Uptake and Intracellular Transport of Acidic Fibroblast Growth Factor: Evidence for Free and Cytoskeleton-anchored Fibroblast Growth Factor Receptors

Endocytic uptake and intracellular transport of acidic FGF was studied in cells transfected with FGF receptor 4 (FGFR4). Acidification of the cytosol to block endocytic uptake from coated pits did not inhibit endocytosis of the growth factor in COS cells transfected with FGFR4, indicating that it is to a large extent taken up by an alternative endocytic pathway. Fractionation of the cells demonstrated that part of the growth factor receptor was present in a low-density, caveolin-containing fraction, but we were unable to demonstrate binding to caveolin in immunoprecipitation studies. Upon treatment of the cells with acidic FGF, the activated receptor, together with the growth factor, moved to a juxtanuclear compartment, which was identified as the recycling endosome compartment. When the cells were lysed with Triton X-100, 3-([3-chloramidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate, or 2-octyl glucoside, almost all surface-exposed and endocytosed FGFR4 was solubilized, but only a minor fraction of the total FGFR4 in the cells was found in the soluble fraction. The data indicate that the major part of FGFR4 is anchored to detergent-insoluble structures, presumably cytoskeletal elements associated with the recycling endosome compartment.

Identification of surface residues mediating tissue factor binding and catalytic function of the serine protease factor VIIa

Factor VIIa (VIIa), the serine protease that initiates the coagulation pathways, is catalytically activated upon binding to its cell surface receptor and cofactor tissue factor (TF). This study provides a comprehensive analysis of the functional surface of VIIa by alanine scanning mutagenesis of 112 residues. Residue side chains were defined which contribute to TF binding and factor X hydrolysis. Energetically important binding contacts at the interface with TF were identified in the first epidermal growth factor domain of VIIa (Gln-64, Ile-69, Phe-71, Arg-79) and in the protease domain (Arg-277, Met-306, Asp-309). The observed energetic defects are in good agreement with the corresponding residues in TF, suggesting that the VIIa light chain plays a prominent role in high affinity binding of cofactor. Mutation of protease domain interface residues indicated that TF allosterically influences the active site of VIIa. Stabilization of a labile zymogen to enzyme transition could explain the activating effect of TF on VIIa catalytic function. Residues important for factor X hydrolysis were found in three regions of the protease domain: (i) specificity determinants in the catalytic cleft and adjacent loops, (ii) an exosite near the TF binding site, and (iii) a large electronegative exosite which is in a position analogous to the basic exosite I of thrombin. TF regions involved in factor X activation are positioned on the same face of the TF·VIIa complex as the two exosites identified on the protease domain surface, providing evidence for an extended interaction of TF·VIIa with macromolecular substrate.

Mannose-6-phosphate/insulin-like growth factor-II receptor is a receptor for retinoic acid

Retinoic acid (RA) exerts diverse biological effects in the control of cell growth in embryogenesis and oncogenesis. These effects of RA are thought to be mediated by the nuclear retinoid receptors. Mannose-6-phosphate (M6P)/insulin-like growth factor-II (IGF-II) receptor is a multifunctional membrane glycoprotein that is known to bind both M6P and IGF-II and function primarily in the binding and trafficking of lysosomal enzymes, the activation of transforming growth factor-β, and the degradation of IGF-II. M6P/IGF-II receptor has recently been implicated in fetal development and carcinogenesis. Despite the functional similarities between RA and the M6P/IGF-II receptor, no direct biochemical link has been established. Here, we show that the M6P/IGF-II receptor also binds RA with high affinity at a site that is distinct from those for M6P and IGF-II, as identified by a photoaffinity labeling technique. We also show that the binding of RA to the M6P/IGF-II receptor enhances the primary functions of this receptor. The biological consequence of the interaction appears to be the suppression of cell proliferation and/or induction of apoptosis. These findings suggest that the M6P/IGF-II receptor mediates a RA response pathway that is important in cell growth regulation. This discovery of the interaction of RA with the M6P/IGF-II receptor may have important implications for our understanding of the roles of RA and the M6P/IGF-II receptor in development, carcinogenesis, and lysosomal enzyme-related diseases.

Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF

The x-ray structure of a C-terminal fragment of the RAP74 subunit of human transcription factor (TF) IIF has been determined at 1.02-Å resolution. The α/β structure is strikingly similar to the globular domain of linker histone H5 and the DNA-binding domain of hepatocyte nuclear factor 3γ (HNF-3γ), making it a winged-helix protein. The surface electrostatic properties of this compact domain differ significantly from those of bona fide winged-helix transcription factors (HNF-3γ and RFX1) and from the winged-helix domains found within the RAP30 subunit of TFIIF and the β subunit of TFIIE. RAP74 has been shown to interact with the TFIIF-associated C-terminal domain phosphatase FCP1, and a putative phosphatase binding site has been identified within the RAP74 winged-helix domain.

Analysis of transforming activity of human synovial sarcoma-associated chimeric protein SYT-SSX1 bound to chromatin remodeling factor hBRM/hSNF2α

Human synovial sarcoma has been shown to exclusively harbor the chromosomal translocation t(X;18) that produces the chimeric gene SYT-SSX. However, the role of SYT-SSX in cellular transformation remains unclear. In this study, we have established 3Y1 rat fibroblast cell lines that constitutively express SYT, SSX1, and SYT-SSX1 and found that SYT-SSX1 promoted growth rate in culture, anchorage-independent growth in soft agar, and tumor formation in nude mice. Deletion of the N-terminal 181 amino acids of SYT-SSX1 caused loss of its transforming activity. Furthermore, association of SYT-SSX1 with the chromatin remodeling factor hBRM/hSNF2α, which regulates transcription, was demonstrated in both SYT-SSX1-expressing 3Y1 cells and in the human synovial sarcoma cell line HS-SY-II. The binding region between the two molecules was shown to reside within the N-terminal 181 amino acids stretch (aa 1–181) of SYT-SSX1 and 50 amino acids (aa 156–205) of hBRM/hSNF2α and we found that the overexpression of this binding region of hBRM/hSNF2α significantly suppressed the anchorage-independent growth of SYT-SSX1-expressing 3Y1 cells. To analyze the transcriptional regulation by SYT-SSX1, we established conditional expression system of SYT-SSX1 and examined the gene expression profiles. The down-regulation of potential tumor suppressor DCC was observed among 1,176 genes analyzed by microarray analysis, and semi-quantitative reverse transcription–PCR confirmed this finding. These data clearly demonstrate transforming activity of human oncogene SYT-SSX1 and also involvement of chromatin remodeling factor hBRM/hSNF2α in human cancer.

X-ray emission from clusters and groups of galaxies

Recent major advances in x-ray imaging and spectroscopy of clusters have allowed the determination of their mass and mass profile out to ≈1/2 the virial radius. In rich clusters, most of the baryonic mass is in the gas phase, and the ratio of mass in gas/stars varies by a factor of 2–4. The baryonic fractions vary by a factor of ≈3 from cluster to cluster and almost always exceed 0.09 h50−[3/2] and thus are in fundamental conflict with the assumption of Ω = 1 and the results of big bang nucleosynthesis. The derived Fe abundances are 0.2–0.45 solar, and the abundances of O and Si for low redshift systems are 0.6–1.0 solar. This distribution is consistent with an origin in pure type II supernova. The amount of light and energy produced by these supernovae is very large, indicating their importance in influencing the formation of clusters and galaxies. The lack of evolution of Fe to a redshift of z ≈ 0.4 argues for very early enrichment of the cluster gas. Groups show a wide range of abundances, 0.1–0.5 solar. The results of an x-ray survey indicate that the contribution of groups to the mass density of the universe is likely to be larger than 0.1 h50−2. Many of the very poor groups have large x-ray halos and are filled with small galaxies whose velocity dispersion is a good match to the x-ray temperatures.

Mutations within a furin consensus sequence block proteolytic release of ectodysplasin-A and cause X-linked hypohidrotic ectodermal dysplasia

