Role of FGFs in skeletal muscle and limb development

Fibroblast growth factors (FGFs) are a family of nine proteins that bind to three distinct types of cell surface molecules: (i) FGF receptor tyrosine kinases (FGFR-1 through FGFR-4); (ii) a cysteine-rich FGF receptor (CFR); and (iii) heparan sulfate proteoglycans (HSPGs). Signaling by FGFs requires participation of at least two of these receptors: the FGFRs and HSPGs form a signaling complex. The length and sulfation pattern of the heparan sulfate chain determines both the activity of the signaling complex and, in part, the ligand specificity for FGFR-1. Thus, the heparan sulfate proteoglycans are likely to play an essential role in signaling. We have recently identified a role for FGF in limb bud development in vivo. In the chick limb bud, ectopic expression of the 18 kDa form of FGF-2 or FGF-2 fused to an artificial signal peptide at its amino terminus causes skeletal duplications. These data, and the observations that FGF-2 is localized to the subjacent mesoderm and the apical ectodermal ridge in the early developing limb, suggest that FGF-2 plays an important role in limb outgrowth. We propose that FGF-2 is an apical ectodermal ridgederived factor that participates in limb outgrowth and patterning. © 1994 Wiley-Liss, Inc.     

FGF-20, a novel neurotrophic factor, preferentially expressed in the substantia nigra pars compacta of rat brain

We have isolated cDNA encoding a novel FGF (212 amino acids) from rat brain. Because this is the 20th documented member of the FGF family, we tentatively term it FGF-20. Among FGF family members, FGF-20 is most similar to FGF-9 and FGF-16 (70 and 62% amino acid identity, respectively). Human FGF-20 gene was found in the human genomic sequence mapped to the 8p21.3-p22 region. Human FGF-20 is highly identical to rat FGF-20 (95% amino acid identity). FGF-20 mRNA was preferentially expressed in rat brain among the adult major tissues examined. The localization of FGF-20 mRNA in rat brain was also examined by in situ hybridization. FGF-20 mRNA was preferentially expressed in the substantia nigra pars compacta. To examine the biological activity of FGF-20, recombinant rat FGF-20 was produced by insect cells infected with recombinant baculovirus containing rat FGF-20 cDNA. Recombinant rat FGF-20 enhanced the survival of midbrain dopaminergic neurons. The present results indicate that FGF-20 is a novel neurotrophic factor preferentially expressed in the substantia nigra pars compacta of rat brain.     

Cysteine-rich FGF receptor regulates intracellular FGF-1 and FGF-2 levels

The cysteine-rich FGF receptor (CFR) is a 150-kD membrane-associated glycoprotein that specifically binds FGFs. CFR protein is not detectable at the cell surface and immunocytochemistry with anti-CFR antibodies demonstrates that CFR is concentrated in the Golgi apparatus. These data suggest CFR does not function as a plasma membrane FGF receptor. CFR expressed in chinese hamster ovary cells reduces the intracellular accumulation of exogenously applied FGF-1 and FGF-2. A mutant CFR lacking the juxtamembrane, transmembrane and intracellular domains is unable to alter intracellular FGF levels. Mutant CFR is detected throughout the cell, indicating that the domains absent in mutant CFR are required for appropriate subcellular localization and the regulation of intracellular FGF levels. Although the activation of plasma membrane receptors is necessary for cellular responses to FGFs, a requirement for intracellular FGF has also been proposed. The subcellular localization of CFR and its ability to regulate the levels of intracellular FGFs suggests that CFR may be involved in intracellular FGF trafficking and the regulation of cellular responses to FGFs. J. Cell. Physiol. 170:217–227, 1997. © 1997 Wiley-Liss, Inc.     

Molecular cloning of a novel cytokine cDNA encoding the ninth member of the fibroblast growth factor family, which has a unique secretion property.

