Dissecting the Interaction of FGF8 with Receptor FGFRL1.

In mammals, the novel protein fibroblast growth factor receptor-like 1 (FGFRL1) is involved in the development of metanephric kidneys. It appears that this receptor controls a crucial transition of the induced metanephric mesenchyme to epithelial renal vesicles, which further develop into functional nephrons. FGFRL1 knockout mice lack metanephric kidneys and do not express any fibroblast growth factor (FGF) 8 in the metanephric mesenchyme, suggesting that FGFRL1 and FGF8 play a decisive role during kidney formation. FGFRL1 consists of three extracellular immunoglobulin (Ig) domains (Ig1-Ig2-Ig3), a transmembrane domain and a short intracellular domain. We have prepared the extracellular domain (Ig123), the three individual Ig domains (Ig1, Ig2, Ig3) as well as all combinations containing two Ig domains (Ig12, Ig23, Ig13) in recombinant form in human cells. All polypeptides that contain the Ig2 domain (Ig123, Ig12, Ig23, Ig2) were found to interact with FGF8 with very high affinity, whereas all constructs that lack the Ig2 domain (Ig1, Ig3, Ig13) poorly interacted with FGF8 as shown by ELISA and surface plasmon resonance. It is therefore likely that FGFRL1 represents a physiological receptor for FGF8 in the kidney and that the ligand primarily binds to the Ig2 domain of the receptor. With Biacore experiments, we also measured the affinity of FGF8 for the different constructs. All constructs containing the Ig2 domain showed a rapid association and a slow dissociation phase, from which a KD of 2-3 × 10-9 M was calculated. Our data support the hypothesis that binding of FGF8 to FGFRL1 could play an important role in driving the formation of nephrons in the developing kidney.

A Novel Mutation in the IL6R Gene Identified in a Family with Asthma Patients.

Background: Allergic diseases, including asthma, atopic dermatitis, allergic rhinitis, and food allergies, are caused by both environmental and genetic factors. The allergic condition, where genetic factors make up the largest proportion (up to 95%), is asthma. Aim: To identify polymorphisms and mutations in potentially disease-causing genes in a family affected with allergic asthma. Methods: Whole exome sequencing of the index patient was performed via next-generation sequencing. Variants in known allergy-associated susceptibility genes were identified by comparison with the reference genome GRChr37. Results: Seven common polymorphisms and three rare mutations were identified in the allergy-susceptibility genes of the index patient. Only four of these variants co-segregated with a second patient in the same family. These variants occurred in the TENS1, NPSR1, RAD50, and IL6R genes. Discussion: The variants observed in TENS1 and NPSR1 are relatively common (minor allele frequency, MAF ∼0.4), whereas the mutation in RAD50 is rare (MAF 0.0035). The mutation identified in IL6R (S409P) has never been found before. IL6R encodes an important receptor of the inflammatory system. The mutation occurs in the intracellular domain within a tyrosine-based motif, which is required for sorting of the IL6R protein to the basolateral side of polarized cells. It is likely that this rare mutation contributes-together with the other variants-to the predisposition to asthma and other allergic diseases.

Functional domains of the FgfrL1 receptor.

FgfrL1 is a novel growth factor receptor that is primarily expressed in musculoskeletal tissues and the kidney. FgfrL1-deficient mice have a malformed diaphragm and no kidneys. Such animals die immediately after birth because they are not able to inflate their lungs. The FgfrL1 molecule is composed of three extracellular Ig domains, a transmembrane helix and a short intracellular domain. To investigate the contribution of each of these domains to the function of the novel receptor, we generated mice with deletions of the individual domains. Mice lacking the intracellular domain are viable and phenotypically normal. Mice lacking the first (N-terminal) Ig domain are also viable and normal, but have a reduced life span. Mice lacking the Ig2 or the Ig3 domain are born alive, but die within 24 â€‹h after birth. Ig2-deficient animals exhibit substantially smaller kidneys than wild-type littermates and contain a lower number of glomeruli. Ig3-deficient mice completely lack metanephric kidneys. Interestingly, both the Ig2 and the Ig3-deficient animals show only minor alterations in the diaphragm, which still enables them to inflate their lungs after birth. Our results demonstrate that the principal function of the FgfrL1 receptor is to control the growth of the metanephric kidneys by regulating nephrogenesis. It appears that this function is primarily accomplished by the Ig3 domain with some contribution of the Ig2 domain. It is conceivable that the two domains interact with an Fgf ligand and another molecule from the surface of neighboring cells to induce condensation of the metanephric mesenchyme to renal epithelia and glomeruli.

