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Isolated C-terminal tail of FGF23 alleviates hypophosphatemia by inhibiting FGF23-FGFR-Klotho complex formation Regina Goetza,1, Yuji Nakadab,1, Ming Chang Huc,1, Hiroshi Kurosub, Lei Wangb, Teruyo Nakatanid, Mingjun Shic, Anna V. Eliseenkovaa, Mohammed S. Razzaqued,e, Orson W. Moec,2, Makoto Kuro-ob,2, and Moosa Mohammadia,2 aDepartment of Pharmacology, New York University School of Medicine, New York, NY 10016; bDepartment of Pathology and cDepartment of Internal Medicine, Department of Physiology, and Charles and Jane Pak Center of Mineral Metabolism, University of Texas Southwestern Medical Center, Dallas,TX 75390; dDepartment of Oral and Developmental Biology, Harvard School of Dental Medicine, Boston, MA 02115; and eDepartment of Pathology, Riyadh Armed Forces Hospital, Riyadh, Kingdom of Saudi Arabia Edited by John T. Potts, Jr., Massachusetts General Hospital, Charlestown, MA, and approved November 5, 2009 (received for review February 23, 2009) Fibroblast growth factor (FGF) 23 inhibits renal phosphate reabsorp- and complications include “overshoot” hyperphosphatemia, hy- tion by activating FGF receptor (FGFR) 1c in a Klotho-dependent pocalcemia, and metastatic calcification (3, 7). In addition, pa- fashion. The phosphaturic activity of FGF23 is abrogated by proteo- renteral regimens are not practical for chronic disorders. Most lytic cleavage at the RXXR motif that lies at the boundary between importantly, replacement therapy alone is never adequate when the FGF core homology domain and the 72-residue-long C-terminal there is significant renal phosphate wasting. Therefore, novel tail of FGF23. Here, we show that the soluble ectodomains of FGFR1c strategies for the treatment of hypophosphatemia are needed. and Klotho are sufficient to form a ternary complex with FGF23 in Fibroblast growth factor (FGF) 23, originally identified as the vitro. The C-terminal tail of FGF23 mediates binding of FGF23 to a de mutated gene in patients with the phosphate wasting disorder novo site generated at the composite FGFR1c-Klotho interface. autosomal dominant hypophosphatemic rickets (ADHR) (11) Consistent with this finding, the isolated 72-residue-long C-terminal and as the causative factor of tumor-induced osteomalacia (12, tail of FGF23 impairs FGF23 signaling by competing with full-length 13), is an endocrine regulator of phosphate homeostasis. FGF23 ligand for binding to the binary FGFR-Klotho complex. Injection of inhibits reabsorption of phosphate in the renal proximal tubule the FGF23 C-terminal tail peptide into healthy rats inhibits renal by decreasing the abundance of the type II sodium-dependent phosphate excretion and induces hyperphosphatemia. In a mouse phosphate transporters NaPi-2A and NaPi-2C in the apical brush model of renal phosphate wasting attributable to high FGF23, the border membrane (14–16). FGF23 activity is regulated by a FGF23 C-terminal peptide reduces phosphate excretion, leading to an proteolytic cleavage at the 176RXXR179 motif, located at the increase in serum phosphate concentration. Our data indicate that boundary between the FGF core homology domain and the 72- proteolytic cleavage at the RXXR motif abrogates FGF23 activity by a residue-long C-terminal tail of FGF23 (17, 18). The proteolytic dual mechanism: by removing the binding site for the binary FGFR- cleavage generates an inactive N-terminal fragment (Y25 to Klotho complex that resides in the C-terminal region of FGF23, and R179, the FGF core homology domain) and a C-terminal frag- by generating an endogenous inhibitor of FGF23. We propose that ment (S180 to I251) (19) (Fig. 1A). Missense mutations of either peptides derived from the C-terminal tail of FGF23 or peptidomi- R176 or R179 of the 176RXXR179 motif inhibit this proteolytic metics and small-molecule organomimetics of the C-terminal tail can cleavage (17, 18) and lead to accumulation of full-length, bio- be used as therapeutics to treat renal phosphate wasting. active FGF23, thereby inducing renal phosphate wasting in ADHR patients (11). To exert its phosphaturic activity, FGF23 FGF23 antagonist | endogenous inhibitor of FGF23 | FGF23 C-terminal requires Klotho as an obligate coreceptor (20, 21), a protein first peptide | binary FGF receptor 1c-Klotho complex | composite FGF receptor described as an aging suppressor (22). The dependency on PHARMACOLOGY 1c-Klotho interface Klotho compensates for the poor binding affinity of FGF23 to both FGF receptor (FGFR) and heparan sulfate (19). Klotho norganic phosphate plays a key role in a myriad of biological constitutively binds the cognate FGFRs of FGF23, and the bi- Iprocesses, including bone