Glia-activating factor (GAF) is a novel heparin-binding growth factor purified from the culture supernatant of a human glioma cell line. It shows a spectrum of activity slightly different from those of other known growth factors. We have isolated the cDNA which encodes human GAF. A homology search revealed that GAF would be the ninth member of the FGF family, and we therefore call it FGF-9. The human FGF-9 cDNA cloned by using oligonucleotide probes encoded a polypeptide consisting of 208 amino acids. Sequence similarity to other members of the FGF family was estimated to be around 30%. Two cysteine residues and other consensus sequences in family members were also well conserved in the FGF-9 sequence. FGF-9 was found to have no typical signal sequence in its N terminus like those in acidic FGF and basic FGF. Acidic FGF and basic FGF are known not to be secreted from cells in a conventional manner. However, FGF-9 was found to be secreted from cells after synthesis despite its lack of a typical signal sequence. It could be detected exclusively in the culture medium of cDNA-transfected COS cells. The amino acid sequence of proteins purified from culture supernatant of the CHO cell line, which was cDNA transfected and selected as a high producer of FGF-9, showed that no peptides were cleaved from the N terminus except the initiation methionine. The rat FGF-9 cDNA was also cloned, and the structural analysis indicated that the PGF-9 gene is highly conserved. Expression of the FGF-9 gene could be detected in the brain and kidney of the adult rat. Restricted gene expression in organs and the unique secretion nature of the protein suggest that FGF-9 plays a physiological role which differs from those of well-characterized acidic FGF and basic FGF.     

Identification of a novel FGF, FGF-21, preferentially expressed in the liver

We isolated cDNA encoding a novel FGF (210 amino acids) from mouse embryos. As this is the 21st documented FGF, we tentatively term it FGF-21. FGF-21 has a hydrophobic amino terminus ( approximately 30 amino acids), which is a typical signal sequence, and appears to be a secreted protein. The expression of FGF-21 mRNA in mouse adult tissues was examined by Northern blotting analysis. FGF-21 mRNA was most abundantly expressed in the liver, and also expressed in the thymus at lower levels. We also isolated human cDNA encoding FGF-21 (209 amino acids). Human FGF-21 is highly identical ( approximately 75% amino acid identity) to mouse FGF-21. Among human FGF family members, FGF-21 is most similar ( approximately 35% amino acid identity) to FGF-19.     

High affinity immunoreactive FGF receptors in the extracellular matrix of vascular endothelial cells--implications for the modulation of FGF-2

We recently characterized three FGF-binding proteins (FGF-BPs) which are soluble forms of the extracellular domains of the high affinity FGF receptors (Hanneken, A. M., W. Ying, N. Ling, and A. Baird. Proc. Natl. Acad. Sci. USA. 1994. 91:9170-9174). These proteins circulate in blood and have been proposed to modulate the biological activity of the FGF family of proteins. Immunohistochemical studies now demonstrate that these soluble, truncated FGF receptors are also present in the basement membranes of retinal vascular endothelial cells. These immunoreactive proteins can be detected with antibodies raised to the extracellular domain of FGFR-1 but not with antibodies raised to either the juxtamembrane domain or the cytoplasmic domain of FGFR-1. Western blotting of human retinal extracts with the antibody raised to the extracellular domain of FGFR-1 detects specific, low molecular mass proteins at 85 kD and 55 kD, corresponding in size to the FGF-BPs, which are not detected with antibodies against the cytoplasmic domain of the receptor. The interaction of this receptor with the extracellular matrix is not dependent on the presence of FGF-2. Immunoreactive receptors are still detected in vascular basement membranes after the removal of FGF-2 with heparitinase. In addition, the recombinant extracellular domain of FGFR-1 continues to bind to corneal endothelial cell matrix after endogenous FGF-2 has been removed with 2 M NaCl. Acid treatment, which has been shown to disrupt protein interactions with the extracellular matrix, leads to a significant reduction in the presence of the matrix form of the FGF receptor. This loss can be restored with exogenous incubations of the recombinant extracellular domain of FGFR-1. This report is the first demonstration that a truncated form of a high affinity growth factor receptor can be localized to the extracellular matrix. These findings add to the list of binding proteins associated with the extracellular matrix (IGFBP-5) and suggest a potentially new regulatory mechanism for controlling the biological availability of FGF, and other peptide growth factors, in the extracellular matrix.     

cDNA cloning and developmental expression of fibroblast growth factor receptors from Xenopus laevis.

