Regulation of Receptor Binding Specificity of FGF9 by an Autoinhibitory Homodimerization.

The epithelial fibroblast growth factor 9 (FGF9) subfamily specifically binds and activates the mesenchymal "c" splice isoform of FGF receptors 1-3 (FGFR1-3) to regulate organogenesis and tissue homeostasis. The unique N and C termini of FGF9 subfamily ligands mediate a reversible homodimerization that occludes major receptor binding sites within the ligand core region. Here we provide compelling X-ray crystallographic, biophysical, and biochemical data showing that homodimerization controls receptor binding specificity of the FGF9 subfamily by keeping the concentration of active FGF9 monomers at a level, which is sufficient for a normal FGFR "c" isoform binding/signaling, but is insufficient for an illegitimate FGFR "b" isoform binding/signaling. We show that deletion of the N terminus or alanine substitutions in the C terminus of FGF9 skews the delicate ligand equilibrium toward active FGF9 monomers causing off-target binding and activation of FGFR b isoforms. Our study is the first to implicate ligand homodimerization in the regulation of ligand-receptor specificity.

The alternatively spliced acid box region plays a key role in FGF receptor autoinhibition.

Uncontrolled fibroblast growth factor (FGF) signaling can lead to human malignancies necessitating multiple layers of self-regulatory control mechanisms. Fibroblast growth factor receptor (FGFR) autoinhibition mediated by the alternatively spliced immunoglobulin (Ig) domain 1 (D1) and the acid box (AB)-containing linker between D1 and Ig domain 2 (D2) serves as the first line of defense to minimize inadvertent FGF signaling. In this report, nuclear magnetic resonance and surface plasmon resonance spectroscopy are used to demonstrate that the AB subregion of FGFR electrostatically engages the heparan sulfate (HS)-binding site on the D2 domain in cis to directly suppress HS-binding affinity of FGFR. Furthermore, the cis electrostatic interaction sterically autoinhibits ligand-binding affinity of FGFR because of the close proximity of HS-binding and primary ligand-binding sites on the D2 domain. These data, together with the strong amino acid sequence conservation of the AB subregion among FGFR orthologs, highlight the universal role of the AB subregion in FGFR autoinhibition.

Plasticity in interactions of fibroblast growth factor 1 (FGF1) N terminus with FGF receptors underlies promiscuity of FGF1.

Tissue-specific alternative splicing in the second half of Ig-like domain 3 (D3) of fibroblast growth factor receptors 1-3 (FGFR1 to -3) generates epithelial FGFR1b-FGFR3b and mesenchymal FGFR1c-FGFR3c splice isoforms. This splicing event establishes a selectivity filter to restrict the ligand binding specificity of FGFRb and FGFRc isoforms to mesenchymally and epithelially derived fibroblast growth factors (FGFs), respectively. FGF1 is termed the "universal FGFR ligand" because it overrides this specificity barrier. To elucidate the molecular basis for FGF1 cross-reactivity with the "b" and "c" splice isoforms of FGFRs, we determined the first crystal structure of FGF1 in complex with an FGFRb isoform, FGFR2b, at 2.1 Å resolution. Comparison of the FGF1-FGFR2b structure with the three previously published FGF1-FGFRc structures reveals that plasticity in the interactions of the N-terminal region of FGF1 with FGFR D3 is the main determinant of FGF1 cross-reactivity with both isoforms of FGFRs. In support of our structural data, we demonstrate that substitution of three N-terminal residues (Gly-19, His-25, and Phe-26) of FGF2 (a ligand that does not bind FGFR2b) for the corresponding residues of FGF1 (Phe-16, Asn-22, and Tyr-23) enables the FGF2 triple mutant to bind and activate FGFR2b. These findings taken together with our previous structural data on receptor binding specificity of FGF2, FGF8, and FGF10 conclusively show that sequence divergence at the N termini of FGFs is the primary regulator of the receptor binding specificity and promiscuity of FGFs.

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

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.

Differential interactions of FGFs with heparan sulfate control gradient formation and branching morphogenesis.