mineralization, reversible regulation nary FGFR-Klotho complexes exhibit enhanced binding affinity of protein function by phosphorylation, and production of adeno- for FGF23 (20, 21). sine triphosphate. Plasma levels of phosphate range between 2.2 We have recently shown that in contrast to full-length FGF23, and 4.9 mg/dL (1, 2), and are primarily regulated by modifying renal the inactive N-terminal fragment of proteolytic cleavage fails to tubular reabsorption. Because of phosphate’s pleiotropic activity, coimmunoprecipitate with binary FGFR-Klotho complexes, imbalances in phosphate homeostasis adversely affect essentially suggesting that the 72-residue-long C-terminal tail of FGF23 every major tissue/organ. Hypophosphatemia is a common clinical mediates binding of FGF23 to its cognate FGFR-Klotho com- condition with an incidence ranging from 0.2% to 3.1% in all plexes (19). Here, we demonstrate that the C-terminal tail of hospital admissions to 21.5% to 80% in specific subgroups of hospitalized patients (3, 4). Clinical manifestations of hypo- phosphatemia include respiratory failure, cardiac arrhythmia, he- Author contributions: R.G., Y.N., M.C.H., H.K., L.W., M.S.R., O.W.M., M.K., and M.M. molysis, rhabdomyolysis, seizures, and coma acutely and myalgia designed research; R.G., Y.N., M.C.H., H.K., L.W., T.N., and M.S. performed research; R.G. and A.V.E. contributed new reagents/analytic tools; R.G., Y.N., M.C.H., H.K., L.W., T.N., M. and osteomalacia chronically (3). Hypophosphatemia originates S., M.S.R., O.W.M., M.K., and M.M. analyzed data; and R.G., O.W.M., M.K., and M.M. from diverse pathophysiologic mechanisms, most importantly from wrote the paper. renal phosphate wasting, an inherited or acquired condition in The authors declare no conflict of interest. which renal tubular reabsorption of phosphate is impaired (5, 6). This article is a PNAS Direct Submission. Oral or i.v. administration of inorganic phosphate salts cur- 1R.G., Y.N., and M.C.H. contributed equally to this work. rently is the mainstay for the management of hypophosphatemia. 2To whom correspondence may be addressed. E-mail: [email protected], Oral phosphate therapy requires high doses, which frequently lead [email protected], or [email protected]. to diarrhea or gastric irritation (7). For i.v. phosphate therapy, This article contains supporting information online at www.pnas.org/cgi/content/full/ the response to any given dose is sometimes unpredictable (8–10), 0902006107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0902006107 PNAS | January 5, 2010 | vol. 107 | no. 1 | 407–412 Downloaded by guest on September 30, 2021 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 (KD) was calculated from the saturation binding curve. (C and D) Representative SPR sensorgrams illustrating binding of FGF2328-251 (C) and FGF23180-251 (D) to the binary FGFR1c-Klotho complex. FGF2328-251 and FGF23180-251 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 FGF23180-251 (E)orFGF23180-205 (F) of FGFR1c-Klotho binding to FGF2328-251 immobilized on a biosensor chip. Increasing concentrations of either FGF23180-251 or FGF23180-205 were mixed with a fixed concentration of FGFR1c-Klotho complex, and the mixtures were passed over a FGF23 chip. FGF23 mediates binding of FGF23 to a de novo site at the Klotho bound FGFR1c with high affinity (KD = 72 nM; Fig. 1B). composite FGFR-Klotho interface. We exploit this finding for Because Klotho harbors a high-affinity binding site for FGFR1c, therapeutic purposes and show that the isolated C-terminal tail we reasoned that Klotho might also possess a distinct high-af- of FGF23 can compete with full-length ligand for binding to the finity binding site for FGF23 and promote FGF23-FGFR1c FGFR-Klotho complex, and hence can antagonize the phos- binding by engaging FGF23 and FGFR1c simultaneously. To test phaturic activity of FGF23 in vivo, both in healthy rats and in a this, FGF2328-251 was coupled to a biosensor chip, and increasing mouse model of phosphate wasting disorders. concentrations of Klotho ectodomain were passed over the chip. As shown in Fig. S1C, Klotho bound poorly to FGF2328-251. Results These data demonstrate that the Klotho ectodomain contains a The Ternary FGF23-FGFR1c-Klotho Complex Can Be Reconstituted in high-affinity binding site for FGFR1c but not for FGF23. Solution by Using Recombinant Soluble Ectodomains of FGFR1c and Next, we measured binding of FGF23 to FGFR1c by injecting Klotho. To understand how FGF23, FGFR, and Klotho interact increasing concentrations of FGFR1c over the FGF23 chip. As to form a ternary complex, we decided to reconstitute the ternary shown in Fig.

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