The heparin-binding growth factors constitute a family of homologous polypeptides including basic and acidic fibroblast growth factors (FGFs). These factors participate in a variety of processes, including wound healing, angiogenesis, neuronal survival, and inductive events in the early amphibian embryo. We have isolated three closely related species of cDNA clones for Xenopus FGF receptors. One of these, designated XFGFR-A1, encodes an open reading frame of 814 amino acids. A second class encodes an identical amino acid sequence with the exception of an 88-amino-acid deletion near the 5' end. This species probably arises through alternative splicing. A third class of cDNA corresponding to the shorter form of XFGFR-A1 was isolated and shown to be 95% homologous and is designated XFGFR-A2. Xenopus FGF receptors are similar to FGF receptors from other species in that they contain a transmembrane domain, a tyrosine kinase domain split by a 14-amino-acid insertion, and a unique conserved stretch of eight acidic residues in the extracellular domain. Overexpression of Xenopus FGF receptor protein by transfection of COS1 cells with the corresponding cDNA in a transient expression vector leads to the appearance of new FGF binding sites on transfected cells, consistent with these cDNAs encoding for FGF receptors. RNA gel blot analysis demonstrates that Xenopus FGF receptor mRNA is a maternal message and is expressed throughout early development. When blastula-stage ectoderm is cultured in control amphibian salt solutions, Xenopus FGF receptor mRNA declines to undetectable levels by late neurula stages. However, when cultured in the presence of FGF of XTC mesoderm-inducing factor, Xenopus FGF receptor RNA expression is maintained.     

Identification of a novel fibroblast growth factor, FGF-23, preferentially expressed in the ventrolateral thalamic nucleus of the brain

We isolated mouse cDNA encoding a novel FGF (251 amino acids). As this is the 23rd documented FGF, we termed it FGF-23. FGF-23 has a hydrophobic amino terminus ( approximately 24 amino acids), which is a typical signal sequence. As expected, recombinant mouse FGF-23 was efficiently secreted by High Five insect cell-infected recombinant baculovirus containing the cDNA, indicating that FGF-23 is a secreted protein. We also isolated human cDNA encoding FGF-23 (251 amino acids), which is highly identical ( approximately 72% amino acid identity) to mouse FGF-23. Of human FGF family members, FGF-23 is most similar to FGF-21 and FGF-19 ( approximately 24% and approximately 22% amino acid identities, respectively). Human FGF-23 gene was localized on the chromosome 12p13 and found to be tandem linked (within 5.5 kb) to human FGF-6 gene. The expression of FGF-23 mRNA in mouse adult tissues was examined by real-time quantitative polymerase chain reaction. FGF-23 mRNA was mainly expressed in the brain and thymus at low levels. The localization of FGF-23 mRNA in the brain was examined by in situ hybridization. FGF-23 mRNA in the brain was found to be preferentially expressed in the ventrolateral thalamic nucleus. Therefore, FGF-23 is expected a unique FGF that plays roles in the function of the ventrolateral thalamic nucleus.     

Expression and structure of the human NGF receptor

The nucleotide sequence for the human nerve growth factor (NGF) receptor has been determined. The 3.8 kb receptor mRNA encodes a 427 amino acid protein containing a 28 amino acid signal peptide, an extracellular domain containing four 40 amino acid repeats with six cysteine residues at conserved positions followed by a serine/threonine-rich region, a single transmembrane domain, and a 155 amino acid cytoplasmic domain. The sequence of the extracellular domain of the NGF receptor predicts a highly ordered structure containing a negatively charged region that may serve as the ligand-binding site. This domain is conserved through evolution. Transfection of a full-length cDNA in mouse fibroblasts results in stable expression of NGF receptors that are recognized by monoclonal antibodies to the human NGF receptor and that bind [125I]NGF.     

