Conversion of a paracrine fibroblast growth factor into an endocrine fibroblast growth factor

Abstract

FGFs 19, 21, and 23 are hormones that regulate in a Klotho co-receptor-dependent fashion major metabolic processes such as glucose and lipid metabolism (FGF21) and phosphate and vitamin D homeostasis (FGF23). The role of heparan sulfate glycosaminoglycan in the formation of the cell surface signaling complex of endocrine FGFs has remained unclear. Here we show that heparan sulfate is not a component of the signal transduction unit of FGF19 and FGF23. In support of our model, we convert a paracrine FGF into an endocrine ligand by diminishing heparan sulfate-binding affinity of the paracrine FGF and substituting its C-terminal tail for that of an endocrine FGF containing the Klotho co-receptor-binding site to home the ligand into the target tissue. In addition to serving as a proof of concept, the ligand conversion provides a novel strategy for engineering endocrine FGF-like molecules for the treatment of metabolic disorders, including global epidemics such as type 2 diabetes and obesity.

FIGURE 1.

Side by side comparison of the HS-binding site of FGF2, FGF19, and FGF23 and working model of the endocrine FGF signaling complex. A, interactions of FGF2 (shown as cartoon representation) with a heparin hexasaccharide (shown as sticks) as observed in the crystal structure of the 2:2 FGF2-FGFR1c dimer (Protein Data Bank code 1FQ9) (31). The heparin hexasaccharide consists of three disaccharide units of 1→4-linked N-sulfated-6-O-sulfated d-glucosamine and 2-O-sulfated l-iduronic acid. Note that the heparin hexasaccharide interacts with both side chain and backbone atoms of residues in the HS-binding site of FGF2. Dashed lines denote hydrogen bonds. Lys¹²⁸, Arg¹²⁹, and Lys¹³⁴, which make the majority of hydrogen bonds with the heparin hexasaccharide, are boxed. The β-strand nomenclature follows the original FGF1 and FGF2 crystal structures (–28). Please note that compared with the prototypical β-trefoil fold seen in soybean trypsin inhibitor (Protein Data Bank code 1TIE) (64) and interleukin 1β (Protein Data Bank code 1I1B) (65), the β10-β11 strand pairing in FGF2 and other paracrine FGFs is less well defined. B and C, cartoon representation of the crystal structures of FGF19 (Protein Data Bank code 2P23) (29) (B) and FGF23 (Protein Data Bank code 2P39) (29) (C) shown in the same orientation as the FGF2 structure in A. The side chains of residues that map to the corresponding HS-binding sites of these ligands are shown as sticks. Residues selected for mutagenesis to knock out residual HS binding in FGF19 and FGF23 are boxed. NT and CT indicate N and C termini of the FGFs. D, schematic of the endocrine FGF-FGFR-Klotho signal transduction unit. For comparison, a schematic of the paracrine FGF-FGFR-HS signaling unit is shown that was made based on the crystal structure of the 2:2:2 FGF2-FGFR1c-HS complex (Protein Data Bank code 1FQ9) (31). HS engages both paracrine FGF and receptor to enhance binding of FGF to its primary and secondary receptors thus promoting receptor dimerization. A question mark denotes whether or not HS is also a component of the endocrine FGF signaling complex.

FIGURE 2.

Sequence alignment of the endocrine FGFs. The amino acid sequences of the mature human FGF19, FGF21, and FGF23 ligands are aligned. Also included in the alignment is the human sequence of FGF2, a prototypical paracrine FGF, which was used in this study to convert into endocrine FGF ligands. Residue numbers are in parentheses to the left of the alignment. Secondary structure elements are labeled, and residues containing these elements for known secondary structures are boxed. Gaps (dashes) were introduced to optimize the sequence alignment. The β-trefoil core domain for known FGF crystal structures is shaded gray. Dark gray bars on top of the alignment indicate the location of the HS-binding regions. HS-binding residues selected for mutagenesis are shaded dark gray.

FIGURE 3.

Knock-out of residual heparin binding in FGF19 and FGF23 by site-directed mutagenesis. A, overlay of SPR sensorgrams illustrating heparin binding of FGF2, FGF19, FGF21, and FGF23 (left panel) and zoomed-in view of the binding responses for FGF19-, FGF21-, and FGF23-heparin interactions (right panel). Heparin was immobilized on a biosensor chip, and 400 nm of FGF2, FGF19, FGF21, or FGF23 was passed over the chip. Note that FGF19, FGF21, and FGF23 exhibit measurable, residual heparin binding and that differences in heparin binding exist between these three endocrine FGFs. B and C, SPR sensorgrams illustrating binding of FGF19 to heparin (B) and lack of interaction between the FGF19^K149A mutant and heparin (C). Heparin was immobilized on a biosensor chip, and increasing concentrations of FGF19 were passed over the chip. Thereafter, FGF19^K149A was injected over the heparin chip at the highest concentration tested for the wild-type ligand. D and E, SPR sensorgrams illustrating binding of FGF23 to heparin (D) and lack of interaction between the FGF23^R140A/R143A mutant and heparin (E). Heparin was immobilized on a biosensor chip, and increasing concentrations of FGF23 were passed over the chip. FGF23^R140A/R143A was then injected over the heparin chip at the highest concentration tested for the wild-type ligand.

