betaKlotho is required for fibroblast growth factor (FGF) 21 signaling through FGF receptor (FGFR) 1c and FGFR3c

Fibroblast growth factor (FGF) 21, a structural relative of FGF23 that regulates phosphate homeostasis, is a regulator of insulin-independent glucose transport in adipocytes and plays a role in the regulation of body weight. It also regulates ketogenesis and adaptive responses to starvation. We report that in a reconstituted receptor activation assay system using BaF3 cells, which do not endogenously express any type of FGF receptor (FGFR) or heparan sulfate proteoglycan, FGF21 alone does not activate FGFRs and that betaKlotho is required for FGF21 to activate two specific FGFR subtypes: FGFR1c and FGFR3c. Coexpression of betaKlotho and FGFR1c on BaF3 cells enabled FGF21, but not FGF23, to activate receptor signaling. Conversely, coexpression of FGFR1c and Klotho, a protein related to betaKlotho, enabled FGF23 but not FGF21 to activate receptor signaling, indicating that expression of betaKlotho/Klotho confers target cell specificity on FGF21/FGF23. In all of these cases, heparin enhanced the activation but was not essential. In 3T3-L1 adipocytes, up-regulation of glucose transporter (GLUT) expression by FGF21 was associated with expression of betaKlotho, which was absent in undifferentiated 3T3-L1 fibroblasts. It is thus suggested that betaKlotho expression is a crucial determinant of the FGF21 specificity of the target cells upon which it acts in an endocrine fashion.     

Signaling by fibroblast growth factors (FGF) and fibroblast growth factor receptor 2 (FGFR2)-activating mutations blocks mineralization and induces apoptosis in osteoblasts

Fibroblast growth factors (FGF) play a critical role in bone growth and development affecting both chondrogenesis and osteogenesis. During the process of intramembranous ossification, which leads to the formation of the flat bones of the skull, unregulated FGF signaling can produce premature suture closure or craniosynostosis and other craniofacial deformities. Indeed, many human craniosynostosis disorders have been linked to activating mutations in FGF receptors (FGFR) 1 and 2, but the precise effects of FGF on the proliferation, maturation and differentiation of the target osteoblastic cells are still unclear. In this report, we studied the effects of FGF treatment on primary murine calvarial osteoblast, and on OB1, a newly established osteoblastic cell line. We show that FGF signaling has a dual effect on osteoblast proliferation and differentiation. FGFs activate the endogenous FGFRs leading to the formation of a Grb2/FRS2/Shp2 complex and activation of MAP kinase. However, immature osteoblasts respond to FGF treatment with increased proliferation, whereas in differentiating cells FGF does not induce DNA synthesis but causes apoptosis. When either primary or OB1 osteoblasts are induced to differentiate, FGF signaling inhibits expression of alkaline phosphatase, and blocks mineralization. To study the effect of craniosynostosis-linked mutations in osteoblasts, we introduced FGFR2 carrying either the C342Y (Crouzon syndrome) or the S252W (Apert syndrome) mutation in OB1 cells. Both mutations inhibited differentiation, while dramatically inducing apoptosis. Furthermore, we could also show that overexpression of FGF2 in transgenic mice leads to increased apoptosis in their calvaria. These data provide the first biochemical analysis of FGF signaling in osteoblasts, and show that FGF can act as a cell death inducer with distinct effects in proliferating and differentiating osteoblasts.     

90-kDa ribosomal S6 kinase is a direct target for the nuclear fibroblast growth factor receptor 1 (FGFR1): role in FGFR1 signaling

Fibroblast growth factor receptor 1 (FGFR1) is a transmembrane protein capable of transducing stimulation by secreted FGFs. In addition, newly synthesized FGFR1 enters the nucleus in response to cellular stimulation and during development. Nuclear FGFR1 can transactivate CRE (cAMP responsive element), activate CRE-binding protein (CREB)-binding protein (CBP) and gene activities causing cellular growth and differentiation. Here, a yeast two-hybrid assay was performed to identify FGFR1-binding proteins and the mechanism of nuclear FGFR1 action. Ten FGFR1-binding proteins were identified. Among the proteins detected with the intracellular FGFR1 domain was a 90-kDa ribosomal S6 kinase (RSK1), a regulator of CREB, CBP, and histone phosphorylation. FGFR1 bound to the N-terminal region of RSK1. The FGFR1-RSK1 interaction was confirmed by co-immunoprecipitation and colocalization in the nucleus and cytoplasm of mammalian cells. Predominantly nuclear FGFR1-RSK1 interaction was observed in the rat brain during neurogenesis and in cAMP-stimulated cultured neural cells. In TE671 cells, transfected FGFR1 colocalized and coimmunoprecipitated, almost exclusively, with nuclear RSK1. Nuclear RSK1 kinase activity and RSK1 activation of CREB were enhanced by transfected FGFR1. In contrast, kinase-deleted FGFR1 (TK-), which did not bind to RSK1 failed to stimulate nuclear RSK1 activity or RSK1 activation of CREB. Kinase inactive FGFR1 (K514A) bound effectively to nuclear RSK1, but it failed to stimulate RSK1. Thus, active FGFR1 kinase regulates the functions of nuclear RSK1. The interaction of nuclear FGFR1 with pluripotent RSK1 offers a new mechanism through which FGFR1 may control fundamental cellular processes.     