The developmental activities of morphogens depend on the gradients that they form in the extracellular matrix. Here, we show that differences in the binding of fibroblast growth factor 7 (FGF7) and FGF10 to heparan sulfate (HS) underlie the formation of different gradients that dictate distinct activities during branching morphogenesis. Reducing the binding affinity of FGF10 for HS by mutating a single residue in its HS-binding pocket converted FGF10 into a functional mimic of FGF7 with respect to gradient formation and regulation of branching morphogenesis. In particular, the mutant form of FGF10 caused lacrimal and salivary gland epithelium buds to branch rather than to elongate. In contrast, mutations that reduced the affinity of the FGF10 for its receptor affected the extent, but not the nature, of the response. Our data may provide a general model for understanding how binding to HS regulates other morphogenetic gradients.

Compositional analysis of heparin/heparan sulfate interacting with fibroblast growth factor.fibroblast growth factor receptor complexes.

Heparan sulfate (HS) proteoglycans (PGs) interact with a number of extracellular signaling proteins, thereby playing an essential role in the regulation of many physiological processes. One major function of HS is to interact with fibroblast growth factors (FGFs) and their receptors (FGFRs) and form FGF.HS.FGFR signaling complexes. Past studies primarily examined the selectivity of HS for FGF or FGFR. In this report, we used a new strategy to study the structural specificity of HS binding to 10 different FGF.FGFR complexes. Oligosaccharide libraries prepared from heparin, 6-desulfated heparin, and HS were used for the interaction studies by solution competition surface plasmon resonance (SPR) and filter trapping assays. Specific oligosaccharides binding to FGF.FGFR complexes were subjected to polyacrylamide gel electrophoresis (PAGE) analysis and disaccharide compositional analysis using liquid chromatography and mass spectrometry. The competition SPR studies using sized oligosaccharide mixtures showed that binding of each of the tested FGFs or FGF.FGFR complexes to heparin immobilized to an SPR chip was size-dependent. The 6-desulfated heparin oligosaccharides exhibited a reduced level of inhibition of FGF and FGF.FGFR complex binding to heparin in the competition experiments. Heparin and the 6-desulfated heparin exhibited higher levels of inhibition of the FGF.FGFR complex binding to heparin than of FGF binding to heparin. In the filter trapping experiments, PAGE analysis showed different affinities between the FGF.FGFR complexes and oligosaccharides. Disaccharide analysis showed that HS disaccharides with a degree of polymerization of 10 (dp10) had high binding selectivity, while dp10 heparin and dp10 6-desulfated heparin showed reduced or no selectivity for the different FGF.FGFR complexes tested.

Homodimerization controls the fibroblast growth factor 9 subfamily's receptor binding and heparan sulfate-dependent diffusion in the extracellular matrix.

Uncontrolled fibroblast growth factor (FGF) signaling can lead to human diseases, necessitating multiple layers of self-regulatory control mechanisms to keep its activity in check. Herein, we demonstrate that FGF9 and FGF20 ligands undergo a reversible homodimerization, occluding their key receptor binding sites. To test the role of dimerization in ligand autoinhibition, we introduced structure-based mutations into the dimer interfaces of FGF9 and FGF20. The mutations weakened the ability of the ligands to dimerize, effectively increasing the concentrations of monomeric ligands capable of binding and activating their cognate FGF receptor in vitro and in living cells. Interestingly, the monomeric ligands exhibit reduced heparin binding, resulting in their increased radii of heparan sulfate-dependent diffusion and biologic action, as evidenced by the wider dilation area of ex vivo lung cultures in response to implanted mutant FGF9-loaded beads. Hence, our data demonstrate that homodimerization autoregulates FGF9 and FGF20's receptor binding and concentration gradients in the extracellular matrix. Our study is the first to implicate ligand dimerization as an autoregulatory mechanism for growth factor bioactivity and sets the stage for engineering modified FGF9 subfamily ligands, with desired activity for use in both basic and translational research.

Crystal structure of a fibroblast growth factor homologous factor (FHF) defines a conserved surface on FHFs for binding and modulation of voltage-gated sodium channels.

