Klotho coreceptors inhibit signaling by paracrine fibroblast growth factor 8 subfamily ligands

Abstract

It has been recently established that Klotho coreceptors associate with fibroblast growth factor (FGF) receptor tyrosine kinases (FGFRs) to enable signaling by endocrine-acting FGFs. However, the molecular interactions leading to FGF-FGFR-Klotho ternary complex formation remain incompletely understood. Here, we show that in contrast to αKlotho, βKlotho binds its cognate endocrine FGF ligand (FGF19 or FGF21) and FGFR independently through two distinct binding sites. FGF19 and FGF21 use their respective C-terminal tails to bind to a common binding site on βKlotho. Importantly, we also show that Klotho coreceptors engage a conserved hydrophobic groove in the immunoglobulin-like domain III (D3) of the "c" splice isoform of FGFR. Intriguingly, this hydrophobic groove is also used by ligands of the paracrine-acting FGF8 subfamily for receptor binding. Based on this binding site overlap, we conclude that while Klotho coreceptors enhance binding affinity of FGFR for endocrine FGFs, they actively suppress binding of FGF8 subfamily ligands to FGFR.

Fig 1

βKlotho possesses distinct high-affinity binding sites for FGF19/21 and FGFR. (A and B) Overlays of SPR sensorgrams illustrating binding of FGFR1c (A) and FGFR4 (B) to βKlotho and fitted saturation binding curves. βKlotho ectodomain was immobilized on a biosensor chip, and increasing concentrations of the ligand-binding domain of FGFR1c or FGFR4 were passed over the chip. The dissociation constants (K_Ds) were calculated from the saturation binding curve. (C and D) Overlays of SPR sensorgrams illustrating binding of βKlotho to FGF19 (C) and FGF21 (D). FGF19 and FGF21 were immobilized on a biosensor chip, and increasing concentrations of βKlotho ectodomain were passed over the chip. Note that for any given concentration of βKlotho, the binding response is greater on the FGF19 chip surface than on the FGF21 chip surface. Also note that the FGF19-βKlotho complex dissociates more slowly than the FGF21-βKlotho complex (compare the dissociation phases of the sensorgrams shown in panels C and D). (E) Overlay of SPR sensorgrams showing no interaction between βKlotho and FGF23. FGF23 was immobilized on a biosensor chip, and increasing concentrations of βKlotho ectodomain were passed over the chip. (F and G) Overlays of SPR sensorgrams showing no interaction between αKlotho and FGF19 (F) or FGF21 (G). FGF19 and FGF21 were immobilized on a biosensor chip, and increasing concentrations of αKlotho ectodomain were passed over the chip. The data shown in each figure panel are representative of two to three independent experiments.

Fig 2

The C-terminal tail peptides of FGF19 and FGF21 bind to a common site on βKlotho, albeit with different affinities. (A) Alignment of the C-terminal tail sequences of human FGF19 and FGF21. Residue numbers are in parentheses to the left of the alignment. Gaps (dashes) were introduced to optimize the sequence alignment. Residues that are identical between human FGF19 and FGF21 are shaded gray. Note that the greatest degree of sequence identity (40%) is confined to the 20 most C-terminal residues. (B and C) Overlays of SPR sensorgrams illustrating inhibition by FGF19^C-tail (B) or FGF21^C-tail (C) of βKlotho binding to FGF19. FGF19 was immobilized on a biosensor chip, and mixtures of a fixed concentration of βKlotho ectodomain with increasing concentrations of either FGF19^C-tail or FGF21^C-tail were passed over the chip. (D and E) Overlays of SPR sensorgrams illustrating inhibition by FGF21^C-tail (D) or FGF19^C-tail (E) of βKlotho binding to FGF21. FGF21 was immobilized on a biosensor chip, and mixtures of a fixed concentration of βKlotho ectodomain with increasing concentrations of either FGF19^C-tail or FGF21^C-tail were passed over the chip. Note that FGF19^C-tail is more potent than FGF21^C-tail at inhibiting βKlotho binding to FGF19 or FGF21 (compare panels B and E with panels C and D). (F) Overlay of SPR sensorgrams illustrating inhibition by FGF21^29–190/FGF19^197–216 or FGF21 of βKlotho binding to FGF21 immobilized on a biosensor chip. βKlotho ectodomain alone and 1:1 mixtures of βKlotho ectodomain with either FGF21^29–190/FGF19^197–216 or FGF21 were passed over a FGF21 chip. Note that the FGF21^29–190/FGF19^197–216 chimera is a more potent competitor for βKlotho binding than is native FGF21. The data shown in figure panels B to F are representative of two to three independent experiments.

Fig 3

The C-terminal tail peptides of FGF19 and FGF21 are interchangeable in inhibiting the signaling of FGF19. (A) Immunoblot analysis of phosphorylation of FRS2α (pFRS2α) and 44/42 MAP kinase (p44/42 MAPK) in the rat hepatoma cell line H4IIE, which had been pretreated with FGF19^C-tail and then stimulated with FGF19. As controls, cells were stimulated with FGF19 or FGF19^C-tail alone. (B) Immunoblot analysis of phosphorylation of FRS2α (pFRS2α) and 44/42 MAP kinase (p44/42 MAPK) in the rat hepatoma cell line H4IIE, which had been pretreated with FGF21^C-tail and then stimulated with FGF19. As controls, cells were stimulated with FGF19 or FGF21^C-tail alone. Numbers above the lanes give the amounts of protein/peptide added in ng ml⁻¹. To control for equal sample loading, the protein blots were probed with an antibody recognizing both phosphorylated and nonphosphorylated 44/42 MAP kinase (44/42 MAPK). The data shown in each figure panel are representative of two independent experiments.