FIGURE 4.

HS is dispensable for the metabolic activity of FGF19 and FGF23. A, immunoblot analysis of phosphorylation of FRS2α (pFRS2α) and 44/42 MAP kinase (p44/42 MAPK) in H4IIE hepatoma cells following stimulation with the FGF19^K149A mutant or wild-type FGF19. The numbers above the lanes give the amounts of protein added in ng ml⁻¹. Total 44/42 MAPK protein expression was used as a loading control. B, immunoblot analysis of phosphorylation of FRS2α (pFRS2α) and 44/42 MAP kinase (p44/42 MAPK) in a HEK293-αKlotho cell line following stimulation with the FGF23^R140A/R143A mutant or wild-type FGF23. The numbers above the lanes give the amounts of protein added in ng ml⁻¹. Total 44/42 MAPK and αKlotho protein expression were used as loading controls. C, quantitative analysis of CYP7A1 and CYP8B1 mRNA expression in liver tissue from mice treated with FGF19^K149A, FGF19, or vehicle. 1 mg of protein/kg of body weight was given. The data are presented as the means ± S.E. ***, p < 0.001 by Student's t test. D, analysis of serum phosphate concentrations (serum P_i) in mice before and 8 h after intraperitoneal injection of FGF23^R140A/R143A, FGF23, or vehicle. 0.29 mg of protein/kg of body weight was given. The data are presented as the means ± S.E. *, p < 0.05; **, p < 0.01 by analysis of variance.

FIGURE 5.

Design of the conversion of FGF2 into an endocrine ligand. A, schematic of human FGF2, FGF21, FGF23, and engineered FGF2-FGF21 and FGF2-FGF23 chimeras. Amino acid boundaries of each ligand and of each component of the chimeras are labeled with residue letter and number. The β-trefoil core domain for the known ligand crystal structures is shaded gray. HS-binding residues mutated in the FGF2 portion of chimeras are labeled with residue letter and number. Also labeled are the arginine residues of the proteolytic cleavage site in the C-terminal region of FGF23 that were mutated to glutamine in both FGF23 and the FGF2-FGF23 chimeras. B and C, overlays of SPR sensorgrams illustrating binding of FGF2^WTcore-FGF21^C-tail (B) and FGF2^ΔHBScore-FGF21^C-tail (C) to heparin and fitted saturation binding curves. Heparin was immobilized on a biosensor chip, and increasing concentrations of FGF2^WTcore-FGF21^C-tail or FGF2^ΔHBScore-FGF21^C-tail were passed over the chip. Dissociation constants (K_D values) were derived from the saturation binding curves. D and E, overlays of SPR sensorgrams illustrating binding of FGF2^WTcore-FGF23^C-tail (D) and FGF2^ΔHBScore-FGF23^C-tail (E) to heparin. Increasing concentrations of FGF2^WTcore-FGF23^C-tail or FGF2^ΔHBScore-FGF23^C-tail were passed over a chip containing immobilized heparin. F and G, immunoblot analysis for Egr1 expression in HEK293 cells following stimulation with chimeras or native FGFs as denoted. The numbers above the lanes give the amounts of protein added in nanomolar. GAPDH protein expression was used as a loading control.

FIGURE 6.