Voltage-gated sodium channels (Nav) produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. Fibroblast growth factor homologous factors (FHFs) bind to the intracellular C-terminal region of the Nav alpha subunit to modulate fast inactivation of the channel. In this study we solved the crystal structure of a 149-residue-long fragment of human FHF2A which unveils the structural features of the homology core domain of all 10 human FHF isoforms. Through analysis of crystal packing contacts and site-directed mutagenesis experiments we identified a conserved surface on the FHF core domain that mediates channel binding in vitro and in vivo. Mutations at this channel binding surface impaired the ability of FHFs to co-localize with Navs at the axon initial segment of hippocampal neurons. The mutations also disabled FHF modulation of voltage-dependent fast inactivation of sodium channels in neuronal cells. Based on our data, we propose that FHFs constitute auxiliary subunits for Navs.

Loss-of-function fibroblast growth factor receptor-2 mutations in melanoma.

We report that 10% of melanoma tumors and cell lines harbor mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. These novel mutations include three truncating mutations and 20 missense mutations occurring at evolutionary conserved residues in FGFR2 as well as among all four FGFRs. The mutation spectrum is characteristic of those induced by UV radiation. Mapping of these mutations onto the known crystal structures of FGFR2 followed by in vitro and in vivo studies show that these mutations result in receptor loss of function through several distinct mechanisms, including loss of ligand binding affinity, impaired receptor dimerization, destabilization of the extracellular domains, and reduced kinase activity. To our knowledge, this is the first demonstration of loss-of-function mutations in a class IV receptor tyrosine kinase in cancer. Taken into account with our recent discovery of activating FGFR2 mutations in endometrial cancer, we suggest that FGFR2 may join the list of genes that play context-dependent opposing roles in cancer.

A crystallographic snapshot of tyrosine trans-phosphorylation in action.

Tyrosine trans-phosphorylation is a key event in receptor tyrosine kinase signaling, yet, the structural basis for this process has eluded definition. Here, we present the crystal structure of the FGF receptor 2 kinases caught in the act of trans-phosphorylation of Y769, the major C-terminal phosphorylation site. The structure reveals that enzyme- and substrate-acting kinases engage each other through elaborate and specific interactions not only in the immediate vicinity of Y769 and the enzyme active site, but also in regions that are as much of 18 A away from D626, the catalytic base in the enzyme active site. These interactions lead to an unprecedented level of specificity and precision during the trans-phosphorylation on Y769. Time-resolved mass spectrometry analysis supports the observed mechanism of trans-phosphorylation. Our data provide a molecular framework for understanding the mechanism of action of Kallmann syndrome mutations and the order of trans-phosphorylation reactions in FGFRs. We propose that the salient mechanistic features of Y769 trans-phosphorylation are applicable to trans-phosphorylation of the equivalent major phosphorylation sites in many other RTKs.

Decreased FGF8 signaling causes deficiency of gonadotropin-releasing hormone in humans and mice.

Idiopathic hypogonadotropic hypogonadism (IHH) with anosmia (Kallmann syndrome; KS) or with a normal sense of smell (normosmic IHH; nIHH) are heterogeneous genetic disorders associated with deficiency of gonadotropin-releasing hormone (GnRH). While loss-of-function mutations in FGF receptor 1 (FGFR1) cause human GnRH deficiency, to date no specific ligand for FGFR1 has been identified in GnRH neuron ontogeny. Using a candidate gene approach, we identified 6 missense mutations in FGF8 in IHH probands with variable olfactory phenotypes. These patients exhibited varied degrees of GnRH deficiency, including the rare adult-onset form of hypogonadotropic hypogonadism. Four mutations affected all 4 FGF8 splice isoforms (FGF8a, FGF8b, FGF8e, and FGF8f), while 2 mutations affected FGF8e and FGF8f isoforms only. The mutant FGF8b and FGF8f ligands exhibited decreased biological activity in vitro. Furthermore, mice homozygous for a hypomorphic Fgf8 allele lacked GnRH neurons in the hypothalamus, while heterozygous mice showed substantial decreases in the number of GnRH neurons and hypothalamic GnRH peptide concentration. In conclusion, we identified FGF8 as a gene implicated in GnRH deficiency in both humans and mice and demonstrated an exquisite sensitivity of GnRH neuron development to reductions in FGF8 signaling.