Identification of a MAFB mutation in a patient with multicentric carpotarsal osteolysis.

Multicentric carpotarsal osteolysis (MCTO) is an autosomal dominant disease of the skeleton characterised by progressive destruction of carpal and tarsal bones. Recently, it has been demonstrated that this disease is caused by heterozygous mutations in the gene for the transcriptional repressor MAFB. We analysed genomic DNA and RNA from leucocytes of a female patient diagnosed with MCTO. We identified the mutation c.161C>T in the genomic sequence and in the expressed messenger RNA for MAFB. This is the second report of the c.161C>T mutation in a MCTO patient. Since the parents do not possess this mutation, the daughter must have acquired a de novo mutation. At the level of the gene, this mutation is found at a CpG dinucleotide sequence, suggesting that DNA methylation was involved in the occurrence of the DNA aberration. At the level of the protein, the mutation exchanges a serine with a leucine residue at a position on MAFB that can become phosphorylated in the wild-type protein. MAFB negatively regulates the RANKL-dependent differentiation of monocytes into osteoclasts. It is likely that the mutation will affect the phosphorylation status of the protein and its biological activity. When the activity of the transcriptional repressor is reduced, osteoclastogenesis will be increased, which might explain the carpotarsal bone destruction observed in the patient.

Evolution of the fusogenic activity of the receptor FGFRL1.

FGFRL1 is a transmembrane receptor that can induce the fusion of CHO cells to multinucleated syncytia. This cell fusion activity has been attributed to the extracellular Ig3 domain of the receptor. We investigated how the fusogenic activity evolved during the evolution of animals. We found that the Ig3 domain from humans, mice, chicken and fish stimulates fusion of CHO cells, while the Ig3 domain from lancelet and sea urchin does not. It is therefore conceivable that the fusogenic activity of FGFRL1 developed during the evolution of vertebrates. Bony fish contain two copies of the FGFRL1 gene because they have undergone a whole-genome duplication. One of the corresponding proteins (FGFRL1a) induces cell-cell fusion, while the other (FGFRL1b) does not. Analysis of chimeric constructs and in vitro mutagenesis suggested that FGFRL1b has lost its fusogenic activity after duplication. A rescue experiment supported this conclusion. When four amino acids were changed, the Ig3 domain of FGFRL1b was converted into an active, fusogenic protein comparable to FGFRL1a. The four amino acids are located in a hydrophobic pocket of the Ig3 domain. It is likely that this hydrophobic pocket interacts with a target molecule on the membrane of adjacent cells to induce cell-cell fusion.

Receptor FGFRL1 does not promote cell proliferation but induces cell adhesion.

Fibroblast growth factor receptor (FGFR)-like protein 1 (FGFRL1) is the most recently discovered member of the FGFR family. Owing to the fact that it interacts with FGF ligands, but lacks the intracellular tyrosine kinase domain, several researchers have speculated that it may function as a decoy receptor and exert a negative effect on cell proliferation. In this study, we performed overexpression experiments with TetOn­inducible cell clones and downregulation experiments with siRNA oligonucleotides, and found that FGFRL1 had absolutely no effect on cell growth and proliferation. Likewise, we did not observe any influence of FGFRL1 on ERK1/2 activation and on the phosphorylation of 250 other signaling proteins analyzed by the Kinexus antibody microarray. On the other hand, with bacterial petri dishes, we observed a clear effect of FGFRL1 on cell adhesion during the initial hours after cell seeding. Our results suggest that FGFRL1 is a cell adhesion protein similar to the nectins rather than a signaling receptor similar to FGFR1-FGFR4.