Genomic organization of the human fibroblast growth factor receptor 2 (FGFR2) gene and comparative analysis of the human FGFR gene family

The human fibroblast growth factor receptor (FGFR) genes play important roles in normal vertebrate development. Mutations in the human FGFR2 gene have been associated with many craniosynostotic syndromes and malformations, including Crouzon, Pfeiffer, Apert, Jackson-Weiss, Beare-Stevenson cutis gyrata, and Antley-Bixler syndromes, and Kleeblaatschadel (cloverleaf skull) deformity. The mutations identified to date are concentrated in the previously characterized region of FGFR2 that codes for the extracellular IgIII domain of the receptor protein. The search for mutations in other regions of the gene, however, has been hindered by lack of knowledge of the genomic structure. Using a combination of genomic library screening, long-range PCR, and genomic walking, we have characterized the genomic structure of nearly the entire human FGFR2 gene, including a delineation of the organization and size of all introns and exons and determination of the DNA sequences at the intron/exon boundaries. Comparative analysis of the human FGFR gene family reveals that the genomic organization of the FGFRs is relatively conserved. Moreover, alignment of the amino acid sequences shows that the four corresponding proteins share 46% identity overall, with up to 70% identity between individual pairs of FGFR proteins. However, the FGFR2 gene contains an additional exon not found in other members of the family, and it also has much larger intronic sequences throughout the gene. Remarkable similarities in genomic organization, intron/exon boundaries, and intron sizes are found between the human and mouse FGFR2 genes. Knowledge gained from this study of the human FGFR2 gene structure may prove useful in future screening studies designed to find additional mutations associated with craniosynostotic syndromes, and in understanding the molecular and cell biology of this receptor family.     

Co-purification of proteases with basic fibroblast growth factor (FGF)

Acidic and basic fibroblast growth factors (FGFs) are proteins of 16-18 kDa. Other forms of 25-30 kDa related to this growth factor family have recently been described. All these components bind tightly to heparin-Sepharose, a property that allows the purification of several FGF-related proteins. During the purification of acidic and basic FGFs from bovine pituitary glands, we detected the presence of 28-30 kDa components that are immunoreactive against anti-basic FGF antisera. However, microsequencing analysis revealed that the 28-30 kDa components are lysosomal proteases that co-elute with basic FGF from heparin-Sepharose columns. The involvement of these proteases in the etiology of microheterogenous forms of FGFs and/or release of FGFs from the extracellular matrix is discussed.     

Study of the interaction of the Ig2 module of the fibroblast growth factor receptor, FGFR Ig2, with the fibroblast growth factor 1, FGF1, by means of NMR spectroscopy

Fibroblast growth factor (FGF) receptor (FGFR) consists extracellularly of three immunoglobulin (Ig) modules (Ig1-3). Currently, there are two competing models (symmetric and asymmetric) of the FGF-FGFR-heparin complex based on crystal structures. Indirect evidence exists in support of both models. However, it is not clear which model is physiologically relevant. Our aim was to obtain direct, non-crystallographic evidence in support of them. We found by nuclear magnetic resonance that Ig2 could bind to FGF1 not only via the primary site (present in both models), but also via the secondary site (present only in the symmetric model). Thus, our data support the symmetric model.     

A naturally occurring secreted form of fibroblast growth factor (FGF) receptor 1 binds basic FGF in preference over acidic FGF.

Alternatively spliced variants of fibroblast growth factor receptor 1 mRNA are predicted to encode secreted forms and membrane-bound forms of receptors. The predicted amino acid sequences of these receptor variants differ in a portion of the extracellular region. In this study, we characterized the function of one of these splice variants which was predicted by its cDNA to be a secreted FGF receptor. We expressed this secreted form of the human FGFR1 (sFGFR1) in Chinese hamster ovary cells. The sFGFR1 protein oligomerized upon ligand binding. Surprisingly, the sFGFR1 preferentially bound basic FGF over acidic FGF. In cross-linking experiments, the sFGFR1 showed higher binding affinity for basic FGF (Kd approximately 30 nM) than for acidic FGF (Kd greater than 300 nM). These results suggest that this secreted form of FGF receptor has an unusual ligand binding specificity that may be important for its biological role in vivo.     

