RESUMEN
Here we show a complete list of the P-type ATPase genes in Caenorhabditis elegans and Drosophila melanogaster. A detailed comparison of the deduced amino-acid sequences in combination with phylogenetic and chromosomal analyses has revealed the following: (1) The diversity of this gene family has been achieved by two major evolutionary steps; the establishment of the major P-type ATPase subgroups with distinct substrate (ion) specificities in a common ancestor of vertebrate and invertebrate, followed by the evolution of multiple isoforms occurring independently in vertebrate and invertebrate phyla. (2) Pairs of genes that have intimate phylogenetic relationship are frequently found in proximity on the same chromosome. (3) Some of the Na,K- and H,K-ATPase isoforms in D. melanogaster and C. elegans lack motifs shown to be important for alpha/beta-subunit assembly, suggesting that such alpha- and beta-subunits might exist by themselves (lonely subunits). The mutation rates for these subunits are much faster than those for the subunits with recognizable assembly domains. (4) The lonely alpha-subunits also lack the major site for ouabain binding that apparently arose before the separation of vertebrates and invertebrates and thus well before the separation of vertebrate Na,K-ATPases and H,K-ATPases. These findings support the idea that a relaxation of functional constraints would increase the rate of evolution and provide clues for identifying the origins of inhibitor sensitivity, subunit assembly, and separation of Na,K- and H,K-ATPases.
Asunto(s)
Caenorhabditis elegans/genética , Drosophila melanogaster/genética , Evolución Molecular , Perfilación de la Expresión Génica/métodos , ATPasa Intercambiadora de Hidrógeno-Potásio/genética , ATPasa Intercambiadora de Sodio-Potasio/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/química , Caenorhabditis elegans/enzimología , Análisis Mutacional de ADN/métodos , Drosophila melanogaster/química , Drosophila melanogaster/enzimología , ATPasa Intercambiadora de Hidrógeno-Potásio/química , ATPasa Intercambiadora de Hidrógeno-Potásio/clasificación , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Datos de Secuencia Molecular , Fenotipo , Subunidades de Proteína , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/clasificación , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismo , Alineación de Secuencia/métodos , Análisis de Secuencia de Proteína/métodos , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/clasificación , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Especificidad de la EspecieRESUMEN
The mechanisms by which receptor tyrosine kinases (RTKs) utilize intracellular signaling pathways to direct gene expression and cellular response remain unclear. A current question is whether different RTKs within a single cell target similar or different sets of genes. In this study we have used the ErbB receptor network to explore the relationship between RTK activation and gene expression. We profiled growth factor-stimulated signaling pathway usage and broad gene expression patterns in two human mammary tumor cell lines expressing different complements of ErbB receptors. Although the growth factors epidermal growth factor (EGF) and neuregulin (NRG) 1 similarly stimulated Erk1/2 in MDA-MB-361 cells, EGF acting through an EGF receptor/ErbB2 heterodimer preferentially stimulated protein kinase C, and NRG1beta acting through an ErbB2/ErbB3 heterodimer preferentially stimulated Akt. The two growth factors regulated partially overlapping yet distinct sets of genes in these cells. In MDA-MB-453 cells, NRG1beta acting through an ErbB2/ErbB3 heterodimer stimulated prolonged signaling of all pathways examined relative to NRG2beta acting through the same heterodimeric receptor species. Surprisingly, NRG1beta and NRG2beta also regulated partially overlapping but distinct sets of genes in these cells. These results demonstrate that the activation of different RTKs, or activation of the same RTKs with different ligands, can lead to distinct profiles of gene regulation within a single cell type. Our observations also suggest that the identity and kinetics of signaling pathway usage by RTKs may play a role in the selection of regulated genes.