Somatic FGF9 mutations in colorectal and endometrial carcinomas associated with membranous beta-catenin.

We previously described striking molecular features including high frequency of membranous beta-catenin in subsets of familial colon cancers with as yet unknown predisposition. We hypothesized that such tumors might carry mutations in Wnt/beta-catenin target genes. Fibroblast growth factor 9 (FGF9) was an attractive target, as it maps to a common area of loss of heterozygosity (LOH) in colorectal carcinomas on 13q12.11. Here, we report, for the first time, the occurrence of FGF9 mutations in human cancers. We found a total of six distinct FGF9 mutations including one frameshift, four missense, and one nonsense, in 10 (six colorectal and four endometrial) out of 203 tumors and cell lines. The frameshift mutation was detected in five different tumors. Mapping of these mutations onto the crystal structure of FGF9 predicted that they should all lead to loss of function albeit through variable mechanisms. The p.R173K mutation should diminish ligand affinity for heparin/heparan sulfate, the p.V192M, p.D203G, and p.L188YfsX18 (FGF9(Delta205-208)) mutations should negatively impact ligand's interaction with receptor, while p.G84E and p.E142X (FGF9(Delta142-208)) mutations should interfere with ligand folding. Consistent with these structural predictions, the p.V192M, p.D203G, and p.L188YfsX18 (FGF9(Delta205-208)) mutations impaired the ability of ligand to activate mitogen-activated protein kinase (MAPK) cascade in cultured cells expressing FGF receptors. LOH was observed in seven out of nine FGF9 mutant tumors, supporting the predicted loss of function. Interestingly, eight out of 10 (80%) of the FGF9 mutant tumors showed normal membranous beta-catenin expression and the absence of mutation in the beta-catenin gene (CTNNB1). These data suggest that FGF9 plays a role in colorectal and endometrial carcinogenesis.

A molecular brake in the kinase hinge region regulates the activity of receptor tyrosine kinases.

Activating mutations in the tyrosine kinase domain of receptor tyrosine kinases (RTKs) cause cancer and skeletal disorders. Comparison of the crystal structures of unphosphorylated and phosphorylated wild-type FGFR2 kinase domains with those of seven unphosphorylated pathogenic mutants reveals an autoinhibitory "molecular brake" mediated by a triad of residues in the kinase hinge region of all FGFRs. Structural analysis shows that many other RTKs, including PDGFRs, VEGFRs, KIT, CSF1R, FLT3, TEK, and TIE, are also subject to regulation by this brake. Pathogenic mutations activate FGFRs and other RTKs by disengaging the brake either directly or indirectly.

Tissue-specific expression of betaKlotho and fibroblast growth factor (FGF) receptor isoforms determines metabolic activity of FGF19 and FGF21.

The fibroblast growth factor (FGF) 19 subfamily of ligands, FGF19, FGF21, and FGF23, function as hormones that regulate bile acid, fatty acid, glucose, and phosphate metabolism in target organs through activating FGF receptors (FGFR1-4). We demonstrated that Klotho and betaKlotho, homologous single-pass transmembrane proteins that bind to FGFRs, are required for metabolic activity of FGF23 and FGF21, respectively. Here we show that, like FGF21, FGF19 also requires betaKlotho. Both FGF19 and FGF21 can signal through FGFR1-3 bound by betaKlotho and increase glucose uptake in adipocytes expressing FGFR1. Additionally, both FGF19 and FGF21 bind to the betaKlotho-FGFR4 complex; however, only FGF19 signals efficiently through FGFR4. Accordingly, FGF19, but not FGF21, activates FGF signaling in hepatocytes that primarily express FGFR4 and reduces transcription of CYP7A1 that encodes the rate-limiting enzyme for bile acid synthesis. We conclude that the expression of betaKlotho, in combination with particular FGFR isoforms, determines the tissue-specific metabolic activities of FGF19 and FGF21.

BetaKlotho is required for metabolic activity of fibroblast growth factor 21.