Atrial natriuretic peptide clearance receptor. Complete sequence and functional expression of cDNA clones

The major class of atrial natriuretic peptide (ANP) receptors was isolated from cultured vascular smooth muscle cells, and a partial amino acid sequence was obtained. This allowed the isolation of cDNA clones from which the entire amino acid sequence was established. The smooth muscle cell ANP receptor appears to be synthesized as a 537-amino acid precursor with an N-terminal membrane translocation signal. The mature form consists of 496 amino acids with a single potential transmembrane domain predicting a 37-amino acid cytoplasmic domain and a large, acidic, extracellular domain low in cysteine and probably containing attached carbohydrate. The receptor is therefore similar in structure to the growth factor receptors but notably lacks repetitive cysteine-rich domains and has a relatively small intracellular domain. Expression of the cloned receptor in Xenopus oocytes elicited high affinity, membrane-associated binding sites for ANP and for truncated and internally deleted analogs of ANP. These results reflect the ligand binding specificity found for the major class of ANP receptors on smooth muscle cells and thus provide additional evidence that two distinct ANP receptors exist since ANP receptor-coupled guanylate cyclase activity exhibits a very different ANP analog specificity.     

Identification of a Frizzled-like cysteine rich domain in the extracellular region of developmental receptor tyrosine kinases

In Drosophila, members of the Frizzled family of tissue-polarity genes encode proteins that appear to function as cell-surface receptors for Wnts. The Frizzled genes belong to the seven transmembrane class of receptors (7TMR) and have on their extracellular region a cysteine-rich domain that has been implicated as the Wnt binding domain. This region has a characteristic spacing of ten cysteines, which has also been identified in FrzB (a secreted antagonist of Wnt signaling) and Smoothened (another 7TMR, which is involved in the hedgehog signalling pathway). We have identified, using BLAST, sequence similarity between the cysteine-rich domain of Frizzled and several receptor tyrosine kinases, which have roles in development. These include the muscle-specific receptor tyrosine kinase (MuSK), the neuronal specific kinase (NSK2), and ROR1 and ROR2. At present, the ligands for these developmental tyrosine kinases are unknown. Our results suggest that Wnt-like ligands may bind to these developmental tyrosine kinases     

Identification of a Frizzled-like cysteine rich domain in the extracellular region of developmental receptor tyrosine kinases.

In Drosophila, members of the Frizzled family of tissue-polarity genes encode proteins that appear to function as cell-surface receptors for Wnts. The Frizzled genes belong to the seven transmembrane class of receptors (7TMR) and have on their extracellular region a cysteine-rich domain that has been implicated as the Wnt binding domain. This region has a characteristic spacing of ten cysteines, which has also been identified in FrzB (a secreted antagonist of Wnt signaling) and Smoothened (another 7TMR, which is involved in suppression of the hedgehog pathway). We have identified, using BLAST, sequence similarity between the cysteine-rich domain of Frizzled and several receptor tyrosine kinases, which have roles in development. These include the muscle-specific receptor tyrosine kinase (MuSK), the neuronal specific kinase (NSK2), and ROR1 and ROR2. At present, the ligands for these developmental tyrosine kinases are unknown. Our results suggest that Wnt-like ligands may bind to these developmental tyrosine kinases.     

Protein sequence of endothelial glycoprotein IIIa derived from a cDNA clone. Identity with platelet glycoprotein IIIa and similarity to "integrin"

Platelet membrane glycoprotein (GP) IIIa forms a Ca2+-dependent heterodimer complex with GP IIb. The GP IIb-IIIa complex constitutes the fibrinogen and fibronectin receptor on stimulated platelets. A biochemically and immunologically similar membrane glycoprotein complex is present on endothelial cells. A human umbilical vein endothelial cell cDNA library was screened using oligonucleotide probes designed from peptide sequences obtained from platelet GP IIIa. A cDNA clone was sequenced and found to encode a protein of 84.5 kDa. The translated endothelial cDNA contained five sequences that corresponded to peptide sequences in platelet GP IIIa, including the amino-terminal 19 residues. Thus, the endothelial and platelet forms of GP IIIa are apparently identical. Glycoprotein IIIa consists of a long amino-terminal extracellular domain with several potential N-linked glycosylation sites and four cysteine-rich tandem repeats, a 29-residue hydrophobic transmembrane segment, and a short carboxyl-terminal cytoplasmic domain. Glycoprotein IIIa has a 47% amino acid sequence homology to "integrin," a fibronectin receptor from chicken embryo fibroblasts. This homology suggests that GP IIIa is a member of a family of cell-surface adhesion receptors.     