Asunto(s)
Factor de Crecimiento Epidérmico/metabolismo , Expresión Génica/fisiología , Neurregulina-1/metabolismo , Proteínas Tirosina Quinasas Receptoras/fisiología , Transducción de Señal/fisiología , Animales , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Tumorales CultivadasRESUMEN
Cytosine methylation of mammalian DNA is essential for the proper epigenetic regulation of gene expression and maintenance of genomic integrity. To define the mechanism through which demethylated cells die, and to establish a paradigm for identifying genes regulated by DNA methylation, we have generated mice with a conditional allele for the maintenance DNA methyltransferase gene Dnmt1. Cre-mediated deletion of Dnmt1 causes demethylation of cultured fibroblasts and a uniform p53-dependent cell death. Mutational inactivation of Trp53 partially rescues the demethylated fibroblasts for up to five population doublings in culture. Oligonucleotide microarray analysis showed that up to 10% of genes are aberrantly expressed in demethylated fibroblasts. Our results demonstrate that loss of Dnmt1 causes cell-type-specific changes in gene expression that impinge on several pathways, including expression of imprinted genes, cell-cycle control, growth factor/receptor signal transduction and mobilization of retroelements.
Asunto(s)
Apoptosis , Metilación de ADN , Regulación de la Expresión Génica , Genoma , Impresión Genómica , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Virales , Alelos , Animales , Sitios de Ligazón Microbiológica/genética , División Celular , Línea Celular Transformada , Células Cultivadas , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Evolución Molecular , Fibroblastos , Eliminación de Gen , Perfilación de la Expresión Génica , Genes de Partícula A Intracisternal/genética , Integrasas/genética , Integrasas/metabolismo , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/análisis , ARN Mensajero/genética , Recombinación Genética/genética , Células Madre/enzimología , Células Madre/metabolismoRESUMEN
Lysosome associated membrane proteins (LAMPs) constitute a family of vertebrate proteins located predominantly in lysosomes, with lesser amounts present in endosomes and at the cell surface. Macrosialin/CD68s are similar to LAMPs in their subcellular distribution and amino acid sequence and presumed structure across the carboxyl terminal two thirds of their length. The functions of LAMPs and CD68s are not known. In the present study, a bioinformatics approach was used to identify a Caenorhabditis elegans protein (LMP-1) with sequence and presumed structural similarity to LAMPs and CD68s. LMP-1 appears to be the only membrane protein in C. elegans that carries a GYXX(phi) vertebrate lysosomal targeting sequence at its C terminus (where (phi) is a large, hydrophobic residue). LMP-1 was found to be present from early embryonic stages through adulthood and to be predominantly localized at the periphery of a population of large, membrane-bound organelles, called granules, that are seen throughout the early embryo but in later stages are restricted to the cells of the intestine. Analysis of an LMP-1 deficient C. elegans mutant revealed that LMP-1 is not required for viability under laboratory conditions, but the absence of LMP-1 leads to an alteration in intestinal granule populations, with apparent loss of one type of granule.
Asunto(s)
Antígenos CD/genética , Antígenos de Diferenciación Mielomonocítica/genética , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/genética , Proteínas del Helminto/química , Proteínas del Helminto/genética , Proteínas del Helminto/fisiología , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Secuencias de Aminoácidos , Animales , Antígenos CD/química , Antígenos de Diferenciación Mielomonocítica/química , Caenorhabditis elegans/citología , Caenorhabditis elegans/crecimiento & desarrollo , Clonación Molecular , Biología Computacional , Gránulos Citoplasmáticos/química , Gránulos Citoplasmáticos/ultraestructura , Etiquetas de Secuencia Expresada , Intestinos/química , Intestinos/citología , Intestinos/ultraestructura , Proteínas de Membrana de los Lisosomas , Datos de Secuencia Molecular , Fenotipo , Estructura Terciaria de Proteína , ARN Bicatenario/genética , Eliminación de Secuencia/genética , Homología de Secuencia de AminoácidoRESUMEN
Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease of the kidney, characterized by cystic enlargement of renal tubules, aberrant epithelial proliferation, and ion and fluid secretion into the lumen. Previous studies have shown abnormalities in polarization of membrane proteins, including mislocalization of the NaK-ATPase to the apical plasma membranes of cystic epithelia. Apically located NaK-ATPase has previously been shown to be fully functional in vivo and in membrane-grown ADPKD epithelial cells in vitro, where basal-to-apical (22)Na transport was inhibited by application of ouabain to the apical membrane compartment. Studies were conducted with polymerase chain reaction-generated specific riboprobes and polyclonal peptide antibodies against human sequences of alpha1, alpha3, beta1, and beta2 subunits of NaK-ATPase. High levels of expression of alpha1 and beta1 messenger RNA were detected in ADPKD and age-matched normal adult kidneys in vivo, whereas beta2 messenger RNA was detected only in ADPKD kidneys. Western blot analysis and immunocytochemical studies showed that, in normal adult kidneys, peptide subunit-specific antibodies against alpha1 and beta1 localized to the basolateral membranes of normal renal tubules, predominantly thick ascending limbs of Henle's loop. In ADPKD kidneys, alpha1 and beta2 subunits were localized to the apical epithelial cell membranes, whereas beta1 was distributed throughout the cytoplasm and predominantly in the endoplasmic reticulum, but was not seen associated with cystic epithelial cell membranes or in cell membrane fractions. Polarizing, renal-derived epithelial Madin Darby canine kidney cells, stably expressing normal or N-terminally truncated chicken beta1 subunits, showed selective accumulation in the basolateral Madin Darby canine kidney cell surface, whereas c-myc epitope-tagged chicken beta2 or human beta2 subunits accumulated selectively in the apical cell surface. Similarly, human ADPKD epithelial cell lines, which endogenously expressed alpha1 and beta2 NaK-ATPase subunits, showed colocalization at the apical cell surface and coassociation by immunoprecipitation analysis. These results are consistent with a model in which the additional transcription and translation of the beta2 subunit of NaK-ATPase may result in the apical mislocalization of NaK-ATPase in ADPKD cystic epithelia.
Asunto(s)
Isoenzimas/metabolismo , Enfermedades Renales Poliquísticas/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Polaridad Celular , Células Cultivadas , Perros , Células Epiteliales/enzimología , Humanos , Isoenzimas/genética , Riñón/citología , Riñón/enzimología , Riñón/patología , Enfermedades Renales Poliquísticas/patología , ATPasa Intercambiadora de Sodio-Potasio/genética , Distribución Tisular , Transcripción Genética/fisiologíaRESUMEN
A systematic study was conducted on the requirements at the C-terminal position for the targeting of LAMPs to lysosomes, examining the hypothesis that a bulky hydrophobic residue is required. Mutations deleting or replacing the C-terminal valine with G, A, C, L, I, M, K, F, Y, or W were constructed in a reporter protein consisting of the lumenal/extracellular domain of avian LAMP-1 fused to the transmembrane and cytoplasmic domains of LAMP-2b. The steady-state distribution of each mutant form in mouse L-cells was assessed by quantitative antibody binding assays and immunofluorescence microscopy; efficiency of internalization from the plasma membrane and delivery to the lysosome were also estimated. It is found that (a) only C-terminal V, L, I, M, and F mediated efficient targeting to lysosomes, demonstrating the importance hydrophobicity and an optimal size of the C-terminal residue in targeting; (b) efficiency of lysosomal targeting generally correlated with efficiency of internalization; and (c) mutant forms that did not target well to lysosomes showed unique distributions in cells rather than simply default accumulation in the plasma membrane. Interactions of the targeting signals with adaptor subunits were measured using a yeast two-hybrid assay. The results are consistent with the hypothesis that trafficking of LAMP forms in cells through the indirect pathway is determined by the affinities of their targeting signals, predominantly for the mu2 and mu3 adaptors involved at plasma membrane and endosomal cellular sorting sites, respectively.