Fibroblast growth factor 21 (FGF21) is a liver-derived endocrine factor that stimulates glucose uptake in adipocytes. Here, we show that FGF21 activity depends on betaKlotho, a single-pass transmembrane protein whose expression is induced during differentiation from preadipocytes to adipocytes. BetaKlotho physically interacts with FGF receptors 1c and 4, thereby increasing the ability of these FGF receptors to bind FGF21 and activate the MAP kinase cascade. Knockdown of betaKlotho expression by siRNA in adipocytes diminishes glucose uptake induced by FGF21. Importantly, administration of FGF21 into mice induces MAP kinase phosphorylation in white adipose tissue and not in tissues without betaKlotho expression. Thus, betaKlotho functions as a cofactor essential for FGF21 activity.

Molecular insights into the klotho-dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members.

Unique among fibroblast growth factors (FGFs), FGF19, -21, and -23 act in an endocrine fashion to regulate energy, bile acid, glucose, lipid, phosphate, and vitamin D homeostasis. These FGFs require the presence of Klotho/betaKlotho in their target tissues. Here, we present the crystal structures of FGF19 alone and FGF23 in complex with sucrose octasulfate, a disaccharide chemically related to heparin. The conformation of the heparin-binding region between beta strands 10 and 12 in FGF19 and FGF23 diverges completely from the common conformation adopted by paracrine-acting FGFs. A cleft between this region and the beta1-beta2 loop, the other heparin-binding region, precludes direct interaction between heparin/heparan sulfate and backbone atoms of FGF19/23. This reduces the heparin-binding affinity of these ligands and confers endocrine function. Klotho/betaKlotho have evolved as a compensatory mechanism for the poor ability of heparin/heparan sulfate to promote binding of FGF19, -21, and -23 to their cognate receptors.

Digenic mutations account for variable phenotypes in idiopathic hypogonadotropic hypogonadism.

Idiopathic hypogonadotropic hypogonadism (IHH) due to defects of gonadotropin-releasing hormone (GnRH) secretion and/or action is a developmental disorder of sexual maturation. To date, several single-gene defects have been implicated in the pathogenesis of IHH. However, significant inter- and intrafamilial variability and apparent incomplete penetrance in familial cases of IHH are difficult to reconcile with the model of a single-gene defect. We therefore hypothesized that mutations at different IHH loci interact in some families to modify their phenotypes. To address this issue, we studied 2 families, one with Kallmann syndrome (IHH and anosmia) and another with normosmic IHH, in which a single-gene defect had been identified: a heterozygous FGF receptor 1 (FGFR1) mutation in pedigree 1 and a compound heterozygous gonadotropin-releasing hormone receptor (GNRHR) mutation in pedigree 2, both of which varied markedly in expressivity within and across families. Further candidate gene screening revealed a second heterozygous deletion in the nasal embryonic LHRH factor (NELF) gene in pedigree 1 and an additional heterozygous FGFR1 mutation in pedigree 2 that accounted for the considerable phenotypic variability. Therefore, 2 different gene defects can synergize to produce a more severe phenotype in IHH families than either alone. This genetic model could account for some phenotypic heterogeneity seen in GnRH deficiency.

Mutations in fibroblast growth factor receptor 1 cause both Kallmann syndrome and normosmic idiopathic hypogonadotropic hypogonadism.

Mutations in KAL1 and FGFR1 cause Kallmann syndrome (KS), whereas mutations in the GNRHR and GPR54 genes cause idiopathic hypogonadotropic hypogonadism with normal olfaction (nIHH). Mixed pedigrees containing both KS and nIHH have also been described; however, the genetic cause of these rare cases is unknown. We examined the FGFR1 gene in seven nIHH subjects who either belonged to a mixed pedigree (n = 5) or who had associated midline defects (n = 2). Heterozygous FGFR1 mutations were found in three of seven unrelated nIHH probands with normal MRI of the olfactory system: (i) G237S in an nIHH female and a KS brother; (ii) (P722H and N724K) in an nIHH male missing two teeth and his mother with isolated hyposmia; and (iii) Q680X in a nIHH male with cleft lip/palate and missing teeth, his brother with nIHH, and his father with delayed puberty. We show that these mutations lead to receptor loss-of-function. The Q680X leads to an inactive FGFR1, which lacks a major portion of the tyrosine kinase domain (TKD). The G237S mutation inhibits proper folding of D2 of the FGFR1 and likely leads to the loss of cell-surface expression of FGFR1. In contrast, the (P722H and N724K) double mutation causes structural perturbations in TKD, reducing the catalytic activity of TKD. We conclude that loss-of-function mutations in FGFR1 cause nIHH with normal MRI of the olfactory system. These mutations also account for some of the mixed pedigrees, thus challenging the current idea that KS and nIHH are distinct entities.