Identification of a novel fibroblast growth factor, FGF-22, preferentially expressed in the inner root sheath of the hair follicle

We isolated cDNA encoding a novel fibroblast growth factor (FGF-22) (170 amino acids) from human placenta. Of the FGF family members, FGF-22, which appears to be a secreted protein, is most similar to FGF-10 and FGF-7 (approximately 46% and approximately 40% amino acid identities, respectively). The human FGF-22 gene was localized on chromosome 19p13.3. We also isolated mouse cDNA encoding FGF-22 (162 amino acids) from the skin. Mouse FGF-22 shows high homology (87% amino acid identity) to human FGF-22. Mouse FGF-22 mRNA was found to be preferentially expressed in the skin among the mouse adult tissues examined by Northern blotting analysis. By in situ hybridization, FGF-22 mRNA in the skin was found to be preferentially expressed in the inner root sheath of the hair follicle. Therefore, FGF-22 is expected to be a unique FGF that plays a role in hair development.     

FGFR-4, a novel acidic fibroblast growth factor receptor with a distinct expression pattern.

We have previously identified two novel members of the fibroblast growth factor receptor (FGFR) gene family expressed in K562 erythroleukemia cells. Here we report cDNA cloning and analysis of one of these genes, named FGFR-4. The deduced amino acid sequence of FGFR-4 is 55% identical with both previously characterized FGFRs, flg and bek, and has the structural characteristics of a FGFR family member including three immunoglobulin-like domains in its extracellular part. Antibodies raised against the carboxy terminus of FGFR-4 detected 95 and 110 kd glycoproteins with a protein backbone of 88 kd in COS cells transfected with a FGFR-4 cDNA expression vector. The FGFR-4 protein expressed in COS cells could also be affinity-labeled with radioiodinated acidic FGF. Furthermore, ligand binding experiments demonstrated that FGFR-4 binds acidic FGF with high affinity but does not bind basic FGF. FGFR-4 is expressed as a 3.0 kb mRNA in the adrenal, lung, kidney, liver, pancreas, intestine, striated muscle and spleen tissues of human fetuses. The expression pattern of FGFR-4 is distinct from that of flg and bek and the yet additional member of the same gene family, FGFR-3, which we have also cloned from the K562 leukemia cells. Our results suggest that FGFR-4 along with other fibroblast growth factor receptors performs cell lineage and tissue-specific functions.     

Fibroblast growth factors and their receptors in the central nervous system

Fibroblast growth factors (FGFs) and their receptors constitute an elaborate signaling system that participates in many developmental and repair processes of virtually all mammalian tissues. Among the 23 FGF members, ten have been identified in the brain. Four FGF receptors (FGFRs), receptor tyrosine kinases, are known so far. Ligand binding of these receptors greatly depends on the presence of heparan sulfate proteoglycans, which act as low affinity FGFRs. Ligand binding specificity of FGFRs depends on the third extracellular Ig-like domain, which is subject to alternative splicing. Activation of FGFRs triggers several intracellular signaling cascades. These include phosphorylation of src and PLC leading finally to activation of PKC, as well as activation of Crk and Shc. SNT/FRS2 serves as an alternative link of FGFRs to the activation of PKC and, in addition, activates the Ras signaling cascade. In the CNS, FGFs are widely expressed; FGF-2 is predominantly synthesized by astrocytes, whereas other FGF family members, e.g., FGF-5, FGF-8, and FGF-9, are primarily synthesized by neurons. During CNS development FGFs play important roles in neurogenesis, axon growth, and differentiation. In addition, FGFs are major determinants of neuronal survival both during development and during adulthood. Adult neurogenesis depends greatly on FGF-2. Finally, FGF-1 and FGF-2 seem to be involved in the regulation of synaptic plasticity and processes attributed to learning and memory.     

