Biochemical analysis of pathogenic ligand-dependent FGFR2 mutations suggests distinct pathophysiological mechanisms for craniofacial and limb abnormalities - PubMed (original) (raw)

. 2004 Oct 1;13(19):2313-24.

doi: 10.1093/hmg/ddh235. Epub 2004 Jul 28.

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Biochemical analysis of pathogenic ligand-dependent FGFR2 mutations suggests distinct pathophysiological mechanisms for craniofacial and limb abnormalities

Omar A Ibrahimi et al. Hum Mol Genet. 2004.

Abstract

Gain-of-function missense mutations in FGF receptor 2 (FGFR2) are responsible for a variety of craniosynostosis syndromes including Apert syndrome (AS), Pfeiffer syndrome (PS) and Crouzon syndrome (CS). Unlike the majority of FGFR2 mutations, S252W and P253R AS mutations and a D321A PS mutation retain ligand-dependency and are also associated with severe limb pathology. In addition, a recently identified ligand-dependent S252L/A315S double mutation in FGFR2 was shown to cause syndactyly in the absence of craniosynostosis. Here, we analyze the effect of the canonical AS mutations, the D321A PS mutation and the S252L/A315S double mutation on FGFR2 ligand binding affinity and specificity using surface plasmon resonance. Both AS mutations and the D321A PS mutation, but not the S252L/A315S double mutation, increase the binding affinity of FGFR2c to multiple FGFs expressed in the cranial suture. Additionally, all four pathogenic mutations also violate FGFR2c ligand binding specificity and enable this receptor to bind FGF10. Based on our data, we propose that an increase in mutant FGFR2c binding to multiple FGFs results in craniosynostosis, whereas binding of mutant FGFR2c to FGF10 results in severe limb pathology. Structural and biophysical analysis shows that AS mutations in FGFR2b also enhance and violate FGFR2b ligand binding affinity and specificity, respectively. We suggest that elevated AS mutant FGFR2b signaling may account for the dermatological manifestations of AS.

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Figures

Figure 1

Figure 1

Mapping of pathogenic FGFR2 mutations. D1, D2, D3 represent immunoglobulin (Ig)-like domain 1, 2 and 3; S represents the signal peptide; AB represents the acid box; TM represents the transmembrane helix; TK1 and TK2 represent the split-kinase domain, which is interrupted by the kinase insert; J represents the juxtamembrane region. The heparin-binding site (HBS) in D2 is marked by a thickened black line. The alternatively spliced region (encoded by either exon ‘b’ or ‘c’ in a tissue specific manner) in D3 is represented by a thickened gray line. The location of AS S252W and P253R mutations, the D321A PS mutation and the S252L/A315S double mutation are indicated by arrows. The D321A PS mutation and the S252L/A315S double mutation manifest only in the ‘c’ isoform of FGFR2.

Figure 2

Figure 2

Surface plasmon resonance analysis of wild-type and mutant FGFR2c-FGF interactions. Sensorgrams of representative analyte injections of wild-type, S252W, P253R, D321A and S252L/A315S FGFR2c binding to (A) FGF2 (at 12.5 nM), (B) FGF4 (at 6.25 nM), (C) FGF5 (at 200 nM), (D) FGF10 (at 400 nM), (E) FGF16 (at 200 nM), (F) FGF18 (at 400 nM), (G) FGF19 (at 400 nM), and (H) FGF23 (at 400 nM). Analyte injections are colored as follows: wild-type FGFR2c in black, S252W FGFR2c in blue, P253R FGFR2c in green, D321A FGFR2c in yellow and S252L/A315S in red. The biosensor chip response is indicated on the _y_-axis (ΔRU) as a function of time (_x_-axis) at 25°C. Kinetic data are summarized in Table 1.

Figure 3

Figure 3

Structural and biophysical analysis of wild-type and AS mutant FGFR2b-FGF interactions. (A) Gain-of-function contact in the S252W FGFR2b-FGF10 complex. D2 and D3 of FGFR2b are shown in green and cyan, respectively. The alternatively spliced region of D3 is colored purple. The D2–D3 linker is colored gray. FGF10 is shown in orange. (Right) View of whole structure in the exact orientation as the detailed view is shown, with the region of interest boxed. (B) Gain-of-function hydrogen bonds in the P253R FGFR2b-FGF1 complex. Coloring is as in (A). Dotted lines represent hydrogen bonds and the hydrogen-bonding distances are indicated. (Right) View of whole structure in the exact orientation as the detailed view is shown, with the region of interest boxed. (C, D, E and F) Sensorgrams of representative analyte injections of wild-type and AS mutant FGFR2b binding to (C) FGF10 (at 100 nM), (D) FGF1 (at 400 nM), (E) FGF2 (at 400 nM), and (F) FGF8 (at 800 nM). Analyte injections are colored as follows: wild-type FGFR2b in black, S252W FGFR2b in blue, and P253R FGFR2b in green. The biosensor chip response is indicated on the _y_-axis (ΔRU) as a function of time (_x_-axis) at 25°C. Kinetic data are summarized in Table 2.

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