Fig 4

A chimeric FGF21 protein in which the FGF21 C-terminal tail had been swapped with that of FGF19 exhibits FGF21-like activity. (A) Immunoblot analysis of Egr1 expression in HEK293-βKlotho cells stimulated with FGF21 or FGF21^29–167/FGF19^169–216. Numbers above the lanes give the amounts of protein added in ng ml⁻¹. To control for equal sample loading, the protein blots were probed with an antibody to GAPDH. Note that the FGF21^29–167/FGF19^169–216 chimera is more potent than native FGF21 at inducing Egr1 expression, which is consistent with the SPR data shown in Fig. 2F. The data are representative of two independent experiments. (B) Analysis of blood glucose concentrations in mice before and at the indicated time points after intraperitoneal injection of insulin alone, insulin plus FGF21^29–167/FGF19^169–216, insulin plus FGF21, or vehicle alone. Insulin and FGF21 ligand were injected at 0.5 units per kg of body weight and 0.3 mg per kg of body weight, respectively. Blood glucose concentrations are expressed as percentages of preinjection values. Data are presented as means ± SEM. (C) Changes in plasma insulin concentrations in mice in response to a single intraperitoneal injection of FGF21^29–167/FGF19^169–216, FGF21, or vehicle. Three different doses of protein were tested, and the numbers below the x axis give each dose of protein injected in mg per kg of body weight. Plasma insulin concentrations are expressed as percentages of preinjection values. Data are presented as means ± SEM. *, P < 0.05; **, P < 0.01 (both by Student's t test).

Fig 5

Comprehensive analysis of FGFR binding specificity of Klotho coreceptors. (A and B) Overlays of SPR sensorgrams illustrating the FGFR binding specificity profile of αKlotho (A) and βKlotho (B). The ectodomains of αKlotho and βKlotho were immobilized on a biosensor chip, and increasing concentrations of the ligand-binding domain of each of the seven principal FGFRs were passed over the chip. Where possible, equilibrium dissociation constants (K_Ds) were derived from fitted saturation binding curves. The data are representative of two to five independent experiments. To give a complete view of the FGFR binding specificity of Klotho coreceptors, the sensorgrams illustrating βKlotho binding to FGFR1c and FGFR4 from Fig. 1 are shown again in panel B of this figure.

Fig 6

Alignment of the D3 domain sequences of the seven principal human FGFRs. Residue numbers are in parentheses to the left of the alignment. Gaps (dashes) were introduced to optimize the alignment. Bars on top of the alignment indicate the location of the β strands in FGFR1c. A dashed line across the alignment marks the junction between the constant N-terminal half and the alternatively spliced C-terminal half of the D3 domain. Residues that form the hydrophobic groove in D3 of FGFR1c are shaded gray. Note that four of these residues map to the alternatively spliced region of D3. The positions of pathogenic mutations in the D3 domain of FGFR1 are indicated by boxes.

Fig 7

Klotho proteins and FGF8b engage the same hydrophobic groove in D3 of FGFR1c. (A) Structural model of the FGF8b-FGFR1c complex. The model was created by superimposing the ligand-binding domain of FGFR1c from the FGF2-FGFR1c crystal structure (PDB ID, 1CVS [55]) onto the ligand-binding domain of FGFR2c in the FGF8b-FGFR2c crystal structure (PDB ID, 2FDB [50]). On the left is a view of the whole model, and on the right is a close-up view of the ligand-receptor D3 interface. FGF8b is shown as a ribbon diagram, and FGFR1c is shown as a space-filling molecular surface. Note that F32 and V36 of the N-terminal g helix and F93 of the β4-β5 loop of FGF8b bind to a hydrophobic groove in the D3 domain of FGFR1c formed by L290, L305, P306, V308, T340, L342, L349, and H351. NT and CT denote N and C termini of FGF8b and FGFR1c, respectively. (B, C, and D) Overlays of SPR sensorgrams illustrating binding of wild-type and mutant FGFR1c proteins to αKlotho (B), βKlotho (C), and FGF8b (D). αKlotho ectodomain, βKlotho ectodomain, or FGF8b was immobilized on biosensor chips, and increasing concentrations of either wild-type or mutant FGFR1c ligand-binding domain were passed over the chips. Maximal binding responses of FGFR1c mutants relative to wild-type protein were plotted (25). As we have previously reported (53), the L342S mutation causes a major loss in binding affinity of FGFR1c for FGF8b.

Fig 8

βKlotho inhibits FGF8b signaling. Shown are dose-response curves for Egr1 promoter-driven induction of luciferase activity by FGF8b in HEK293 cells transfected with βKlotho (+) or empty vector (−). Luciferase activity of cells expressing βKlotho was normalized to that of cells transfected with empty vector. Data points represent mean values of three independent experiments each performed in triplicate, and error bars denote standard deviations.