RESUMEN
Fibroblast growth factor receptor-1 (FGFR1) activity at the plasma membrane is tightly controlled by the availability of co-receptors and competing receptor isoforms. We have previously shown that FGFR1 activity in pancreatic beta-cells modulates a wide range of processes, including lipid metabolism, insulin processing, and cell survival. More recently, we have revealed that co-expression of FGFR5, a receptor isoform that lacks a tyrosine-kinase domain, influences FGFR1 responses. We therefore hypothesized that FGFR5 is a co-receptor to FGFR1 that modulates responses to ligands by forming a receptor heterocomplex with FGFR1. We first show here increased FGFR5 expression in the pancreatic islets of nonobese diabetic (NOD) mice and also in mouse and human islets treated with proinflammatory cytokines. Using siRNA knockdown, we further report that FGFR5 and FGFR1 expression improves beta-cell survival. Co-immunoprecipitation and quantitative live-cell imaging to measure the molecular interaction between FGFR5 and FGFR1 revealed that FGFR5 forms a mixture of ligand-independent homodimers (â¼25%) and homotrimers (â¼75%) at the plasma membrane. Interestingly, co-expressed FGFR5 and FGFR1 formed heterocomplexes with a 2:1 ratio and subsequently responded to FGF2 by forming FGFR5/FGFR1 signaling complexes with a 4:2 ratio. Taken together, our findings identify FGFR5 as a co-receptor that is up-regulated by inflammation and promotes FGFR1-induced survival, insights that reveal a potential target for intervention during beta-cell pathogenesis.
Asunto(s)
Citocinas/inmunología , Diabetes Mellitus/genética , Células Secretoras de Insulina/inmunología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/genética , Animales , Diabetes Mellitus/inmunología , Dimerización , Femenino , Factor 2 de Crecimiento de Fibroblastos/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/química , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/inmunología , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/química , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/inmunología , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Células Gigantes/metabolismo , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Fusión Celular , Clonación Molecular , Cricetulus , Evolución Molecular , Células Gigantes/citología , Humanos , Dominios Proteicos , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/química , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/genética , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Ratones/genética , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/genética , Animales , Histidina/genética , Humanos , Invertebrados , Mutación , Estructura Terciaria de Proteína , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/química , Análisis de Secuencia de ADN , VertebradosRESUMEN
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.
Asunto(s)
Modelos Moleculares , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/química , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Células CHO , Fusión Celular , Cricetinae , Cricetulus , Células HEK293 , Humanos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
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.
Asunto(s)
Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/genética , Animales , Membrana Celular/metabolismo , Femenino , Técnicas de Sustitución del Gen , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Insulina/biosíntesis , Células Secretoras de Insulina/metabolismo , Riñón/citología , Riñón/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Estructura Terciaria de Proteína , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/química , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The fusion of mammalian cells into syncytia is a developmental process that is tightly restricted to a limited subset of cells. Besides gamete and placental trophoblast fusion, only macrophages and myogenic stem cells fuse into multinucleated syncytia. In contrast to viral cell fusion, which is mediated by fusogenic glycoproteins that actively merge membranes, mammalian cell fusion is poorly understood at the molecular level. A variety of mammalian transmembrane proteins, among them many of the immunoglobulin superfamily, have been implicated in cell-cell fusion, but none has been shown to actively fuse cells in vitro. Here we report that the FGFRL1 receptor, which is up-regulated during the differentiation of myoblasts into myotubes, fuses cultured cells into large, multinucleated syncytia. We used luciferase and GFP-based reporter assays to confirm cytoplasmic mixing and to identify the fusion inducing domain of FGFRL1. These assays revealed that Ig-like domain III and the transmembrane domain are both necessary and sufficient to rapidly fuse CHO cells into multinucleated syncytia comprising several hundred nuclei. Moreover, FGFRL1 also fused HEK293 and HeLa cells with untransfected CHO cells. Our data show that FGFRL1 is the first mammalian protein that is capable of inducing syncytium formation of heterologous cells in vitro.