Deletion of exon 8 from the EXT1 gene causes multiple osteochondromas (MO) in a family with three affected members.

Multiple osteochondromas (also called hereditary multiple exostoses) is an autosomal dominant disorder characterized by multiple cartilaginous tumors, which are caused by mutations in the genes for exostosin-1 (EXT1) and exostosin-2 (EXT2). The goal of this study was to elucidate the genetic alterations in a family with three affected members. Isolation of RNA from the patients' blood followed by reverse transcription and PCR amplification of selected fragments showed that the three patients lack a specific region of 90 bp from their EXT1 mRNA. This region corresponds to the sequence of exon 8 from the EXT1 gene. No splice site mutation was found around exon 8. However, long-range PCR amplification of the region from intron 7 to intron 8 indicated that the three patients contain a deletion of 4318 bp, which includes exon 8 and part of the flanking introns. There is evidence that the deletion was caused by non-homologous end joining because the breakpoints are not located within a repetitive element, but contain multiple copies of the deletion hotspot sequence TGRRKM. Exon 8 encodes part of the active site of the EXT1 enzyme, including the DXD signature of all UDP-sugar glycosyltransferases. It is conceivable that the mutant protein exerts a dominant negative effect on the activity of the EXT glycosyltransferase since it might interact with normal copies of the enzyme to form an inactive hetero-oligomeric complex. We suggest that sequencing of RNA might be superior to exome sequencing to detect short deletions of a single exon.

Receptor FGFRL1 acts as a tumor suppressor in nude mice when overexpressed in HEK 293 Tet-On cells.

Fibroblast growth factor receptor-like 1 (FGFRL1) is a transmembrane receptor that interacts with heparin and FGF ligands. In contrast to the classical FGF receptors, FGFR1 to FGFR4, it does not appear to affect cell growth and proliferation. In the present study, an inducible gene expression system was utilized in combination with a xenograft tumor model to investigate the effects of FGFRL1 on cell adhesion and tumor formation. It was determined that recombinant FGFRL1 promotes the adhesion of HEK 293 Tet-On® cells in vitro. Moreover, when such cells are induced to express FGFRL1ΔC they aggregate into huge clusters. If injected into nude mice, the cells form large tumors. Notably, this tumor growth is completely inhibited when the expression of FGFRL1 is induced. The forced expression of FGFRL1 in the tumor tissue may restore contact inhibition, thereby preventing growth of the cells in nude mice. The results of the present study demonstrate that FGFRL1 acts as a tumor suppressor similar to numerous other cell adhesion proteins. It is therefore likely that FGFRL1 functions as a regular cell-cell adhesion protein.

Phylogenetic analysis of receptor FgfrL1 shows divergence of the C-terminal end in rodents.

FGFRL1 is a member of the fibroblast growth factor receptor (FGFR) family. Similar to the classical receptors FGFR1-FGFR4, it contains three extracellular Ig-like domains and a single transmembrane domain. However, it lacks the intracellular tyrosine kinase domain that would be required for signal transduction, but instead contains a short intracellular tail with a peculiar histidine-rich motif. This motif has been conserved during evolution from mollusks to echinoderms and vertebrates. Only the sequences of FgfrL1 from a few rodents diverge at the C-terminal region from the canonical sequence, as they appear to have suffered a frameshift mutation within the histidine-rich motif. This mutation is observed in mouse, rat and hamster, but not in the closely related rodents mole rat (Nannospalax) and jerboa (Jaculus), suggesting that it has occurred after branching of the Muridae and Cricetidae from the Dipodidae and Spalacidae. The consequence of the frameshift is a deletion of a few histidine residues and an extension of the C-terminus by about 40 unrelated amino acids. A similar frameshift mutation has also been observed in a human patient with a craniosynostosis syndrome as well as in several patients with colorectal cancer and bladder tumors, suggesting that the histidine-rich motif is prone to mutation. The reason why this motif was conserved during evolution in most species, but not in mice, is not clear.