Acidic fibroblast growth factor (FGF) potentiates glial-mediated neurotoxicity by activating FGFR2 IIIb protein

Previous studies indicate that astrocytes are the brain cells that express acidic fibroblast growth factor (aFGF) and that the expression is increased upon activation. However, there has been no study investigating the significance of this phenomenon. Here we report that aFGF treatment of IFNγ-stimulated human astrocytes, and LPS/IFNγ-stimulated human microglia, enhances their secretion of inflammatory cytokines and other materials toxic to human neuroblastoma SH-SY5Y cells. The mechanism of aFGF enhancement involves stimulation of the receptor FGFR2 IIIb. We show by RT-PCR that this receptor, but not other FGF receptors, is robustly expressed by astrocytes and microglia. We establish by Western blotting, and immunohistochemistry on postmortem human brain tissue that the FGFR2 IIIb protein is expressed by both of these glial cell types. We blocked the inflammatory stimulant action of aFGF by transfecting microglia and astrocytes with a small inhibitory RNA (siRNA) to FGFR2 IIIb as well as by removal of aFGF using an anti-aFGF antibody. Treatment with bFGF in combination with the stimulants was without effect, but together with aFGF, it partially counteracted the action of aFGF, indicating that it may be a weak antagonist of FGFR2 IIIb. The inflammatory effect was also attenuated by treatment with inhibitors of protein kinase C, Src tyrosine kinase, and MEK-1/2 indicating the involvement of these intracellular pathways. Our data suggest that inhibition of expression or release of aFGF could have therapeutic potential by inhibiting inflammation in neurodegenerative diseases such as Alzheimer disease where many neuroinflammatory molecules are prominently expressed.     

Structural insights into the human D1 and D2 dopamine receptor signaling complexes

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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.     

A light scattering study of the interaction of fibroblast growth factor (FGF) with its receptor

Light scattering technique has been used to study the interaction between fibroblast growth factor (FGF) and its receptor. In this study, a general mathematical model has been developed where the concentration of product formed by the interaction of two proteins and its dependence on the initial concentration of interacting proteins have been determined using laser light scattering. Calculated hydrodynamic diameters reveal that both human fibroblast growth factor (hFGF-1) and its receptor domain (D2 domain) exist as monomers in solution. Titration of hFGF-1 and the D2 domain of FGFR show that they interact in a 1:1 stoichiometry in solution. The binding stoichiometry does not depend on the concentrations of the interacting proteins. The results of this study, for the first time to our knowledge, provide an unambiguous evidence that the 2:2 binary complex of FGF and FGFR observed in the crystal structures of the FGF-FGFR complex (in the absence of heparin) is possibly a crystallization artifact.     

Acidic fibroblast growth factor (FGF) from bovine brain: amino-terminal sequence and comparison with basic FGF.

Acidic fibroblast growth factor (FGF) from bovine brain has been isolated by a combination of salt precipitation, ion-exchange chromatography, heparin-Sepharose affinity chromatography and reverse phase h.p.l.c. The amino acid composition of the mitogen is indistinguishable from that of acidic FGF previously purified. The amino-terminal sequence of acidic FGF was established as Phe-Asn-Leu- Pro-Gly-Asn-Tyr-Lys-Pro-Lys-Leu-X-Tyr-X-Ser-Asn-Gly-X-Tyr-Phe-Leu-Arg-Il e-Leu-Pro-Asp-Gly. Acidic FGF is structurally different from basic FGF as judged by mol. wt., amino acid composition and sequence. In vitro biological comparison of the two growth factors indicates that acidic and basic FGFs possess the same intrinsic activities to stimulate the proliferation of aorta, vein or capillary endothelial cells and adrenal cortex cells, but acidic FGF is 30-100 times less potent, depending on the cell type.     