Asunto(s)
Antígenos CD/metabolismo , Lisosomas/fisiología , Glicoproteínas de Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD/química , Antígenos CD/genética , Sitios de Unión de Anticuerpos , Butiratos/farmacología , Membrana Celular/fisiología , Cicloheximida/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Células L , Proteínas de Membrana de los Lisosomas , Lisosomas/ultraestructura , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Ratones , Microscopía Confocal , Fragmentos de Péptidos/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Antibodies were used to examine the expression patterns of Antennapedia (Antp), Ultrabithorax (Ubx), Ubx and abdominal-A combined (Ubx/abd-A), and Distalless (Dll) in the embryos of the moth Manduca sexta. We found that the spatial and temporal pattern of Antp expression in Manduca was correlated with the anterior migration of two patches of epithelium that include the anterior-most tracheal pits, and with the development of functional spiracles. Ubx expression showed an intricate pattern which suggests complex regulation during development. Throughout Manduca embryogenesis the expression of Ubx/Abd-A and Dll was similar to that reported for other insects. However, there was no apparent reduction in Ubx/Abd-A expression in the Manduca abdominal proleg primordia that expressed Dll. The expression of these four proteins was also examined in embryos of the Manduca homozygous homeotic mutant Octopod (Octo). The Octo mutation results in the transformation of A1 and A2 in the anterior direction, with homeotic legs appearing on A1 and occasionally A2. Our results suggest that in Octo animals there is a reduction in the level of Ubx protein expression throughout its domain. Based on homeotic gene expression in wild-type and mutant Manduca and in other insects, we discuss potential roles of homeotic genes in insect morphological evolution.
Asunto(s)
Proteínas de Drosophila , Genes Homeobox , Genes de Insecto , Manduca/genética , Proteínas Nucleares , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Proteína con Homeodominio Antennapedia , Clonación Molecular , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/análisis , Proteínas de Homeodominio/genética , Inmunohistoquímica , Proteínas de Insectos/análisis , Proteínas de Insectos/genética , Manduca/embriología , Datos de Secuencia MolecularRESUMEN
We sought to explore the relationship between receptor tyrosine kinase (RTK) activated signaling pathways and the transcriptional induction of immediate early genes (IEGs). Using global expression monitoring, we identified 66 fibroblast IEGs induced by platelet-derived growth factor beta receptor (PDGFRbeta) signaling. Mutant receptors lacking binding sites for activation of the PLCgamma, PI3K, SHP2, and RasGAP pathways still retain partial ability to induce 64 of these IEGs. Removal of the Grb2-binding site further broadly reduces induction. These results suggest that the diverse pathways exert broadly overlapping effects on IEG induction. Interestingly, a mutant receptor that restores the RasGAP-binding site promotes induction of an independent group of genes, normally induced by interferons. Finally, we compare the PDGFRbeta and fibroblast growth factor receptor 1; each induces essentially identical IEGs in fibroblasts.
Asunto(s)
Regulación de la Expresión Génica , Genes Inmediatos-Precoces , Genes Sobrepuestos , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Células 3T3 , Animales , Línea Celular Transformada , Fibroblastos/citología , Humanos , Interferón gamma/metabolismo , Interferón gamma/farmacología , Ratones , Mutagénesis , Fenilalanina/genética , Fenilalanina/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/genética , Tirosina/genética , Tirosina/metabolismoRESUMEN
This study explored whether Dictyostelium discoideum can be used to express the avian Na,K-ATPase, a heterodimeric membrane protein. Dictyostelium was able to express mRNAs encoding the avian Na, K-ATPase subunits. However, Dictyostelium expressed avian Na, K-ATPase protein when only when a Dictyostelium consensus ribosomal binding sequence, AAAATAAA, was inserted in front of the open reading frames of the alpha1- and beta1-subunit cDNAs and the first eight codons following the start-translation codons were changed to Dictyostelium preferred codons. These modified mRNAs appeared to be much less stable than the forms that were not readily translated. Dictyostelium could express the avian beta-subunit alone but only expressed the alpha1-subunit when the beta1-subunit was co-expressed. Subunit assembly occurred in cells expressing both alpha1- and beta1-subunits. The bulk of the exogenously expressed sodium pump subunits remained in an intracellular compartment, presumed to be the endoplasmic reticulum. Dictyostelium exported little or no Na, K-ATPase or free beta-subunit to the plasma membrane.
