Human fibroblast growth factor receptor 3 gene (FGFR3): genomic sequence and primer set information for gene analysis

We have determined the nucleotide sequence of the human fibroblast growth factor receptor 3 (FGFR3) gene, including 800 bp of the 5′-flanking region and compared the sequence with the previously published murine Fgfr3 gene. The organization of the gene is highly conserved between man and mouse. We used the intron sequences to design a set of primers that allow amplification of the 17 exons (2–18) that encode the complete open reading frame. Using these primers the FGFR3 gene can be amplified at the genomic level, which significantly facilitates mutational screening. Received: 27 December 1996 / Accepted: 6 March 1997     

FRS2 family docking proteins with overlapping roles in activation of MAP kinase have distinct spatial-temporal patterns of expression of their transcripts

FRS2alpha and FRS2beta, two members of the FRS2 family of docking proteins, become tyrosine phosphorylated in response to fibroblast growth factor (FGF) or nerve growth factor (NGF) stimulation. Tyrosine phosphorylated FRS2alpha serves as a platform for the recruitment of multiple signaling proteins for activation of the Ras-mitogen-activated protein (MAP) kinase signaling cascade. We report that Frs2alpha and Frs2beta have distinct spatio-temporal expression patterns in mouse embryos. We further show that FRS2beta can compensate for the loss of FRS2alpha for activation of MAP kinase when expressed in fibroblasts from Frs2alpha(-/-) mouse embryos. We propose that the FRS2 family proteins have distinct roles in vivo through activation of common signaling proteins including MAP kinase.     

Genomic structure, mapping, and expression analysis of the mammalian Lunatic, Manic, and Radical fringe genes

The three members of the mammalian fringe gene family, Manic fringe (Mfng), Radical fringe (Rfng), and Lunatic fringe (Lfng), were identified on the basis of their similarity to Drosophila fringe (fng) and their participation in the evolutionarily conserved Notch receptor signaling pathway. Fringe genes encode pioneer secretory proteins with weak similarity to glycosyltransferases. Both expression patterns and functional studies support an important role for Fringe genes in patterning during embryonic development and an association with cellular transformation. We have now further characterized the expression and determined the chromosomal localization and genomic structure of the mouse Mfng, Rfng, and Lfng genes; the genomic structure and conceptual open reading frame of the human RFNG gene; and the refined chromosomal localization of the three human fringe genes. The mouse Fringe genes are expressed in the embryo and in adult tissues. The mouse and human Fringe family members map to three different chromosomes in regions of conserved synteny: Mfng maps to mouse Chr 15, and MFNG maps to human Chr 22q13.1 in the region of two cancer-associated loci; Lfng maps to mouse Chr 5, and LFNG maps to human Chr 7p22; Rfng maps to mouse Chr 11, and RFNG maps to human Chr 17q25 in the minimal region for a familial psoriasis susceptibility locus. Characterization of the genomic loci of the Fringe gene family members reveals a conserved genomic organization of 8 exons. Comparative analysis of mammalian Fringe genomic organization suggests that the first exon is evolutionarily labile and that the Fringe genes have a genomic structure distinct from those of previously characterized glycosyltransferases. Received: 19 February 1999 / Accepted: 22 February 1999     

FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors

The docking protein FRS2 was implicated in the transmission of extracellular signals from the fibroblast growth factor (FGF) or nerve growth factor (NGF) receptors to the Ras/mitogen-activated protein kinase signaling cascade. The two members of the FRS2 family, FRS2alpha and FRS2beta, are structurally very similar. Each is composed of an N-terminal myristylation signal, a phosphotyrosine-binding (PTB) domain, and a C-terminal tail containing multiple binding sites for the SH2 domains of the adapter protein Grb2 and the protein tyrosine phosphatase Shp2. Here we show that the PTB domains of both the alpha and beta isoforms of FRS2 bind directly to the FGF or NGF receptors. The PTB domains of the FRS2 proteins bind to a highly conserved sequence in the juxtamembrane region of FGFR1. While FGFR1 interacts with FRS2 constitutively, independent of ligand stimulation and tyrosine phosphorylation, NGF receptor (TrkA) binding to FRS2 is strongly dependent on receptor activation. Complex formation with TrkA is dependent on phosphorylation of Y490, a canonical PTB domain binding site that also functions as a binding site for Shc (NPXpY). Using deletion and alanine scanning mutagenesis as well as peptide competition assays, we demonstrate that the PTB domains of the FRS2 proteins specifically recognize two different primary structures in two different receptors in a phosphorylation-dependent or -independent manner. In addition, NGF-induced tyrosine phosphorylation of FRS2alpha is diminished in cells that overexpress a kinase-inactive mutant of FGFR1. This experiment suggests that FGFR1 may regulate signaling via NGF receptors by sequestering a common key element which both receptors utilize for transmitting their signals. The multiple interactions mediated by FRS2 appear to play an important role in target selection and in defining the specificity of several families of receptor tyrosine kinases.     

