not shown. The direction and degree of rotation between the two domains is shown. D2 of FGFR2b is colored blue. D2 of FGFR2c is colored gray. The βA′ strands of both domains are shown as β-strand arrows. The remainders of the domains are displayed as Cα coils. FGF10 is shown in orange. (b) Interactions at the FGF10-D2 interface. The side chains of interacting residues are displayed. (Right) A view of the FGF10-FGFR2b complex, with the region of interest indicated by a square. FGF10 and FGFR2b are colored as in Fig. 1. Oxygen atoms are colored red, nitrogen blue, and carbon atoms the same color as the molecules to which they belong.

Figure 6

Conformation of the FGFR2b linker region. (a) Interactions of the FGFR-invariant linker Arg-251 with FGF10. The side chains of interacting residues are displayed. (Right) A view of the whole FGF10-FGFR2b structure, with the region of interest indicated by a square. FGF10 and FGFR2b are colored as in Fig. 1. Oxygen atoms are colored red, nitrogen blue, and carbon atoms the same color as the molecules to which they belong. (b) Configuration of the FGFR-invariant linker Pro-253 in the FGF10-FGFR2b and FGF1-FGFR2c-heparin structures. The equivalent D2s from the FGF10-FGFR2b and FGF1-FGFR2c-heparin structures are superimposed (rmsd = 0.652 Å). FGFR2b is colored yellow, and FGFR2c is colored blue. The location of the linker prolines are indicated by arrows. Note the dramatic difference in the position of D3 between the two structures. (c) A FGF10-FGFR2b model with Pro-253 in the cis conformation. This model was generated by separately superimposing FGF10 and FGFR2b-D3 from the FGF10-FGFR2b structure onto FGF1 and FGFR2c-D3 in the FGF1-FGFR2c-heparin structure (rmsd = 0.791 Å and 0.668 Å, respectively). D2 and D3 are shown in green and blue, respectively. The alternatively spliced half of D3 is colored purple. FGF1 is displayed as a black Cα coil, and FGF10 is displayed as a thicker orange coil. FGF10 regions that interact with D3 in the FGF10-FGFR2b structure are colored red. In addition, FGF10 residues whose mutations reduce the ability of FGF10 to activate FGFR2b are rendered in ball-and-stick. The N and C termini of the receptor are labeled NT and CT, respectively.