Asunto(s)
Dictyostelium/genética , ATPasa Intercambiadora de Sodio-Potasio/genética , Animales , Aves , Codón , ADN Complementario/química , ADN Complementario/genética , Dictyostelium/enzimología , Expresión Génica , Proteínas de la Membrana/genética , Péptidos/genética , Biosíntesis de Proteínas , Conformación Proteica , ARN Mensajero/análisis , ARN Mensajero/química , ARN Mensajero/genéticaAsunto(s)
Estructura Secundaria de Proteína , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Pollos , Clonación Molecular , Células HeLa , Humanos , Células L , Sustancias Macromoleculares , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Alineación de Secuencia , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Transcripción Genética , TransfecciónRESUMEN
The extensively glycosylated lysosome-associated membrane proteins (LAMP)-2a, b, and c are derived from a single gene by alternative splicing that produces proteins with differences in the transmembrane and cytosolic domains. The lysosomal targeting signals reside in the cytosolic domain of these proteins. LAMPs are not restricted to lysosomes but can also be found in endosomes and at the cell surface. We investigated the subcellular distribution of chimeras comprised of the lumenal domain of avian LAMP-1 and the alternatively spliced domains of avian LAMP-2. Chimeras with the LAMP-2c cytosolic domain showed predominantly lysosomal distribution, while higher levels of chimeras with the LAMP-2a or b cytosolic domain were present at the cell surface. The increase in cell surface expression was due to differences in the recognition of the targeting signals and not saturation of intracellular trafficking machinery. Site-directed mutagenesis defined the COOH-terminal residue of the cytosolic tail as critical in governing the distributions of LAMP-2a, b, and c between intracellular compartments and the cell surface.
Asunto(s)
Empalme Alternativo/fisiología , Antígenos CD/genética , Lisosomas/química , Glicoproteínas de Membrana/genética , Secuencia de Aminoácidos , Animales , Antígenos CD/análisis , Antígenos CD/química , Antígenos de Superficie/análisis , Transporte Biológico/fisiología , Línea Celular , Técnica del Anticuerpo Fluorescente Directa , Expresión Génica/fisiología , Proteínas de Membrana de los Lisosomas , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/química , Ratones , Datos de Secuencia Molecular , Señales de Clasificación de Proteína/fisiología , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Fracciones Subcelulares/químicaRESUMEN
Subunit interactions of the alpha1- and beta1-subunits of the chicken Na,K-ATPase were explored with the yeast two-hybrid system. Gal4-fusion proteins containing domains of the alpha1- and beta1-subunits were designed for examining both intersubunit and intrasubunit protein-protein interactions. Regions of the alpha- and beta-subunits known to be involved in alpha-beta-subunit assembly were positive in two-hybrid assay, supporting the validity of the assays. A library of beta-subunit ectodomains with C-terminal truncations was screened to find the maximal truncation retaining an interaction with the alpha-subunit extracellular H7H8 loop (where H7 refers to the seventh membrane span, and so on). The maximal truncation removed all the cysteines involved in disulfide bridges, leaving only 63 amino acids of the beta-subunit ectodomain. Scanning alanine mutagenesis led to identification of an evolutionarily conserved sequence of four amino acids (SYGQ) in the extracellular H7H8 loop of the alpha-subunit that is crucial to alpha-beta-intersubunit interactions. Oligomerization studies with single domains failed to detect self-association of either of the two large cytosolic loops (H2H3 and H4H5) within the alpha-subunit. However, evidence was found for an interaction between these two cytoplasmic loops.