Cell-cell fusion induced by the Ig3 domain of receptor FGFRL1 in CHO cells.

FGFRL1 is a single-pass transmembrane protein with three extracellular Ig domains. When overexpressed in CHO cells or related cell types, it induces cell-cell fusion and formation of large, multinucleated syncytia. For this fusion-promoting activity, only the membrane-proximal Ig domain (Ig3) and the transmembrane domain are required. It does not matter whether the transmembrane domain is derived from FGFRL1 or from another receptor, but the distance of the Ig3 domain to the membrane is crucial. Fusion can be inhibited with soluble recombinant proteins comprising the Ig1-Ig2-Ig3 or the Ig2-Ig3 domains as well as with monoclonal antibodies directed against Ig3. Mutational analysis reveals a hydrophobic site in Ig3 that is required for fusion. If a single amino acid from this site is mutated, fusion is abolished. The site is located on a ß-sheet, which is part of a larger ß-barrel, as predicted by computer modeling of the 3D structure of FGFRL1. It is possible that this site interacts with a target protein of neighboring cells to trigger cell-cell fusion.

Targeted disruption of the intracellular domain of receptor FgfrL1 in mice.

FgfrL1 is the fifth member of the fibroblast growth factor receptor (Fgfr) family. Studies with FgfrL1 deficient mice have demonstrated that the gene plays an important role during embryonic development. FgfrL1 knock-out mice die at birth as they have a malformed diaphragm and lack metanephric kidneys. Similar to the classical Fgfrs, the FgfrL1 protein contains an extracellular part composed of three Ig-like domains that interact with Fgf ligands and heparin. However, the intracellular part of FgfrL1 is not related to the classical receptors and does not possess any tyrosine kinase activity. Curiously enough, the amino acid sequence of this domain is barely conserved among different species, with the exception of three motifs, namely a dileucine peptide, a tandem tyrosine-based motif YXXΦ and a histidine-rich sequence. To investigate the function of the intracellular domain of FgfrL1, we have prepared genetically modified mice that lack the three conserved sequence motifs, but instead contain a GFP cassette (FgfrL1ΔC-GFP). To our surprise, homozygous FgfrL1ΔC-GFP knock-in mice are viable, fertile and phenotypically normal. They do not exhibit any alterations in the diaphragm or the kidney, except for a slight reduction in the number of glomeruli that does not appear to affect life expectancy. In addition, the pancreas of both FgfrL1ΔC-GFP knock-in and FgfrL1 knock-out mice do not show any disturbances in the production of insulin, in contrast to what has been suggested by recent studies. Thus, the conserved motifs of the intracellular FgfrL1 domain are dispensable for organogenesis and normal life. We conclude that the extracellular domain of the protein must conduct the vital functions of FgfrL1.

The FgfrL1 receptor is required for development of slow muscle fibers.

FgfrL1, which interacts with Fgf ligands and heparin, is a member of the fibroblast growth factor receptor (Fgfr) family. FgfrL1-deficient mice show two significant alterations when compared to wildtype mice: They die at birth due to a malformed diaphragm and they lack metanephric kidneys. Utilizing gene arrays, qPCR and in situ hybridization we show here that the diaphragm of FgfrL1 knockout animals lacks any slow muscle fibers at E18.5 as indicated by the absence of slow fiber markers Myh7, Myl2 and Myl3. Similar lesions are also found in other skeletal muscles that contain a high proportion of slow fibers at birth, such as the extraocular muscles. In contrast to the slow fibers, fast fibers do not appear to be affected as shown by expression of fast fiber markers Myh3, Myh8, Myl1 and MylPF. At early developmental stages (E10.5, E15.5), FgfrL1-deficient animals express slow fiber genes at normal levels. The loss of slow fibers cannot be attributed to the lack of kidneys, since Wnt4 knockout mice, which also lack metanephric kidneys, show normal expression of Myh7, Myl2 and Myl3. Thus, FgfrL1 is specifically required for embryonic development of slow muscle fibers.