Sulfated glycosaminoglycans are required for specific and sensitive fibroblast growth factor (FGF) 19 signaling via FGF receptor 4 and betaKlotho

Secreted from intestine, human fibroblast growth factor 19 (hFGF19) is an endocrine metabolic regulator that controls bile acid synthesis in the liver. Earlier studies have suggested that hFGF19 at 10-100 nM levels signals through FGF receptor 4 (FGFR4) in the presence of a co-receptor, betaKlotho, but its activity and receptor specificity at physiological concentrations (picomolar levels) remain unclear. Here we report that hFGF19 at picomolar levels require sulfated glycosaminoglycans (sGAGs), such as heparan sulfate, heparin, and chondroitin sulfates, for its signaling via human FGFR4 in the presence of human betaKlotho. Importantly, sGAGs isolated from liver are highly active in enhancing the picomolar hFGF19 signaling. At nanomolar levels, in contrast, hFGF19 activates all types of human FGFRs, i.e. FGFR1c, FGFR2c, FGFR3c, and FGFR4 in the co-presence of betaKlotho and heparin and activates FGFR4 even in the absence of betaKlotho. These results show that sGAGs play crucial roles in specific and sensitive hFGF19 signaling via FGF receptors and suggest that hepatic sGAGs are involved in the highly potent and specific signaling of picomolar hFGF19 through FGFR4 and betaKlotho. The results further suggest that hFGF19 at pathological concentrations may evoke aberrant signaling through various FGF receptors.     

Indirect recruitment of the signalling adaptor Shc to the fibroblast growth factor receptor 2 (FGFR2)

The adaptor protein Shc (Src homology and collagen-containing protein) plays an important role in the activation of signalling pathways downstream of RTKs (receptor tyrosine kinases) regulating diverse cellular functions, such as differentiation, adhesion, migration and mitogenesis. Despite being phosphorylated downstream of members of the FGFR (fibroblast growth factor receptor) family, a direct interaction of Shc with this receptor family has not been described to date. Various studies have suggested potential binding sites for the Shc PTB domain (phosphotyrosine-binding domain) and/or the SH2 (Src homology 2) domain on FGFR1, but no interaction of full-length Shc with these sites has been reported in vivo. In the present study, we investigated the importance of the SH2 domain and the PTB domain in recruitment of Shc to FGFR2(IIIc) to characterize the interaction of these two proteins. Confocal microscopy revealed extensive co-localization of Shc with FGFR2. The PTB domain was identified as the critical component of Shc which mediates membrane localization. Results from FLIM (fluorescence lifetime imaging microscopy) revealed that the interaction between Shc and FGFR2 is indirect, suggesting that the adaptor protein forms part of a signalling complex containing the receptor. We identified the non-RTK Src as a protein which potentially mediates the formation of such a ternary complex. Although an interaction between Src and Shc has been described previously, in the present study we implicate the Shc SH2 domain as a novel mediator of this association. The recruitment of Shc to FGFR2 via an indirect mechanism provides new insight into the regulation of protein assembly and activation of various signalling pathways downstream of this RTK.     

Cooperative dimerization of fibroblast growth factor 1 (FGF1) upon a single heparin saccharide may drive the formation of 2:2:1 FGF1.FGFR2c.heparin ternary complexes

The related glycosaminoglycans heparin and heparan sulfate are essential for the activity of the fibroblast growth factor (FGF) family as they form an integral part of the signaling complex at the cell surface. Using size-exclusion chromatography we have studied the capacities of a variety of heparin oligosaccharides to bind FGF1 and FGFR2c both separately and together in ternary complexes. In the absence of heparin, FGF1 had no detectable affinity for FGFR2c. However, 2:2:1 complexes formed spontaneously in solution between FGF1, FGFR2c, and heparin octasaccharide (dp8). The dp8 sample was the shortest chain length that bound FGFR2c, that dimerized FGF1, and that promoted a strong mitogenic response to FGF1 through FGFR2c. Heparin hexasaccharide and various selectively desulfated heparin dp12s failed to bind FGFR2c and could only interact with FGF1 monomerically. These saccharides formed 1:1:1 complexes with FGF1 and FGFR2c, which had no tendency to self-associate, suggesting that binding of two FGF1 molecules to the same saccharide chain is a prerequisite for subsequent FGFR2c dimerization. We found that FGF1 dimerization upon heparin was favored over monomeric interactions even when a large excess of saccharide was present. A cooperative mechanism of FGF1 dimerization could explain how 2:2:1 signaling complexes form at the cell surface, an environment rich in heparan sulfate.     