X-linked hypohidrotic ectodermal dysplasia (XLHED) is a heritable disorder of the ED-1 gene disrupting the morphogenesis of ectodermal structures. The ED-1 gene product, ectodysplasin-A (EDA), is a tumor necrosis factor (TNF) family member and is synthesized as a membrane-anchored precursor protein with the TNF core motif located in the C-terminal domain. The stalk region of EDA contains the sequence -Arg-Val-Arg-Arg156-Asn-Lys-Arg159-, representing overlapping consensus cleavage sites (Arg-X-Lys/Arg-Arg↓) for the proprotein convertase furin. Missense mutations in four of the five basic residues within this sequence account for ≈20% of all known XLHED cases, with mutations occurring most frequently at Arg156, which is shared by the two consensus furin sites. These analyses suggest that cleavage at the furin site(s) in the stalk region is required for the EDA-mediated cell-to-cell signaling that regulates the morphogenesis of ectodermal appendages. Here we show that the 50-kDa EDA parent molecule is cleaved at -Arg156Asn-Lys-Arg159↓- to release the soluble C-terminal fragment containing the TNF core domain. This cleavage appears to be catalyzed by furin, as release of the TNF domain was blocked either by expression of the furin inhibitor α1-PDX or by expression of EDA in furin-deficient LoVo cells. These results demonstrate that mutation of a functional furin cleavage site in a developmental signaling molecule is a basis for human disease (XLHED) and raise the possibility that furin cleavage may regulate the ability of EDA to act as a juxtacrine or paracrine factor.

Phosphoglycerylethanolamine Posttranslational Modification of Plant Eukaryotic Elongation Factor 1α1

Eukaryotic elongation factor 1α (eEF-1A) is a multifunctional protein. There are three known posttranslational modifications of eEF-1A that could potentially affect its function. Except for phosphorylation, the other posttranslational modifications have not been demonstrated in plants. Using matrix-assisted laser desorption/ionization-mass spectrometry and peptide mass mapping, we show that carrot (Daucus carota L.) eEF-1A contains a phosphoglycerylethanolamine (PGE) posttranslational modification. eEF-1A was the only protein labeled with [14C]ethanolamine in carrot cells and was the predominant ethanolamine-labeled protein in Arabidopsis seedlings and tobacco (Nicotiana tabacum L.) cell cultures. In vivo-labeling studies using [3H]glycerol, [32P]Pi, [14C]myristic acid, and [14C]linoleic acid indicated that the entire phospholipid phosphatidylethanolamine is covalently attached to the protein. The PGE lipid modification did not affect the partitioning of eEF-1A in Triton X-114 or its actin-binding activity in in vitro assays. Our in vitro data indicate that this newly characterized posttranslational modification alone does not affect the function of eEF-1A. Therefore, the PGE lipid modification may work in combination with other posttranslational modifications to affect the distribution and the function of eEF-1A within the cell.

Mechanistic coupling of protease signaling and initiation of coagulation by tissue factor

The crucial role of cell signaling in hemostasis is clearly established by the action of the downstream coagulation protease thrombin that cleaves platelet-expressed G-protein-coupled protease activated receptors (PARs). Certain PARs are cleaved by the upstream coagulation proteases factor Xa (Xa) and the tissue factor (TF)–factor VIIa (VIIa) complex, but these enzymes are required at high nonphysiological concentrations and show limited recognition specificity for the scissile bond of target PARs. However, defining a physiological mechanism of PAR activation by upstream proteases is highly relevant because of the potent anti-inflammatory in vivo effects of inhibitors of the TF initiation complex. Activation of substrate factor X (X) by the TF–VIIa complex is here shown to produce enhanced cell signaling in comparison to the TF–VIIa complex alone, free Xa, or Xa that is generated in situ by the intrinsic activation complex. Macromolecular assembly of X into a ternary complex of TF–VIIa–X is required for proteolytic conversion to Xa, and product Xa remains transiently associated in a TF–VIIa–Xa complex. By trapping this complex with a unique inhibitor that preserves Xa activity, we directly show that Xa in this ternary complex efficiently activates PAR-1 and -2. These experiments support the concept that proinflammatory upstream coagulation protease signaling is mechanistically coupled and thus an integrated part of the TF–VIIa-initiated coagulation pathway, rather than a late event during excessive activation of coagulation and systemic generation of proteolytic activity.

