Insights into the molecular basis for fibroblast growth factor receptor autoinhibition and ligand-binding promiscuity

Abstract

The prototypical fibroblast growth factor receptor (FGFR) extracellular domain consists of three Ig domains (D1-D3) of which the two membrane-proximal D2 and D3 domains and the interconnecting D2-D3 linker bear the determinants of ligand binding and specificity. In contrast, D1 and the D1-D2 linker are thought to play autoinhibitory roles in FGFR regulation. Here, we report the crystal structure of the three-Ig form of FGFR3c in complex with FGF1, an FGF that binds promiscuously to each of the seven principal FGFRs. In this structure, D1 and the D1-D2 linker are completely disordered, demonstrating that these regions are dispensable for FGF binding. Real-time binding experiments using surface plasmon resonance show that relative to two-Ig form, the three-Ig form of FGFR3c exhibits lower affinity for both FGF1 and heparin. Importantly, we demonstrate that this autoinhibition is mediated by intramolecular interactions of D1 and the D1-D2 linker with the minimal FGF and heparin-binding D2-D3 region. As in the FGF1-FGFR2c structure, but not the FGF1-FGFR1c structure, the alternatively spliced betaC'-betaE loop is ordered and interacts with FGF1 in the FGF1-FGFR3c structure. However, in contrast to the FGF1-FGFR2c structure in which the betaC'-betaE loop interacts with the beta-trefoil core region of FGF1, in the FGF1-FGFR3c structure, this loop interacts extensively with the N-terminal region of FGF1, underscoring the importance of the FGF1 N terminus in conferring receptor-binding affinity and promiscuity. Importantly, comparison of the three FGF1-FGFR structures shows that the flexibility of the betaC'-betaE loop is a major determinant of ligand-binding specificity and promiscuity.

Fig. 1.

The FGFR3c–FGF1 structure. (A) A ribbon representation of the FGFR3c–FGF1 complex. FGF1 is orange, FGFR3c D2 is green, D3 is cyan, and the D2–D3 linker is black. The alternatively spliced C-terminal half of D3 is purple. The N and C termini of FGF1 are labeled NT and CT, respectively. The FGF1 N-terminal region not included in the truncated FGF1 construct used in the FGFR1c and FGFR2c structures is gray. (Inset) Protein from FGF1–FGFR3c crystals is indistinguishable from the freshly purified complex. SDS/PAGE analysis of the purified three-Ig form FGFR3c–FGF1 complex (1), the purified two-Ig form FGFR3c–FGF1 complex (2), the protein solution from a FGF1–FGFR3c crystal containing hanging drop (3), and dissolved FGF1–FGFR3c crystals (4) (washed twice). Lanes with molecular mass markers are labeled M, and selected molecular masses are labeled. (B) Structure-based sequence alignment of Ig domain 3 from human FGFRs. The sequence alignment was performed by using clustalw (28). The location and length of the β strands are shown on top of the sequence alignment. Note that the βC′ strand of FGFR3c terminates two residues earlier than those of FGFR1c and FGFR2c, and, therefore, the βC′–βE loop of FGFR3 is 12 residues long (residues 310–322). The different lengths of the βC′–βE loops of FGFR1c and FGFR2c from the FGF1–FGFR structures are indicated by boxes within the alignment. A period indicates sequence identity to FGFR3c. A dash represents a gap introduced to optimize the alignment. The alternatively spliced C-terminal half of D3 is marked by red arrows. FGFR3c residues that interact with FGF1 are red. FGFR1c and FGFR2c residues that interact with FGF in other crystal structures are cyan. (C) Superimposition of FGFR3c and FGFR2c D3. The C^α trace of FGFR2c D3 from the FGFR2c–FGF1 structure (cyan) was superimposed onto the C^α trace of FGFR3c D3 from the FGFR3c–FGF1 structure (orange; rms deviation = 0.831 Å). Residues corresponding to the βC–βC′ and βC′–βE loops were not included in the superimposition. The βB′–βC, βC′–βE, and βF–βG loops of the D3s are marked by an arrowhead. (D) Interactions between the FGF1 N terminus and D3 in the FGFR3c–FGF1 structure. Colors are as in A. Selected residues are labeled and are rendered in a stick format. This figure was created by using the program pymol (29).

Fig. 2.

The affinity and activation of FGFRs by FGF1 is enhanced by the N terminus. (A and B) Sensorgrams of the full-length versus truncated FGF1 interaction with the D123 isoform of FGFR3c. Analyte concentrations are colored as follows: purple (1.6 μM), green (0.8 μM), red (0.4 μM), blue (0.2μM), and gray (0.1 μM). (C–E) Full-length FGF1 activates FGFR3 and FGFR2 more potently than truncated FGF1 on RCS cells (C–D). Analysis of FGFR3 and FGFR2 phosphorylation. Clarified cell lysates of RCSs starved overnight and treated with the indicated concentration of FGF for 5 min were examined by using protein immunoblot. (E) FGFR3 and FGFR2 immunoblots were probed with a phosphotyrosine-specific antibody. Analysis of ERK1/2 phosphorylation. Clarified cell lysates used in A were probed with an antibody specific for anti-phospho-ERK1/2.

Fig. 3.

D1 and the D1–D2 linker negatively regulate ligand- and heparin-binding affinity of FGFR3c. (A and B) Sensorgrams of the two-versus three-Ig form of FGFR3c binding to full-length FGF1. Analyte concentrations are colored as follows: purple (1.6 μM), green (0.8 μM), red (0.4 μM), blue (0.2μM), and gray (0.1 μM). (C and D) Sensorgrams of the two-versus three-Ig form of FGFR3c binding to heparin. Analyte concentrations are colored as follows: purple (5 μM), yellow (2.5 μM), orange (1.25 μM), red (0.625 μM), and cyan (0.313 μM). (E) Sensorgram of D1+ binding to the two-Ig form of FGFR3c. Analyte concentrations are colored as follows: purple (10 μM), pink (5 μM), yellow (2.5 μM), and cyan (1 μM).