Signaling pathways activated by FGFs and FGFRs have been identified in multicellular organisms from Caenorhabditis elegans
to vertebrates. It is now well established that the FGFR family of RTKs and their numerous ligands play crucial roles in many developmental and physiological processes and that a variety of diseases are caused by aberrant signaling induced by FGFs or FGFRs (25
). The biological roles of individual FGFs and FGFRs have been analyzed by targeted disruption in mice of individual or combinations of FGF or FGFR genes or via analysis of disease-causing mutations in humans. In humans, both loss- and gain-of-function heterozygous mutations have been described. Several human skeletal dysplasias are caused by gain-of-function mutations in FGFR1, FGFR2, and FGFR3. Activating mutations mapped in the extracellular ligand binding domain were found in FGFR1 and FGFR2 associated with Pfeiffer, Crouzon, Jackson-Weiss, and Apert syndromes and in the FGFR2 kinase domain associated with Pfeiffer and Crouzon syndromes. Likewise, activating mutations in FGFR3 were mapped to the transmembrane and the tyrosine kinase domains found for achondroplasia, thanatophoric dysplasia type I (TDI), and TDII (reviewed in references 6
, and 36
). Interestingly, the nature and severity of the disease might depend on the mutated amino acid; replacement of the same residue by different amino acids may strongly influence the severity of disease. For example, replacement of lysine 650 in the tyrosine kinase domain of FGFR3 by a methionine results in short limbs and developmental delay (dwarfism, severe achondroplasia with developmental delay and acanthosis nigricans), while replacement of the same lysine by a glutamic acid results in lethality (TDII) (15
). These observations emphasize the complexity of molecular change that takes place as the consequence of mutations in FGFRs that may influence receptor activity, receptor stability, and receptor localization, among other changes. It is striking that all syndromes described above are caused by gain-of-function mutations in the c isoform of FGFRs, although in some cases mutations were also found in a region common to both b and c FGFR isoforms.
Genetic studies of families and patients with sporadic LADD syndrome revealed mutations in the tyrosine kinase domains of FGFR2 and FGFR3 (28
). The three missense mutations identified in FGFR2 are located in catalytic (A628T) and activation (A648T, R649S) loops, and a single mutation was found in the tyrosine kinase domain of FGFR3 (D513N). Several mutations in LADD syndrome patients were identified in FGF10 (C106F, I156R), including a nonsense mutation leading to a premature stop of translation (K137X) (22
). A nonredundant role of the FGF10-FGFR2b signaling pathway in lacrimal and salivary gland development was proposed based on the phenotypes of mice deficient in these genes. Aplasia in the lacrimal gland and hypoplasia in the salivary gland were observed for FGF10+/−
mice as well as for mice heterozygous for FGF10 and FGFR2b (FGF10+/−
). Despite the observation that mutations in either FGF10 or FGFR2 cause LADD syndrome, the underlying mechanism is not clear. Moreover, in the absence of biochemical data, modeling of the mutations in the structures of FGF10 and FGFR2 kinase domain did not provide conclusive insights concerning molecular mechanisms.
To reveal the mechanism underlying the molecular basis of LADD syndrome, we have compared the biochemical and biological properties of FGF10 or FGFR2b LADD mutants to the properties of their normal counterparts. Our results show that each of the three LADD mutations affects FGF10 activity by a different mechanism. While the I156R mutant is deficient in binding to FGFR2b, the C106F mutant is unstable at physiological temperatures and is most likely degraded shortly after synthesis before being delivered to its target cell. The K137X mutant, lacking a large C-terminal part of the molecule, was not produced; if it is produced, this mutant will not have any biological activity because its FGF core will have been severely disrupted.
The biological characterization of the FGF10 LADD mutants shows that the activity of the three LADD mutants is strongly compromised. Haploinsufficiency caused by the severely impaired FGF10 mutant leads to LADD syndrome, as the signal induced by FGF10 coded by the normal allele of LADD syndrome patients is not sufficient for mediating the normal development of the salivary and lacrimal glands. This conclusion is supported by genetic studies with mice demonstrating that two copies of FGF10 are required for the normal development of the salivary and lacrimal glands (5
). Moreover, the description of two additional FGF10 mutants (R80S and G138E) for patients with ALSG further emphasizes the critical and nonredundant role of FGF10 in salivary and lacrimal gland development (4
). The reason why FGF10 haploinsufficiency causes ALSG and the more severe LADD syndrome remains to be elucidated.