Asunto(s)
ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Pollos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae , ATPasa Intercambiadora de Sodio-Potasio/químicaRESUMEN
The Drosophila Na,K-ATPase (or sodium pump) alpha-subunit gene was found to contain 10 exons and span approx. 25 kb. Two nearly adjacent transcriptional initiation sites were identified, and the 2085-nucleotide sequence upstream of the first transcriptional start was analysed for promoter activity in transfected Drosophila SL2 cells. This region was found to contain many cis-acting elements that influence promoter activity, including elements that confer 2- to 3-fold higher activity in SL2 cells cultured at 30 degrees C versus 22 degrees C. Temperature-sensitive transcriptional regulation of the Na,K-ATPase alpha-subunit in Drosophila is a plausible mechanistic candidate for the factor driving temperature-dependent up-regulation of the Na,K-ATPase alpha-subunit described here for fly strains homozygous for single P-element insertions in the alpha-subunit gene. Four new P-element insertion strains were identified in this study, each insertion site lying within the first intron of the Na,K-ATPase alpha-subunit gene. The insertion in strain 0462 resulted in cold-sensitive recessive lethality; flies homozygous for the 0462 mutation could be rescued by growth at 29-30 degrees C, a condition that partially corrected a deficiency in the level of Na,K-ATPase alpha-subunit. The high-temperature rescue of homozygous 0462 flies appeared to result primarily from improved Na,K-ATPase expression rather than an increase in the rate of ion transport per Na,K-ATPase molecule. These observations point to a role for sodium-pump activity in determining the range of temperature tolerance in Drosophila and demonstrate that relatively subtle changes in sodium-pump expression can have major consequences in whole organisms.
Asunto(s)
Drosophila/genética , Genes Letales , Genes Recesivos , Mutación , Regiones Promotoras Genéticas , ATPasa Intercambiadora de Sodio-Potasio/genética , Adaptación Fisiológica , Animales , Secuencia de Bases , Línea Celular , ADN , Huella de ADN , Elementos Transponibles de ADN , Drosophila/enzimología , Drosophila/fisiología , Exones , Heterocigoto , Homocigoto , Intrones , Datos de Secuencia Molecular , Unión Proteica , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Temperatura , Transcripción GenéticaRESUMEN
The Na+,K(+)-ATPase and the sarcoplasmic/endoplasmic reticulum Ca(2+)-(SERCA-) ATPase belong to a family of P-type ATPases that undergo a cycle of conformational changes between the phosphorylated and dephosphorylated stages in an ion-specific manner. The ouabain-inhibitable Na+,K(+)-ATPase activity requires Na+ and K+. On the other hand, the Ca(2+)-dependent and thapsigargin-inhibitable activity of the SERCA-ATPase does not depend upon Na+ and K+ for its basal activity. However, the SERCA-ATPase and Ca(2+)-transport activities can be further activated either by K+ in a two-step fashion with high (ED50 approximately 20 mM) and low affinity (ED50 approximately 70 mM) or by Na+ in a one-step fashion with an ED50 value of approximately 50 mM. A chimera, in which the carboxy-terminal region (Leu861-COOH) of the Na+,K(+)-ATPase alpha 1 subunit replaced the corresponding region (Ser830-COOH) of the SERCA1-ATPase, lacked the low-affinity K+ activation of the SERCA-ATPase but displayed a higher-affinity (ED50 < 10 mM) activation by K+, similar to that of the Na+,K(+)-ATPase, whereas activation by Na+ was not affected. The replacement of the large cytosolic loop (Gly354-Lys712) and the amino-terminal regions (Met1-Asp162) of the SERCA1-ATPase with the corresponding portions of the Na+,K(+)-ATPase alpha 1 subunit did not affect the sensitivity of the SERCA-ATPase activity to K+. Thus, the carboxy-terminal regions of both the SERCA1 and the Na+,K(+)-ATPase alpha 1 subunit are critical for K+ sensitivity. Analysis of additional (Ca2+/Na+,K+)-ATPase chimeras demonstrated that the carboxy-terminal 102 amino acids (Phe920-Tyr1021) of the Na+/K(+)-ATPase alpha 1 subunit are sufficient to shift the K+ affinity for activation of the SERCA-ATPase without the beta subunit. No change in the two-step activation of SERCA-ATPase by K+ was seen when residues Thr871-Thr898 of the SERCA1-ATPase were replaced with residues Asn894-Ala919 of the Na+,K(+)-ATPase alpha 1 subunit, a region known to bind the Na+,K(+)-ATPase beta subunit [Lemas, M. V., et al. (1994) J. Biol. Chem. 269, 8255-8259]. Thus, the Na+,K(+)-ATPase subunit-assembly domain and the K(+)-sensitive region are distinct within the carboxy-terminal 161 amino acids of the Na+,K(+)-ATPase.