Trk receptor binding and neurotrophin/fibroblast growth factor (FGF)-dependent activation of the FGF receptor substrate (FRS)-3

We have investigated the signaling properties of the fibroblast growth factor (FGF) receptor substrate 3 (FRS3), also known as SNT-2 or FRS2beta, in neurotrophin-dependent differentiation in comparison with the related adapter FRS2 (SNT1 or FRS2alpha). We demonstrate that FRS3 binds all neurotrophin Trk receptor tyrosine kinases and becomes tyrosine phosphorylated in response to NGF, BDNF, NT-3 and FGF stimulation in transfected cells and/or primary cortical neurons. Second, the signaling molecules Grb2 and Shp2 bind FRS3 at consensus sites that are highly conserved among FRS family members and that Shp2, in turn, becomes tyrosine phosphorylated. While FRS3 over-expression in PC12 cells neither increases NGF-induced neuritogenesis nor activation of Map kinase/AKT, comparable to previous reports on FRS2, over-expression of a chimeric adapter containing the PH/PTB domains of the insulin receptor substrate (IRS) 2, in place of the PTB domain of FRS3 (IRS2-FRS3) supports insulin-dependent Map kinase activation and neurite outgrowth in PC12 cells. Collectively, these data demonstrate that FRS3 supports ligand-induced Map kinase activation and that the chimeric IRS2-FRS3 adapter is stimulating sufficient levels of activated MapK to support neurite outgrowth in PC12 cells.     

Genomic organization and expression profile of the human and mouse <Emphasis Type="Italic">WAVE</Emphasis> gene family

The WAVE gene family, which contains three members, has been shown to play a major role in the actin polymerization and cytoskeleton organization processes. We have identified the WAVE3 gene from Chromosome (Chr) 13q12, as being involved in one of the breakpoints of a t(1:13)(q21:q12) reciprocal translocation, in a patient with ganglioneuroblastoma (Sossey-Alaoui et al. 2002; Oncogene 21: 5967–5974). We have also reported the cloning of the mouse Wave3. During our analysis of the human gene map, we also noted that WAVE2 maps to Chr region lp35-36, which frequently undergoes loss of heterozygosity and deletion in advanced stage neuroblastoma. These data clearly indicate a possible involvement of the WAVE genes in the pathogenesis of neuroblastoma. In this study, we report the complete genomic organization and expression profile of the three human WAVE genes and their mouse orthologs. We show that the WAVE genes have distinctive expression patterns in both adult and fetal human and mouse tissues. We also show a high level of conservation between these genes, in both the nucleotide and protein sequences. We finally show that the genomic structure is highly conserved among these genes and that the mouse Wave genes map to chromosome regions that have synteny in the human genome. The gene content in these syntenic regions is also conserved, suggesting that the WAVE genes are derived from a common ancient ancestor by genome duplication. The genomic characterization and expression analysis of the WAVE genes provide the basis towards understanding the function of these genes. It also provides the first steps towards the development of mouse models for the role of the WAVE genes in actin and cytoskeleton organization in general, and in the development of neuroblastoma in particular.     

Identification and characterization of two related murine genes, Eat2a and Eat2b, encoding single SH2-domain adapters

Human EAT-2 (SH2D1B) and SLAM-associated protein (SAP) (SH2D1A) are single SH2-domain adapters, which bind to specific tyrosine residues in the cytoplasmic tail of six signaling lymphocytic activation molecule (SLAM) (SLAMF1)-related receptors. Here we report that, unlike in humans, the mouse and rat Eat2 genes are duplicated with an identical genomic organization. The coding regions of the mouse Eat2a and Eat2b genes share 91% identity at the nucleotide level and 84% at the protein level; similarly, segments of introns are highly conserved. Whereas expression of mouse Eat2a mRNA was detected in multiple tissues, Eat2b was only detectable in mouse natural killer cells, CD8⁺ T cells, and ovaries, suggesting a very restricted tissue expression of the latter. Both the EAT-2A and EAT-2B coimmunoprecipitated with mouse SLAM in transfected cells and augmented tyrosine phosphorylation of the cytoplasmic tail of SLAM. Both EAT-2A and EAT-2B bind to the Src-like kinases Fyn, Hck, Lyn, Lck, and Fgr, as determined by a yeast two-hybrid assay. However, unlike SAP, the EAT-2 proteins bind to their kinase domains and not to the SH3 domain of these kinases. Taken together, the data suggest that both EAT-2A and EAT-2B are adapters that recruit Src kinases to SLAM family receptors using a mechanism that is distinct from that of SAP. Electronic supplementary material Supplementary material is available for this article at and accessible for authorised users. S. Calpe and E. Erdős contributed equally to this work     