FGF2^ΔHBScore-FGF23^C-tail chimera exhibits FGF23-like activity. A and B, overlays of SPR sensorgrams illustrating inhibition by FGF2^ΔHBScore-FGF23^C-tail (A) or FGF23 (B) of αKlotho-FGFR1c binding to FGF23 immobilized on a biosensor chip. Increasing concentrations of FGF2^ΔHBScore-FGF23^C-tail or FGF23 were mixed with a fixed concentration of αKlotho-FGFR1c complex, and the mixtures were passed over a FGF23 chip. C, overlay of SPR sensorgrams illustrating failure of FGF2 to inhibit αKlotho-FGFR1c binding to FGF23. FGF2 and αKlotho-FGFR1c complex were mixed at a molar ratio of 15:1, and the mixture was passed over a biosensor chip containing immobilized FGF23. D and E, overlays of SPR sensorgrams illustrating no inhibition by FGF2^ΔHBScore-FGF23^C-tail (D) or FGF23 (E) of βKlotho-FGFR1c binding to FGF21. FGF2^ΔHBScore-FGF23^C-tail or FGF23 were mixed with βKlotho-FGFR1c complex at a molar ratio of 10:1, and the mixtures were passed over a biosensor chip containing immobilized FGF21. F, analysis of serum phosphate concentrations (serum P_i) in mice before and 8 h after intraperitoneal injection of FGF2^ΔHBScore-FGF23^C-tail, FGF2^WTcore-FGF23^C-tail, FGF23, or vehicle. Wild-type mice and αKlotho knock-out mice were given 0.21 and 0.51 mg of protein, respectively, per kg of body weight. The data are presented as the means ± S.E. **, p < 0.01; ***, p < 0.001 by analysis of variance. G, quantitative analysis of CYP27B1 mRNA expression in renal tissue from mice injected with FGF2^ΔHBScore-FGF23^C-tail, FGF2^WTcore-FGF23^C-tail, FGF23, or vehicle. 0.21 mg of protein/kg of body weight was injected. The data are presented as the means ± S.E. ***, p < 0.001 by analysis of variance.

FIGURE 7.

FGF2^ΔHBScore-FGF21^C-tail chimera exhibits FGF21-like activity. A and B, overlays of SPR sensorgrams illustrating inhibition by FGF2^ΔHBScore-FGF21^C-tail (A) or FGF21 (B) of βKlotho-FGFR1c binding to FGF21 immobilized on a biosensor chip. Increasing concentrations of FGF2^ΔHBScore-FGF21^C-tail or FGF21 were mixed with a fixed concentration of βKlotho-FGFR1c complex, and the mixtures were passed over a FGF21 chip. C, overlay of SPR sensorgrams illustrating failure of FGF2 to inhibit βKlotho-FGFR1c binding to FGF21. FGF2 and βKlotho-FGFR1c complex were mixed at a molar ratio of 15:1, and the mixture was passed over a biosensor chip containing immobilized FGF21. D and E, overlays of SPR sensorgrams illustrating no inhibition by FGF2^ΔHBScore-FGF21^C-tail (D) or FGF21 (E) of αKlotho-FGFR1c binding to FGF23. FGF2^ΔHBScore-FGF21^C-tail or FGF21 were mixed with αKlotho-FGFR1c complex at a molar ratio of 10:1, and the mixtures were passed over a biosensor chip containing immobilized FGF23. F, immunoblot analysis for Egr1 expression in HEK293-βKlotho cells stimulated with FGF2^ΔHBScore-FGF21^C-tail or FGF21. Numbers above the lanes give the amounts of protein added in ng ml⁻¹. GAPDH protein expression was used as a loading control. Note that the FGF2^ΔHBScore-FGF21^C-tail chimera is more potent than native FGF21 at inducing Egr1 expression, suggesting that the chimera has agonistic properties. This is expected because the core domain of FGF2 has inherently greater binding affinity for FGFR than the core domain of FGF21 (see Fig. 8, A and C). G, analysis of blood glucose concentrations in mice before and at the indicated time points after intraperitoneal injection of insulin alone, insulin plus FGF2^ΔHBScore-FGF21^C-tail chimera, insulin plus FGF21, or vehicle alone. 0.5 units of insulin/kg of body weight and 0.3 mg of FGF21 ligand/kg of body weight were injected. Blood glucose concentrations are expressed as percentages of preinjection values. The data are presented as the means ± S.E.

FIGURE 8.

Endocrine FGFs have low binding affinity for FGFR1c compared with FGF2. A–D, overlays of SPR sensorgrams illustrating binding of FGFR1c to FGF2 (A), FGF19 (B), FGF21 (C), and FGF23 (D), and fitted saturation binding curves. Increasing concentrations of FGFR1c ligand-binding domain were passed over a biosensor chip containing immobilized FGF2, FGF19, FGF21, or FGF23. E, overlay of SPR sensorgrams illustrating binding of αKlotho-FGFR1c complex to FGF23. Increasing concentrations of αKlotho-FGFR1c complex were passed over a biosensor chip containing immobilized FGF23. F, overlay of SPR sensorgrams showing a lack of interaction between the C-terminal tail peptide of FGF23 and FGFR1c. FGF23^C-tail was immobilized on a biosensor chip, and increasing concentrations of FGFR1c ligand-binding domain were passed over the chip. Dissociation constants (K_D values) given in A–E were derived from the saturation binding curves.