Demonstration of the extrinsic coagulation pathway in teleostei: Identification of zebrafish coagulation factor VII

It is not known whether the mammalian mechanism of coagulation initiation is conserved in fish. Identification of factor VII is critical in providing evidence for such a mechanism. A cDNA was cloned from a zebrafish (teleost) library that predicted a protein with sequence similarity to human factor VII. Factor VII was shown to be present in zebrafish blood and liver by Western blot analysis and immunohistochemistry. Immunodepletion of factor VII from zebrafish plasma selectively inhibited thromboplastin-triggered thrombin generation. Heterologous expression of zebrafish factor VII demonstrated a secreted protein (50 kDa) that reconstituted thromboplastin-triggered thrombin generation in immunodepleted zebrafish plasma. These results suggest conservation of the extrinsic coagulation pathway between zebrafish and humans and add credence to the zebrafish as a model for mammalian hemostasis. The structure of zebrafish factor VIIa predicted by homology modeling was consistent with the overall three-dimensional structure of human factor VIIa. However, amino acid disparities were found in the epidermal growth factor-2/serine protease regions that are present in the human tissue factor–factor VIIa contact surface, suggesting a structural basis for the species specificity of this interaction. In addition, zebrafish factor VII demonstrates that the Gla-EGF-EGF-SP domain structure, which is common to coagulation factors VII, IX, X, and protein C, was present before the radiation of the teleosts from the tetrapods. Identification of zebrafish factor VII significantly narrows the evolutionary window for development of the vertebrate coagulation cascade and provides insight into the structural basis for species specificity in the tissue factor–factor VIIa interaction.

Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1.

The X chromosome-linked transcription factor GATA-1 is expressed specifically in erythroid, mast, megakaryocyte, and eosinophil lineages, as well as in hematopoietic progenitors. Prior studies revealed that gene-disrupted GATA-1- embryonic stem cells give rise to adult (or definitive) erythroid precursors arrested at the proerythroblast stage in vitro and fail to contribute to adult red blood cells in chimeric mice but did not clarify a role in embryonic (or yolk sac derived) erythroid cells. To examine the consequences of GATA-1 loss on embryonic erythropoiesis in vivo, we inactivated the GATA-1 locus in embryonic stem cells by gene targeting and transmitted the mutated allele through the mouse germ line. Male GATA-1- embryos die between embryonic day 10.5 and 11.5 (E10.5-E11.5) of gestation. At E9.5, GATA-1- embryos exhibit extreme pallor yet contain embryonic erythroid cells arrested at an early proerythroblast-like stage of their development. Embryos stain weakly with benzidine reagent, and yolk sac cells express globin RNAs, indicating globin gene activation in the absence of GATA-1. Female heterozygotes (GATA-1+/-) are born pale due to random inactivation of the X chromosome bearing the normal allele. However, these mice recover during the neonatal period, presumably as a result of in vivo selection for progenitors able to express GATA-1. Our findings conclusively establish the essential role for GATA-1 in erythropoiesis within the context of the intact developing mouse and further demonstrate that the block to cellular maturation is similar in GATA-1- embryonic and definitive erythroid precursors. Moreover, the recovery of GATA-1+/- mice from anemia seen at birth provides evidence indicating a role for GATA-1 at the hematopoietic progenitor cell level.

Transcription factor TFIIH and DNA endonuclease Rad2 constitute yeast nucleotide excision repair factor 3: implications for nucleotide excision repair and Cockayne syndrome.

Nucleotide excision repair (NER) of ultraviolet light-damaged DNA in eukaryotes requires a large number of highly conserved protein factors. Recent studies in yeast have suggested that NER involves the action of distinct protein subassemblies at the damage site rather than the placement there of a "preformed repairosome" containing all the essential NER factors. Neither of the two endonucleases, Rad1-Rad10 and Rad2, required for dual incision, shows any affinity for ultraviolet-damaged DNA. Rad1-Rad10 forms a ternary complex with the DNA damage recognition protein Rad14, providing a means for targeting this nuclease to the damage site. It has remained unclear how the Rad2 nuclease is targeted to the DNA damage site and why mutations in the human RAD2 counterpart, XPG, result in Cockayne syndrome. Here we examine whether Rad2 is part of a higher order subassembly. Interestingly, we find copurification of Rad2 protein with TFIIH, such that TFIIH purified from a strain that overexpresses Rad2 contains a stoichiometric amount of Rad2. By several independent criteria, we establish that Rad2 is tightly associated with TFIIH, exhibiting an apparent dissociation constant < 3.3 x 10(-9) M. These results identify a novel subassembly consisting of TFIIH and Rad2, which we have designated as nucleotide excision repair factor 3. Association with TFIIH provides a means of targeting Rad2 to the damage site, where its endonuclease activity would mediate the 3' incision. Our findings are important for understanding the manner of assembly of the NER machinery and they have implications for Cockayne syndrome.