Fibroblast growth factor receptor 1 (FGFR1) tyrosine phosphorylation regulates binding of FGFR substrate 2alpha (FRS2alpha) but not FRS2 to the receptor

Binding of the fibroblast growth factor (FGF) to the FGF receptor (FGFR) tyrosine kinase leads to receptor tyrosine autophosphorylation as well as phosphorylation of multiple downstream signaling molecules that are recruited to the receptor either by direct binding or through adaptor proteins. The FGFR substrate 2 (FRS2) family consists of two members, FRS2alpha and FRS2beta, and has been shown to recruit multiple signaling molecules, including Grb2 and Shp2, to FGFR1. To better understand how FRS2 interacted with FGFR1, in vivo binding assays with coexpressed FGFR1 and FRS2 recombinant proteins in mammalian cells were carried out. The results showed that the interaction of full-length FRS2alpha, but not FRS2beta, with FGFR1 was enhanced by activation of the receptor kinase. The truncated FRS2alpha mutant that was comprised only of the phosphotyrosine-binding domain (PTB) bound FGFR1 constitutively, suggesting that the C-terminal sequence downstream the PTB domain inhibited the PTB-FGFR1 binding. Inactivation of the FGFR1 kinase and substitutions of tyrosine phosphorylation sites of FGFR1, but not FRS2alpha, reduced binding of FGFR1 with FRS2alpha. The results suggest that although the tyrosine autophosphorylation sites of FGFR1 did not constitute the binding sites for FRS2alpha, phosphorylation of these residues was essential for optimal interaction with FRS2alpha. In addition, it was demonstrated that the Grb2-binding sites of FRS2alpha are essential for mediating signals of FGFR1 to activate the FiRE enhancer of the mouse syndecan 1 gene. The results, for the first time, demonstrate the specific signals mediated by the Grb2-binding sites and further our understanding of FGF signal transmission at the adaptor level.     

Molecular Cloning and Chromosomal Localization of the MouseGpr37Gene Encoding an Orphan G-Protein-Coupled Peptide Receptor Expressed in Brain and Testis

We report the cloning of the mouse ortholog of the humanGPR37gene, which encodes an orphan G-protein-coupled receptor highly expressed in brain tissues and homologous to neuropeptide-specific receptors ([20],Genomics 45:68–77;[45],Biochem. Biophys. Res. Commun. 233:559–567). The genomic organization of theGPR37gene is conserved in both mouse and human species with a single intron interrupting the receptor-coding sequence within the presumed third transmembrane domain. Comparative genetic mapping of theGPR37gene showed that it maps to a conserved chromosomal segment on proximal mouse chromosome 6 and human chromosome 7q31. The mouseGpr37gene contains an open reading frame coding for a 600-amino-acid protein 83% identical to the humanGPR37gene product. The predicted mouse GPR37 protein contains seven putative hydrophobic transmembrane domains, as well as a long (249 amino acid residues), arginine- and proline-rich amino-terminal extracellular domain, which is also a distinctive feature of the human GPR37 receptor. Northern blot analysis of mouse tissues withGpr37-specific probes revealed a main 3.8-kb mRNA and a much less abundant 8-kb mRNA, both expressed in the brain. A 3-kb mRNA is also expressed in the testis. Both the mouse and the humanGPR37genes may belong to a class of highly conserved mammalian genes encoding a novel type of G-protein-coupled receptor predominantly expressed in the brain.     