Analysis of the biological properties of ectopically expressed FGFR2 LADD mutants in the activation loops (A648T and R649S) or in the catalytic loop (A628T) shows that the FGFR2b LADD mutants are deficient in tyrosine kinase activity. The A628T mutant has the weakest activity, the A648T mutant exhibits an intermediate activity, and the R649S mutant has the highest activity, albeit lower than the tyrosine kinase activity of WT FGFR2 following ligand stimulation. Unlike the LADD mutation in the ligand molecule that is caused by the haploinsufficiency of FGF10, the FGFR2 LADD mutation will have a dominant negative effect on signaling mediated via WT FGFR2 expressed in the same cell (Fig. ). Three types of FGFR2 dimers will be formed in cells expressing equal amounts of normal FGFR2 and the FGFR2 LADD mutant in response to FGF10 stimulation (Fig. ): one-fourth of the molecules are homodimers of WT FGFR2 with normal tyrosine kinase activity, one-fourth are homodimers of the FGFR2 LADD mutant with a very weak tyrosine kinase activity, and one-half of the molecules are heterodimers composed of WT and LADD FGFR2 with attenuated tyrosine kinase activities. Since the activation of FGFRs is mediated by ligand-induced receptor dimerization and transphosphorylation, mutant receptors are unable to efficiently phosphorylate WT receptors on autophosphorylation sites in the activation loop of the tyrosine kinase core, a step essential for enhanced and sustained tyrosine kinase activity. Consequently, the defective LADD mutant will exert a dominant inhibitory affect on normal FGFR2, resulting in a strongly attenuated signal.
FIG. 6. A model for the dominant negative effect of the FGFR2 LADD mutant (mut) on activity and signaling by WT FGFR2. FGF10 stimulation of cells coexpressing WT FGFR2b together with the FGFR2b LADD mutant leads to the formation of three populations of receptor (more ...)
It has been reported that FGFR2+/− mice are normal, indicating that a single FGFR2 allele (providing 50% of the signal that take place in normal mice) is sufficient to support normal mouse development, including development at the lacrimal and salivary glands. The attenuated signal transmitted in cells coexpressing an FGFR2 LADD mutant together with WT FGFR2 in response to ligand stimulation (assuming that the WT and the FGFR2 LADD mutant are equally expressed) is expected to be larger than 25% and lower than 50% (25% < signal < 50%) of the signal transmitted by FGFR2 in normal mice. This conclusion underscores the importance of exact doses of receptor signaling in mediating biological responses; a small change in signal strength may have a strong impact on development and homeostasis in cells and tissues that do not possess a redundant signaling pathway.
On the basis of the previous genetic studies with and biochemical characterization of LADD mutations, it is possible to conclude that signaling pathways that are stimulated by FGF10 and mediated by FGFR2b play a critical role in the development and morphogenesis of branching organs such as salivary and lacrimal glands, kidneys, and lungs, which among other organs are affected by LADD syndrome. It has been shown that FGF10 expressed by mesenchymal cells will stimulate FGFR2b expressed in epithelial cells. Activation of FGFR2b in epithelial cells leads to the production of FGF8, which in turn stimulates the activity of FGFR2c and FGFR1c expressed in mesenchymal cells (37
). Attenuation in signaling via FGF10 or FGFR2b will lead to the disruption of an important cell signaling circuit that takes place between epithelial and mesenchymal cells during development. Disruption of the epithelial-mesenchymal cell signaling circuit may lead to the dental and skeletal abnormalities seen for LADD syndrome patients.
We also conclude that normal development of lacrimal glands, salivary glands, ears, skeleton, and other organs relies on a correct dose of FGF10 signaling through FGFR2b and that both copies of the FGF10 gene are required for the normal development of these organs; these requirements are not met in the case of the ear, skeletal, and dental abnormalities associated with LADD syndrome. Unlike FGF10 mutations causing ligand haploinsufficiency without affecting the action of the product of the WT FGF10 allele, mutations in FGFR2 lead to more-severe diseases by exerting a dominant negative effect on WT FGFR2 and potentially also on other FGFRs that are expressed in the same cell.
No specific phenotypic differences were observed for patients with mutations in FGF10 and FGFR2 or by comparing phenotypes caused by different FGFR2 mutations. In general, a wide range of phenotypic variability of symptoms exists in LADD syndrome patients, even those within the same family and carrying the identical mutation. This fact makes genotype-phenotype correlations difficult. We propose that the phenotypic outcome of impaired FGF signaling caused by mutations in LADD genes is further modified by genetic, environmental, and stochastic factors which remain to be elucidated.
Finally, although both FGFR2b and FGFR2c carry the LADD mutations, LADD mutation is primarily mediated by the FGFR2b isoform, implying that the compromised signaling via the FGFR2c mutant seen for LADD syndrome is compensated for by other members of the FGFR family expressed in mesenchymal cells.