Evidence that the novel receptor FGFRL1 signals indirectly via FGFR1.

Fibroblast growth factor (FGF) receptor-like protein 1 (FGFRL1) is a recently discovered member of the FGF receptor (FGFR) family. Similar to the classical FGFRs, it contains three extracellular immunoglobulin-like domains and interacts with FGF ligands. However, in contrast to the classical receptors, it does not contain any intracellular tyrosine kinase domain and consequently cannot signal by transphosphorylation. In mouse kidneys, FgfrL1 is expressed primarily at embryonic stages E14-E15 in regions where nascent nephrons develop. In this study, we used whole-mount in situ hybridization to show the spatial pattern of five different Fgfrs in the developing mouse kidney. We compared the expression pattern of FgfrL1 with that of other Fgfrs. The expression pattern of FgfrL1 closely resembled that of Fgfr1, but clearly differed from that of Fgfr2­Fgfr4. It is therefore conceivable that FgfrL1 signals indirectly via Fgfr1. The mechanisms by which FgfrL1 affects the activity of Fgfr1 remain to be elucidated.

Role of FGFRL1 and other FGF signaling proteins in early kidney development.

The mammalian kidney develops from the ureteric bud and the metanephric mesenchyme. In mice, the ureteric bud invades the metanephric mesenchyme at day E10.5 and begins to branch. The tips of the ureteric bud induce the metanephric mesenchyme to condense and form the cap mesenchyme. Some cells of this cap mesenchyme undergo a mesenchymal-to-epithelial transition and differentiate into renal vesicles, which further develop into nephrons. The developing kidney expresses Fibroblast growth factor (Fgf)1, 7, 8, 9, 10, 12 and 20 and Fgf receptors Fgfr1 and Fgfr2. Fgf7 and Fgf10, mainly secreted by the metanephric mesenchyme, bind to Fgfr2b of the ureteric bud and induce branching. Fgfr1 and Fgfr2c are required for formation of the metanephric mesenchyme, however the two receptors can substitute for one another. Fgf8, secreted by renal vesicles, binds to Fgfr1 and supports survival of cells in the nascent nephrons. Fgf9 and Fgf20, expressed in the metanephric mesenchyme, are necessary to maintain survival of progenitor cells in the cortical region of the kidney. FgfrL1 is a novel member of the Fgfr family that lacks the intracellular tyrosine kinase domain. It is expressed in the ureteric bud and all nephrogenic structures. Targeted deletion of FgfrL1 leads to severe kidney dysgenesis due to the lack of renal vesicles. FgfrL1 is known to interact mainly with Fgf8. It is therefore conceivable that FgfrL1 restricts signaling of Fgf8 to the precise location of the nascent nephrons. It might also promote tight adhesion of cells in the condensed metanephric mesenchyme as required for the mesenchymal-to-epithelial transition.

Comparison of the gene expression profiles from normal and Fgfrl1 deficient mouse kidneys reveals downstream targets of Fgfrl1 signaling.

Fgfrl1 (fibroblast growth factor receptor-like 1) is a transmembrane receptor that is essential for the development of the metanephric kidney. It is expressed in all nascent nephrogenic structures and in the ureteric bud. Fgfrl1 null mice fail to develop the metanephric kidneys. Mutant kidney rudiments show a dramatic reduction of ureteric branching and a lack of mesenchymal-to-epithelial transition. Here, we compared the expression profiles of wildtype and Fgfrl1 mutant kidneys to identify genes that act downstream of Fgfrl1 signaling during the early steps of nephron formation. We detected 56 differentially expressed transcripts with 2-fold or greater reduction, among them many genes involved in Fgf, Wnt, Bmp, Notch, and Six/Eya/Dach signaling. We validated the microarray data by qPCR and whole-mount in situ hybridization and showed the expression pattern of candidate genes in normal kidneys. Some of these genes might play an important role during early nephron formation. Our study should help to define the minimal set of genes that is required to form a functional nephron.