The docking protein FRS2alpha is an essential component of multiple fibroblast growth factor responses during early mouse development

The docking protein FRS2alpha is a major mediator of fibroblast growth factor (FGF) signaling. However, the physiological role of FRS2alpha in vivo remains unknown. In this report, we show that Frs2alpha-null mouse embryos have a defect in anterior-posterior (A-P) axis formation and are developmentally retarded, resulting in embryonic lethality by embryonic day 8. We demonstrate that FRS2alpha is essential for the maintenance of self-renewing trophoblast stem (TS) cells in response to FGF4 in the extraembryonic ectoderm (ExE) that gives rise to tissues of the placenta. By analyzing chimeric embryos, we found that FRS2alpha also plays a role in cell movement through the primitive streak during gastrulation. In addition, experiments are presented demonstrating that Bmp4 expression in TS cells is controlled by mitogen-activated protein kinase-dependent FGF4 stimulation. Moreover, both the expression of Bmp4 in ExE and activation of Smad1/5 in epiblasts are reduced in Frs2alpha-null embryos. These experiments underscore the critical role of FRS2alpha in mediating multiple processes during embryonic development and reveal a potential new link between FGF and Bmp4 signaling pathways in early embryogenesis.     

Molecular characterization of a new member of the immunoglobulin superfamily that potentially functions in T-cell activation and development

 Differential hybridization cloning has been used to isolate a novel chicken thymic activation and developmental sequence (cTADS). The nucleotide sequence of the cTADS cDNA predicts an open reading frame of 439 amino acids. The inferred cTADS protein possesses a hydrophobic membrane-spanning domain and putative intracellular kinase activation domains. Its extracellular domain shares similarities with the immunoglobulin protein superfamily, featuring two conserved immunoglobulin folds that resemble C1 and C2 constant regions. The cTADS sequence shows similarity to a subfamily of proteins involved in cellular adhesion: chicken neural cell adhesion molecule and human opioid-binding adhesion molecule, and to proteins that have a biological role in intracellular signaling: mouse platelet-derived growth factor receptor and human fibroblast growth factor receptor. cTADS is differentially expressed in chicken thymic cells during embryonic development and during activation through the T-cell receptor. Sequence similarities and expression patterns suggest that cTADS could be involved in cell recognition and adhesion, and/or peptide ligand binding. Received: 1 May 1999 / Revised: 1 October 1999     

Fibroblast growth factor-2-induced signaling through lipid raft-associated fibroblast growth factor receptor substrate 2 (FRS2)

The plasma membrane is not homogeneous but contains specific subcompartments characterized by their unique lipid and protein composition. Based on their enrichment in various signaling molecules, these membrane microdomains are recognized to be sites of localized signal transduction for a number of extracellular stimuli. We have previously shown that fibroblast growth factor-2 (FGF2) induced a specific signaling response within a lipid raft membrane microdomain in human neuroblastoma cells characterized by the tyrosine phosphorylation of a p80 phosphoprotein. Herein, we show that this protein is the signaling adaptor FRS2 and that it is localized exclusively to lipid rafts in vitro and in vivo. We have examined how the tyrosine phosphorylation and serine-threonine phosphorylation of FRS2 within lipid rafts affect the response of cells to FGF2 signaling. Our data suggest that activation of protein kinase C, Src family kinases, and MEK1/2 are involved in regulating serine-threonine phosphorylation of FRS2, which can indirectly affect FRS2 phosphotyrosine levels. We also show that Grb2 is recruited to lipid rafts during signaling events and that activation of MEK1/2 by different mechanisms within lipid rafts may lead to different cellular responses. This work suggests that compartmentalized signaling within lipid rafts may provide a level of specificity for growth factor signaling.     

Developmental expression patterns of the signaling adapters FRS-2 and FRS-3 during early embryogenesis

Two fibroblast growth factor (FGF) receptor substrates (FRS2 and FRS3) are involved in downstream signaling from activated FGF receptors and neurotrophin-activated Trk receptors. Despite the importance of signaling from these factors in embryogenesis, FRS2 and FRS3 expression patterns during development are unknown. In this study we characterize the expression of FRS2 and FRS3 from E7 to parturition and in adult murine tissues. Both are first detected in whole E8.5 CD1 mouse embryos. FRS2 is detected as early as E7 in the developing syncytiotrophoblast, later in the neural tube (NT) and in many adult and fetal tissues. FRS3 is more restricted in location than FRS2 (fetal NT, heart, stomach, liver and some adult tissues), and is expressed predominantly in the ventricular layer of the developing NT and brains of murine embryos.     

Genomic Structure and Chromosomal Mapping of the MousenovGene

Thenovgene encodes a cysteine-rich protein that is overexpressed in avian nephroblastomas. It is a member of the CCN family of proteins, all of which are involved in cell growth. Genomic and cDNA clones encompassing the mousenovgene have been isolated and characterized. The mousenovgene is highly conserved with the human and chicknovgenes at the level of nucleotide sequence and genomic organization. The exon structure reflects the modular organization of the NOV protein in a number of structural domains. These are highly conserved with other members of the CCN family, as is the distribution of 38 of its 40 cysteine residues. Thenovgene maps to chromosome 15, between D15 Mit 153 and D15 Mit 183, in a region of conserved synteny with human chromosome 8.     