Saccharomyces cerevisiae Gcs1 is an ADP-ribosylation factor GTPase-activating protein.

Movement of material between intracellular compartments takes place through the production of transport vesicles derived from donor membranes. Vesicle budding that results from the interaction of cytoplasmic coat proteins (coatomer and clathrin) with intracellular organelles requires a type of GTP-binding protein termed ADP-ribosylation factor (ARF). The GTPase cycle of ARF proteins that allows the uncoating and fusion of a transport vesicle with a target membrane is mediated by ARF-dependent GTPase-activating proteins (GAPs). A previously identified yeast protein, Gcs1, exhibits structural similarity to a mammalian protein with ARF-GAP activity in vitro. We show herein that the Gcs1 protein also has ARF-GAP activity in vitro using two yeast Arf proteins as substrates. Furthermore, Gcs1 function is needed for the efficient secretion of invertase, as expected for a component of vesicle transport. The in vivo role of Gcs1 as an ARF GAP is substantiated by genetic interactions between mutations in the ARF1/ARF2 redundant pair of yeast ARF genes and a gcs1-null mutation; cells lacking both Gcs1 and Arf1 proteins are markedly impaired for growth compared with cells missing either protein. Moreover, cells with decreased levels of Arf1 or Arf2 protein, and thus with decreased levels of GTP-Arf, are markedly inhibited for growth by increased GCS1 gene dosage, presumably because increased levels of Gcs1 GAP activity further decrease GTP-Arf levels. Thus by both in vitro and in vivo criteria, Gcs1 is a yeast ARF GAP.

Quantitative trait loci and metabolic pathways: genetic control of the concentration of maysin, a corn earworm resistance factor, in maize silks.

Interpretation of quantitative trait locus (QTL) studies of agronomic traits is limited by lack of knowledge of biochemical pathways leading to trait expression. To more fully elucidate the biological significance of detected QTL, we chose a trait that is the product of a well-characterized pathway, namely the concentration of maysin, a C-glycosyl flavone, in silks of maize, Zea mays L. Maysin is a host-plant resistance factor against the corn earworm, Helicoverpa zea (Boddie). We determined silk maysin concentrations and restriction fragment length polymorphism genotypes at flavonoid pathway loci or linked markers for 285 F2 plants derived from the cross of lines GT114 and GT119. Single-factor analysis of variance indicated that the p1 region on chromosome 1 accounted for 58.0% of the phenotypic variance and showed additive gene action. The p1 locus is a transcription activator for portions of the flavonoid pathway. A second QTL, represented by marker umc 105a near the brown pericarp1 locus on chromosome 9, accounted for 10.8% of the variance. Gene action of this region was dominant for low maysin, but was only expressed in the presence of a functional p1 allele. The model explaining the greatest proportion of phenotypic variance (75.9%) included p1, umc105a, umc166b (chromosome 1), r1 (chromosome 10), and two epistatic interaction terms, p1 x umc105a and p1 x r1. Our results provide evidence that regulatory loci have a central role and that there is a complex interplay among different branches of the flavonoid pathway in the expression of this trait.

Mammalian dwarfins are phosphorylated in response to transforming growth factor beta and are implicated in control of cell growth.

The dwarfin protein family has been genetically implicated in transforming growth factor beta (TGF-beta)-like signaling pathways in Drosophila and Caenorhabditis elegans. To investigate the role of these proteins in mammalian signaling pathways, we have isolated and studied two murine dwarfins, dwarfin-A and dwarfin-C. Using antibodies against dwarfin-A and dwarfin-C, we show that these two dwarfins and an immunogenically related protein, presumably also a dwarfin, are phosphorylated in a time- and dose-dependent manner in response to TGF-beta. Bone morphogenetic protein 2, a TGF-beta superfamily ligand, induces phosphorylation of only the related dwarfin protein. Thus, TGF-beta superfamily members may use overlapping yet distinct dwarfins to mediate their intracellular signals. Furthermore, transient overexpression of either dwarfin-A or dwarfin-C causes growth arrest, implicating the dwarfins in growth regulation. This work provides strong biochemical and preliminary functional evidence that dwarfin-A and dwarfin-C represent prototypic members of a family of mammalian proteins that may serve as mediators of signaling pathways for TGF-beta superfamily members.