Isolated C-terminal tail of FGF23 alleviates hypophosphatemia by inhibiting FGF23-FGFR-Klotho complex formation

Abstract

Fibroblast growth factor (FGF) 23 inhibits renal phosphate reabsorption by activating FGF receptor (FGFR) 1c in a Klotho-dependent fashion. The phosphaturic activity of FGF23 is abrogated by proteolytic cleavage at the RXXR motif that lies at the boundary between the FGF core homology domain and the 72-residue-long C-terminal tail of FGF23. Here, we show that the soluble ectodomains of FGFR1c and Klotho are sufficient to form a ternary complex with FGF23 in vitro. The C-terminal tail of FGF23 mediates binding of FGF23 to a de novo site generated at the composite FGFR1c-Klotho interface. Consistent with this finding, the isolated 72-residue-long C-terminal tail of FGF23 impairs FGF23 signaling by competing with full-length ligand for binding to the binary FGFR-Klotho complex. Injection of the FGF23 C-terminal tail peptide into healthy rats inhibits renal phosphate excretion and induces hyperphosphatemia. In a mouse model of renal phosphate wasting attributable to high FGF23, the FGF23 C-terminal peptide reduces phosphate excretion, leading to an increase in serum phosphate concentration. Our data indicate that proteolytic cleavage at the RXXR motif abrogates FGF23 activity by a dual mechanism: by removing the binding site for the binary FGFR-Klotho complex that resides in the C-terminal region of FGF23, and by generating an endogenous inhibitor of FGF23. We propose that peptides derived from the C-terminal tail of FGF23 or peptidomimetics and small-molecule organomimetics of the C-terminal tail can be used as therapeutics to treat renal phosphate wasting.

Fig. 1.

FGF23 C-terminal tail mediates binding of FGF23 to the binary FGFR-Klotho complex, and the isolated FGF23 C-terminal tail peptide competes with FGF23 for binding to FGFR-Klotho. (A) FGF23 proteins and peptides used in this study. Amino acid boundaries of each protein/peptide are labeled with residue letter and number. The FGF23 core region is shaded gray, and the position of the proteolytic cleavage site RXXR is indicated. (B) Representative SPR sensorgram of FGFR1c binding to Klotho, and fitted saturation binding curve. Klotho ectodomain was immobilized on a biosensor chip, and increasing concentrations of FGFR1c ectodomain were passed over the chip. The dissociation constant (K_D) was calculated from the saturation binding curve. (C and D) Representative SPR sensorgrams illustrating binding of FGF23^28-251 (C) and FGF23^180-251 (D) to the binary FGFR1c-Klotho complex. FGF23^28-251 and FGF23^180-251 were immobilized on a biosensor chip, and increasing concentrations of FGFR1c-Klotho complex were passed over the chip. (E and F) Representative SPR sensorgrams illustrating inhibition by FGF23^180-251 (E) or FGF23^180-205 (F) of FGFR1c-Klotho binding to FGF23^28-251 immobilized on a biosensor chip. Increasing concentrations of either FGF23^180-251 or FGF23^180-205 were mixed with a fixed concentration of FGFR1c-Klotho complex, and the mixtures were passed over a FGF23 chip.

Fig. 2.

FGF23 C-terminal tail peptide inhibits tyrosine phosphorylation of FRS2α and downstream activation of MAP kinase cascade induced by FGF23. Shown is an immunoblot analysis for phosphorylation of FRS2α (pFRS2α) and 44/42 MAP kinase (p44/42 MAPK) in a HEK293 Klotho cell line, which had been stimulated with FGF proteins/peptide as denoted. Numbers above the lanes give the amounts of protein/peptide added in nanomolar. To control for equal sample loading, the protein blots were probed with an antibody to Klotho.

Fig. 3.

FGF23 C-terminal tail peptide antagonizes phosphaturic activity of FGF23 in vivo. FGF23^28-251 (0.1 μg·kg body weight⁻¹) or FGF23^180-251 (0.1 μg·kg body weight⁻¹) were injected i.v. into Sprague–Dawley rats. Serum and urine parameters were measured and calculated before and 3 h after injection. FE P_i, fractional excretion of phosphate; U_PiV, phosphate excretion rate; Cl_Cr, creatinine clearance.

Fig. 4.

FGF23 C-terminal peptides alleviate renal phosphate wasting in Hyp mice. FGF23^180-251 (1 mg), FGF23^180-205 (860 μg), or vehicle were injected i.p. into Hyp mice. Urine phosphate (urinary P_i) and creatinine levels and serum phosphate levels (serum P_i) were measured before and at the indicated time points after the injection. Urinary P_i of Hyp mice treated with FGF23^180-205 was not determined (ND). Bars and error bars are mean + SE. *, P < 0.05 by ANOVA.