Phylogenetic footprinting and genome scanning identify vertebrate BMP response elements and new target genes

The complex gene regulatory networks governed by growth factor signaling are still poorly understood. In order to accelerate the rate of progress in uncovering these networks, we explored the usefulness of interspecies sequence comparison (phylogenetic footprinting) to identify conserved growth factor response elements. The promoter regions of two direct target genes of Bone Morphogenetic Protein (BMP) signaling in Xenopus, Xvent2 and XId3, were compared with the corresponding human and/or mouse counterparts to identify conserved sequences. A comparison between the Xenopus and human Vent2 promoter sequences revealed a highly conserved 21 bp sequence that overlaps the previously reported Xvent2 BMP response element (BRE). Reporter gene assays using Xenopus animal pole ectodermal explants (animal caps) revealed that this conserved 21 bp BRE is both necessary and sufficient for BMP responsiveness. We combine the same phylogenetic footprinting approach with luciferase assays to identify a highly conserved 49 bp BMP responsive region in the Xenopus Id3 promoter. GFP reporters containing multimers of either the Xvent2 or XId3 BREs appear to recapitulate endogenous BMP signaling activity in transgenic Xenopus embryos. Comparison of the Xvent2 and the XId3 BRE revealed core sequence features that are both necessary and sufficient for BMP responsiveness: a Smad binding element (SBE) and a GC-rich element resembling an OAZ binding site. Based on these findings, we have implemented genome scanning to identify over 100 additional putative target genes containing 2 or more BRE-like sequences which are conserved between human and mouse. RT-PCR and in situ analyses revealed that this in silico approach can effectively be used to identify potential BMP target genes.     

Cloning and expression of hbves, a novel and highly conserved mRNA expressed in the developing and adult heart and skeletal muscle in the human

bves is a novel mRNA expressed in the developing heart in chick and mouse. Here we describe hbves, the human homolog of chick and mouse bves. Northern and dot blot analyses reveal restricted expression in the heart and skeletal muscle in the embryo and adult. BLAST searches of the NCBI databases confirm that hbves is novel. Portions of the sequence are an exact match with genomic PAC 52202, which localizes to Chromosome (Chr) 6q21. Presumably, these matches and intervening sequences match the intron-exon borders of the gene. Computer conformation analysis of the derived amino acid predicts three transmembrane helices with an extracellular C-terminus that is conserved in chick, mouse, and human. bves is highly conserved among all three species at the amino acid level with 75% identity and 92% similarity. Received: 15 February 1999 / Accepted: 29 April 1999     

Characterization of isoforms and genomic organization of mouse calumenin

Calumenin is a multiple EF-hand protein located in endo/sarcoplasmic reticulum of mammalian heart and other tissues [J. Biol. Chem. 272 (1997) 18232; Genomics 49 (1998) 331; Biochim. Biophys. Acta 1386 (1998) 121]. In the present study, a new isoform of mouse calumenin (mouse calumenin 2) was cloned by RT-PCR and genomic DNA PCR. The deduced amino acid sequence of mouse calumenin 2 is 315 aa long with the calculated MW of 37,064 and pI of 4.26. It has 92% aa sequence identity to previously identified mouse calumenin [J. Biol. Chem. 272 (1997) 18232] (mouse calumenin 1). The difference in the aa sequence was restricted to the first two EF-hand regions (residues 74-138). Northern blot analysis shows that mouse calumenin 2 is highly expressed in heart, lung, testis and unpregnant uterus. The expression of mouse calumenin 2 appears to decrease when fetal development is progressed. Genomic DNA PCR, sequencing and data mining of mouse genome database were utilized to examine the exon-intron boundaries of mouse calumenin genes. Both mouse calumenin 1 and 2 genes encompass six exons, and five of them (Exon1, 3, 4, 5 and 6) are identical. However, mouse calumenin 1 contains Exon2-1, whereas mouse calumenin 2 contains a neighboring Exon2-2. The calumenin genes are localized on mouse chromosome 6 having conserved synteny with human chromosome 7q32. For comparison, the genomic organization of human calumenin was also examined using the published human genome database (UCSC Genome Bioinformatics at ). Like mouse calumenin genes, two human calumenin genes also consist of five identical exons (Exon1, 3, 4, 5 and 6) and a different Exon2. The present study suggests that the genomic organization of calumenin genes is well conserved between human and mouse.