Structural basis by which alternative splicing modulates the organizer activity of FGF8 in the brain.

Two of the four human FGF8 splice isoforms, FGF8a and FGF8b, are expressed in the mid-hindbrain region during development. Although the only difference between these isoforms is the presence of an additional 11 amino acids at the N terminus of FGF8b, these isoforms possess remarkably different abilities to pattern the midbrain and anterior hindbrain. To reveal the structural basis by which alternative splicing modulates the organizing activity of FGF8, we solved the crystal structure of FGF8b in complex with the "c" splice isoform of FGF receptor 2 (FGFR2c). Using surface plasmon resonance (SPR), we also characterized the receptor-binding specificity of FGF8a and FGF8b, the "b" isoform of FGF17 (FGF17b), and FGF18. The FGF8b-FGFR2c structure shows that alternative splicing permits a single additional contact between phenylalanine 32 (F32) of FGF8b and a hydrophobic groove within Ig domain 3 of the receptor that is also present in FGFR1c, FGFR3c, and FGFR4. Consistent with the structure, mutation of F32 to alanine reduces the affinity of FGF8b toward all these receptors to levels characteristic of FGF8a. More importantly, analysis of the mid-hindbrain patterning ability of the FGF8b(F32A) mutant in chick embryos and murine midbrain explants shows that this mutation functionally converts FGF8b to FGF8a. Moreover, our data suggest that the intermediate receptor-binding affinities of FGF17b and FGF18, relative to FGF8a and FGF8b, also account for the distinct patterning abilities of these two ligands. We also show that the mode of FGF8 receptor-binding specificity is distinct from that of other FGFs and provide the first biochemical evidence for a physiological FGF8b-FGFR1c interaction during mid-hindbrain development. Consistent with the indispensable role of FGF8 in embryonic development, we show that the FGF8 mode of receptor binding appeared as early as in nematodes and has been preserved throughout evolution.

Analysis of mutations in fibroblast growth factor (FGF) and a pathogenic mutation in FGF receptor (FGFR) provides direct evidence for the symmetric two-end model for FGFR dimerization.

Two competing models for fibroblast growth factor (FGF) receptor (FGFR) dimerization have recently emerged based on ternary FGF-FGFR-heparin crystal structures. In the symmetric two-end model, heparin promotes dimerization of two FGF-FGFR complexes by stabilizing bivalent interactions of the ligand and receptor through primary and secondary sites and by stabilizing direct receptor-receptor contacts. In the asymmetric model, there are no protein-protein contacts between the two FGF-FGFR complexes, which are bridged solely by heparin. To identify the correct mode of FGFR dimerization, we abolished interactions at the secondary ligand-receptor interaction site, which are observed only in the symmetric two-end model, using site-directed mutagenesis. Cellular studies and real-time binding assays, as well as matrix-assisted laser desorption ionization-time of flight analysis, demonstrate that loss of secondary ligand-receptor interactions results in diminished FGFR activation due to decreased dimerization without affecting FGF-FGFR binding. Additionally, structural and biochemical analysis of an activating FGFR2 mutation resulting in Pfeiffer syndrome confirms the physiological significance of receptor-receptor contacts in the symmetric two-end model and provides a novel mechanism for FGFR gain of function in human skeletal disorders. Taken together, the data validate the symmetric two-end model of FGFR dimerization and argue against the asymmetric model of FGFR dimerization.