A net-like structure with pores is observed during cell fusion induced by the receptor FGFRL1.

FGFRL1 is the fifth member of the fibroblast growth factor receptor (FGFR) family. Similar to the other members, it harbors three Ig loops in its extracellular domain, but in contrast to the other receptors, it lacks the intracellular protein tyrosine kinase domain that would be required for signaling by transphosphorylation. FGFRL1 is mainly found in the musculoskeletal system, where it appears to inhibit cell proliferation but to induce cell adhesion and differentiation. Mice with a targeted disruption of the FGFRL1 gene die during birth due to a malformed diaphragm muscle, which is not strong enough to inflate the lungs after birth. Expression of FGFRL1 is highly upregulated during the differentiation of myoblasts to multinucleated myotubes, suggesting an important role for FGFRL1 in cell-cell fusion. Recently we showed that FGFRL1 does indeed induce fusion of cultured cells into large syncytia. A reporter gene assay demonstrated that the third Ig domain and the transmembrane domain of FGFRL1 are both necessary and sufficient to fuse CHO cells into syncytia comprising several hundred nuclei. At the contact site, the fusing cells reveal a peculiar net-like structure with pores of about 1 µm diameter. It is possible that these structures represent membrane areas with fusion pores that set in motion the cell-cell fusion process. FGFRL1 is the first mammalian protein that is capable of triggering cell-cell fusion in vitro.

Interaction of the receptor FGFRL1 with the negative regulator Spred1.

FGFRL1 is a member of the fibroblast growth factor receptor family. It plays an essential role during branching morphogenesis of the metanephric kidneys, as mice with a targeted deletion of the Fgfrl1 gene show severe kidney dysplasia. Here we used the yeast two-hybrid system to demonstrate that FGFRL1 binds with its C-terminal, histidine-rich domain to Spred1 and to other proteins of the Sprouty/Spred family. Members of this family are known to act as negative regulators of the Ras/Raf/Erk signaling pathway. Truncation experiments further showed that FGFRL1 interacts with the SPR domain of Spred1, a domain that is shared by all members of the Sprouty/Spred family. The interaction could be verified by coprecipitation of the interaction partners from solution and by codistribution at the cell membrane of COS1 and HEK293 cells. Interestingly, Spred1 increased the retention time of FGFRL1 at the plasma membrane where the receptor might interact with ligands. FGFRL1 and members of the Sprouty/Spred family belong to the FGF synexpression group, which also includes FGF3, FGF8, Sef and Isthmin. It is conceivable that FGFRL1, Sef and some Sprouty/Spred proteins work in concert to control growth factor signaling during branching morphogenesis of the kidneys and other organs.

Biology of FGFRL1, the fifth fibroblast growth factor receptor.

FGFRL1 (fibroblast growth factor receptor like 1) is the most recently discovered member of the FGFR family. It contains three extracellular Ig-like domains similar to the classical FGFRs, but it lacks the protein tyrosine kinase domain and instead contains a short intracellular tail with a peculiar histidine-rich motif. The gene for FGFRL1 is found in all metazoans from sea anemone to mammals. FGFRL1 binds to FGF ligands and heparin with high affinity. It exerts a negative effect on cell proliferation, but a positive effect on cell differentiation. Mice with a targeted deletion of the Fgfrl1 gene die perinatally due to alterations in their diaphragm. These mice also show bilateral kidney agenesis, suggesting an essential role for Fgfrl1 in kidney development. A human patient with a frameshift mutation exhibits craniosynostosis, arguing for an additional role of FGFRL1 during bone formation. FGFRL1 contributes to the complexity of the FGF signaling system.