Asunto(s)
Diferenciación Celular , Expresión Génica , Células Gigantes/citología , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Células CHO , Fusión Celular , Cricetinae , Cricetulus , Células Gigantes/metabolismo , Células HEK293 , Células HeLa , Humanos , Mioblastos/citología , Mioblastos/metabolismo , Estructura Terciaria de Proteína , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/químicaRESUMEN
FGFRL1 (fibroblast growth factor receptor like 1) is the fifth and most recently discovered member of the fibroblast growth factor receptor (FGFR) family. With up to 50% amino acid similarity, its extracellular domain closely resembles that of the four conventional FGFRs. Its intracellular domain, however, lacks the split tyrosine kinase domain needed for FGF-mediated signal transduction. During embryogenesis of the mouse, FGFRL1 is essential for the development of parts of the skeleton, the diaphragm muscle, the heart, and the metanephric kidney. Since its discovery, it has been hypothesized that FGFRL1 might act as a decoy receptor for FGF ligands. Here we present several lines of evidence that support this notion. We demonstrate that the FGFRL1 ectodomain is shed from the cell membrane of differentiating C2C12 myoblasts and from HEK293 cells by an as yet unidentified protease, which cuts the receptor in the membrane-proximal region. As determined by ligand dot blot analysis, cell-based binding assays, and surface plasmon resonance analysis, the soluble FGFRL1 ectodomain as well as the membrane-bound receptor are capable of binding to some FGF ligands with high affinity, including FGF2, FGF3, FGF4, FGF8, FGF10, and FGF22. We furthermore show that ectopic expression of FGFRL1 in Xenopus embryos antagonizes FGFR signaling during early development. Taken together, our data provide strong evidence that FGFRL1 is indeed a decoy receptor for FGFs.
Asunto(s)
Membrana Celular/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Xenopus/embriología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Humanos , Larva/crecimiento & desarrollo , Ligandos , Ratones , Datos de Secuencia Molecular , Mioblastos/citología , Péptido Hidrolasas/metabolismo , Polimorfismo de Nucleótido Simple , Estructura Terciaria de Proteína , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/química , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/genética , Solubilidad , Resonancia por Plasmón de SuperficieRESUMEN
BACKGROUND: FGFRL1, the gene for the fifth member of the fibroblast growth factor receptor (FGFR) family, is found in all vertebrates from fish to man and in the cephalochordate amphioxus. Since it does not occur in more distantly related invertebrates such as insects and nematodes, we have speculated that FGFRL1 might have evolved just before branching of the vertebrate lineage from the other invertebrates (Beyeler and Trueb, 2006). RESULTS: We identified the gene for FGFRL1 also in the sea urchin Strongylocentrotus purpuratus and cloned its mRNA. The deduced amino acid sequence shares 62% sequence similarity with the human protein and shows conservation of all disulfides and N-linked carbohydrate attachment sites. Similar to the human protein, the S. purpuratus protein contains a histidine-rich motif at the C-terminus, but this motif is much shorter than the human counterpart. To analyze the function of the novel motif, recombinant fusion proteins were prepared in a bacterial expression system. The human fusion protein bound to nickel and zinc affinity columns, whereas the sea urchin protein barely interacted with such columns. Direct determination of metal ions by atomic absorption revealed 2.6 mole zinc/mole protein for human FGFRL1 and 1.7 mole zinc/mole protein for sea urchin FGFRL1. CONCLUSION: The FGFRL1 gene has evolved much earlier than previously assumed. A comparison of the intracellular domain between sea urchin and human FGFRL1 provides interesting insights into the shaping of a novel zinc binding domain.
Asunto(s)
Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/química , Erizos de Mar/metabolismo , Zinc/química , Secuencia de Aminoácidos , Animales , Evolución Molecular , Glicosilación , Humanos , Datos de Secuencia Molecular , Filogenia , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ARN Mensajero/metabolismo , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/clasificación , Receptor Tipo 5 de Factor de Crecimiento de Fibroblastos/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Erizos de Mar/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Zinc/metabolismoRESUMEN
Fibroblast growth factor receptor-like 1 (FGFRL1) is a recently discovered transmembrane protein whose functions remain unclear. Since mutations in the related receptors FGFR1-3 cause skeletal malformations, DNA samples from 55 patients suffering from congenital skeletal malformations and 109 controls were searched for mutations in FGFRL1. One patient was identified harboring a frameshift mutation in the intracellular domain of this novel receptor. The patient showed craniosynostosis, radio-ulnar synostosis and genital abnormalities and had previously been diagnosed with Antley-Bixler syndrome. The effect of the FGFRL1 mutation was studied in vitro. In a reporter gene assay, the wild-type as well as the mutant receptor inhibited FGF signaling. However, the mutant protein differed from the wild-type protein in its subcellular localization. Mutant FGFRL1 was mainly found at the plasma membrane where it interacted with FGF ligands, while the wild-type protein was preferentially located in vesicular structures and the Golgi complex. Two motifs from the intracellular domain of FGFRL1 appeared to be responsible for this differential distribution, a tandem tyrosine based motif and a histidine-rich sequence. Deletion of either one led to the preferential redistribution of FGFRL1 to the plasma membrane. It is therefore likely that mutant FGFRL1 contributes to the skeletal malformations of the patient.