Fibrolast growth factors in the nervous system

Fibroblast growth factors (FGFs) exhibit widespread mitogenic and neurotrophic activities. Nine members of the family are currently known, and FGF-1 and FGF-2 are present in relatively high levels in CNS. FGF-1 is expressed by a subset of neuronal populations, while FGF-2 is expressed by astrocytes. FGF-1 and FGF-2 lack signal peptides and appear to be present mainly in inracellular compartmens. This suggests that the factors may act as initiators of a repair response after injury. Support for this notion comes from observations that FGF-1 and FGF-2 levels are low during critical phases of development, but high in the adult CNS. A family of transmembrane tyrosine kinase receptors (FGFRs) mediates the effects of FGFs. Four different genes coding for FGF receptors are currently known, three of which are expressed in cell type-specific patterns in the CNS The main receptor variants present in this tissue, however, can by themselves not distinguish between FGF-1 and FGF-2. Additional selectivity may be established by interaction of the FGFs and their receptors with select heparan proteoglycans (HSPGs). Therefore, the precise physiological role of FGFs is determined by the combination of cell type-specific patterns of expression of FGFs, FGFRs and HSPGs together with the mechanisms that regulate the extracellular availability of FGFs. 1994 John Wiley & Sons, Inc.     

Androgen-induced growth factor and its receptor: Demonstration of the androgen-induced autocrine loop in mouse mammary carcinoma cells

SC-3 cells derived from mouse mammary carcinoma (Shinogi carcinoma 115) exhibit remarkable growth enhancement and cell morphology change in response to androgen stimuli. These events are mediated through an androgen-induced growth factor (AIGF). Amino acid sequence deduced from cDNA reveals that AIGF has 215 amino acids with a signal peptide and scattered regions homologous to fibroblast growth factor (FGF) family proteins. The biological ability of AIGF to stimulate SC-3 cell growth is inhibited by heparin or suramin. More importantly, antisense oligodeoxynucleotide of AIGF can block androgen-induced growth of SC-3 cells. Upon synthesis under the control of androgen, AIGF is immediately secreted into the extracellular space without intracellular accumulation. At the early phase (18–24 h) of androgen stimulation, however, AIGF is mainly associated with the glycosaminoglycan on the cell surface or extracellular matris. In addition, treatment of SC-3 cells with sulfation blocker (chlorate) or heparitinase results in the abolishment of their ability to respond to androgen or AIGF, indicating that heparan sulfate has important roles for condensing AIGF on or near cell surface as well as potentiating the biological activity of AIGF. Then, AIGF can bind to the FGF receptor. Northern blot analysis and cDNA cloning indicate that SC-3 cells predominantly express the FGF receptor 1 with some altered amino acid sequences. Transfection of expression vectors of AIGF and this variant from of FGF receptor 1 into FGF receptor-negative myoblast cells (L 6 cells) confirms that a variant from of FGF receptor 1 is a receptor of AIGF. These results clearly demonstrate that an autocrine mechanism is operating in androgen-induced growth of SC-3 cells.     

Expression cloning of a receptor for human granulocyte-macrophage colony-stimulating factor.

Two cDNA clones encoding a receptor for human granulocyte-macrophage colony-stimulating factor (hGM-CSF-R) were isolated by expression screening of a library made from human placental mRNA. Pools of recombinant plasmid DNA were electroporated into COS cells which were then screened for their capacity to bind radioiodinated hGM-CSF using a sensitive microscopic autoradiographic approach. The cloned GM-CSF-R precursor is a 400 amino acid polypeptide (Mr 45,000) with a single transmembrane domain, a glycosylated extracellular domain and a short (54 amino acids) intracytoplasmic tail. It does not contain a tyrosine kinase domain nor show homology with members of the immunoglobulin super gene family, but does show some significant sequence homologies with receptors for several other haemopoietic growth factors, including those for interleukin-6, erythropoietin and interleukin-2 (beta-chain) and also to the prolactin receptor. When transfected into COS cells the cloned cDNA directed the expression of a GM-CSF-R showing a single class of affinity (KD = 2(-8) nM) and specificity for human GM-CSF but not interleukin-3. Messenger RNA coding for this receptor was detected in a variety of haemopoietic cells known to display hGM-CSF binding, and cross-linking experiments revealed a similar size for the glycosylated receptors in transfected COS and haemopoietic cells.