Molecular networks in FGF signaling: flotillin-1 and cbl-associated protein compete for the binding to fibroblast growth factor receptor substrate 2

Fibroblast growth factor receptor substrate 2 (FRS2α) is a signaling adaptor protein that regulates downstream signaling of many receptor tyrosine kinases. During signal transduction, FRS2 can be both tyrosine and threonine phosphorylated and forms signaling complexes with other adaptor proteins and tyrosine phosphatases. We have here identified flotillin-1 and the cbl-associated protein/ponsin (CAP) as novel interaction partners of FRS2. Flotillin-1 binds to the phosphotyrosine binding domain (PTB) of FRS2 and competes for the binding with the fibroblast growth factor receptor. Flotillin-1 knockdown results in increased Tyr phosphorylation of FRS2, in line with the inhibition of ERK activity in the absence of flotillin-1. CAP directly interacts with FRS2 by means of its sorbin homology (SoHo) domain, which has previously been shown to interact with flotillin-1. In addition, the third SH3 domain in CAP binds to FRS2. Due to the overlapping binding domains, CAP and flotillin-1 appear to compete for the binding to FRS2. Thus, our results reveal a novel signaling network containing FRS2, CAP and flotillin-1, whose successive interactions are most likely required to regulate receptor tyrosine kinase signaling, especially the mitogen activated protein kinase pathway.     

Direct cell cycle regulation by the fibroblast growth factor receptor (FGFR) kinase through phosphorylation-dependent release of Cks1 from FGFR substrate 2

Fibroblast growth factors (FGFs) are upstream activators of the mitogen-activated protein kinase pathway and mitogens in a wide variety of cells. However, whether the mitogen-activated protein kinase pathway solely accounts for the induction of cell cycle or antiapoptotic activity of the FGF receptor (FGFR) tyrosine kinase is not clear. Here we report that cell cycle inducer Cks1, which triggers ubiquitination and degradation of p27(Kip1), associates with the unphosphorylated form of FGFR substrate 2 (FRS2), an adaptor protein that is phosphorylated by FGFR kinases and recruits downstream signaling molecules. FGF-dependent activation of FGFR tyrosine kinases induces FRS2 phosphorylation, causes release of Cks1 from FRS2, and promotes degradation of p27(Kip1) in 3T3 cells. Since degradation of p27(Kip1) is a key regulatory step in activation of the cyclin E/A-Cdk complex during the G(1)/S transition of the cell cycle, the results suggest a novel mitogenic pathway whereby FGF and other growth factors that activate FRS2 directly activate cyclin-dependent kinases.     

Ligand dependent and independent internalization and nuclear translocation of fibroblast growth factor (FGF) receptor 1

Basic fibroblast growth factor (FGF-2) is one of the prototype members of a rapidly expanding family of polypeptides. FGF-2 acts on cells via a dual-receptor system consisting of high-affinity tyrosine kinase receptors (FGFR) and low-affinity receptors comprised of heparan sulfate proteoglycans. Following ligand binding and subsequent internalization, both FGF-2 and FGFR1 are translocated to the nucleus where they have activities distinct from those expressed at the cell surface. Despite the growing number of growth factors and receptors shown to translocate to the nucleus, little is known about the mechanisms of internalization and translocation and how these processes are regulated. In the studies reported in this paper, we examined the roles of clathrin-dependent and -independent endocytosis in the uptake of FGFR1 and one of its ligands, FGF-2. While the uptake of FGF-2 occurred at least partly by a caveolar-dependent mechanism, that of FGFR1 was independent of both caveolae and coated pits. Surprisingly, neither the uptake of FGF-2 nor FGFR1 required the activity of the receptor tyrosine kinase. In addition, we identified a cell cycle-dependent pathway of FGFR1 nuclear translocation that appears to be independent of ligand binding.     

Lacrimo-auriculo-dento-digital syndrome is caused by reduced activity of the fibroblast growth factor 10 (FGF10)-FGF receptor 2 signaling pathway

Lacrimo-auriculo-dento-digital (LADD) syndrome is characterized by abnormalities in lacrimal and salivary glands, in teeth, and in the distal limbs. Genetic studies have implicated heterozygous mutations in fibroblast growth factor 10 (FGF10) and in FGF receptor 2 (FGFR2) in LADD syndrome. However, it is not clear whether LADD syndrome mutations (LADD mutations) are gain- or loss-of-function mutations. In order to reveal the molecular mechanism underlying LADD syndrome, we have compared the biological properties of FGF10 LADD and FGFR2 LADD mutants to the activities of their normal counterparts. These experiments show that the biological activities of three different FGF10 LADD mutants are severely impaired by different mechanisms. Moreover, haploinsufficiency caused by defective FGF10 mutants leads to LADD syndrome. We also demonstrate that the tyrosine kinase activities of FGFR2 LADD mutants expressed in transfected cells are strongly compromised. Since tyrosine kinase activity is stimulated by ligand-induced receptor dimerization, FGFR2 LADD mutants may also exert a dominant inhibitory effect on signaling via wild-type FGFR2 expressed in the same cell. These experiments underscore the importance of signal strength in mediating biological responses and that relatively small changes in receptor signaling may influence the outcome of developmental processes in cells or organs that do not possess redundant signaling pathway.