Asunto(s)
ATPasas Transportadoras de Calcio/química , ATPasas Transportadoras de Calcio/metabolismo , Potasio/farmacología , Conformación Proteica , Retículo Sarcoplasmático/enzimología , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , ATPasas Transportadoras de Calcio/aislamiento & purificación , Membrana Celular/enzimología , Pollos , Cromatografía de Afinidad , Cinética , Células L , Ratones , Modelos Biológicos , Modelos Estructurales , Ouabaína/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Sodio/farmacología , ATPasa Intercambiadora de Sodio-Potasio/aislamiento & purificación , Tapsigargina/farmacología , TransfecciónRESUMEN
In the Drosophila embryo, at specific choice points along the major motor nerves, subsets of motor axons defasciculate and then steer into their muscle target regions. Here we describe the analysis of beaten path (beat), a gene required for the selective defasciculation of motor axons at these choice points. In beat mutant embryos, motor axons fail to defasciculate and bypass their targets. This phenotype is suppressed by mutations in FasII and conn, two genes encoding cell adhesion molecules expressed on motor axons, suggesting that beat provides an antiadhesive function. beat encodes a novel secreted protein that is expressed by motoneurons during outgrowth. Rescue and ectopic expression experiments support the model that Beat protein is secreted by motor axons where it functions to regulate their selective defasciculation at specific choice points.
Asunto(s)
Axones/fisiología , Proteínas de Drosophila , Drosophila/genética , Neuronas Motoras/fisiología , Proteínas del Tejido Nervioso/genética , Secuencia de Aminoácidos , Animales , Axones/química , Axones/metabolismo , Adhesión Celular/fisiología , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Mapeo Cromosómico , Clonación Molecular , Conectina , Regulación del Desarrollo de la Expresión Génica/fisiología , Genes de Insecto/fisiología , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Neuronas Motoras/química , Neuronas Motoras/ultraestructura , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculos/química , Músculos/inervación , Músculos/fisiología , Mutagénesis/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuritas/química , Neuritas/metabolismo , Neuritas/fisiología , Fenotipo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , ARN Mensajero/metabolismoRESUMEN
Although the Na+/K(+)-ATPase alpha 1-subunit gene is ubiquitously expressed in vertebrates, its level of expression varies among tissue and cell types. In spite of similar mRNA distribution in tissues of mammals and birds, the 5'-flanking regions of alpha 1-subunit genes exhibit remarkable diversity; i.e., the core promoter activity of the TATA-less chicken alpha 1 gene strongly depends upon multiple Sp1-based regulation (six Sp1 sites), whereas the promoter activity of the TATA-like rat alpha 1-subunit gene relies on the two Sp1 and additional positive regulatory factors. Further analysis of the regulatory regions of the Na+/K(+)-ATPase alpha 1-subunit genes revealed that the vertebrate alpha 1-subunit genes may share common inhibitory mechanisms for subtle transcriptional regulation; the core promoter activities can be either enhanced or repressed depending on the availability of inhibitory factors. Two potential candidates for such inhibitory elements in both avian and mammalian Na+/K(+)-ATPase alpha 1-subunit genes are (1) a newly identified element, GCCCTC, and (2) a GCF-binding sequence, NN[G/c]CG[G/c][G/c][G/c]CN, or its reverse complement. Gel retardation assays using the inhibitory region of the chicken gene and crude nuclear extracts from tissue-cultured chicken and mouse cells showed the existence of a set of proteins that bind to this region. The amounts of individual regulatory proteins in different cell types seem to vary, resulting in differential formation of DNA/protein complexes in different cell types. Thus, the regulation of Na+/K(+)-ATPase alpha 1-subunit gene expression under different cellular environment as well as in different cell types can be achieved by a shared mechanism; modulation of the ratio of the abundance of individual inhibitory factors.
Asunto(s)
Regulación de la Expresión Génica/genética , Regiones Promotoras Genéticas/genética , ATPasa Intercambiadora de Sodio-Potasio/química , Transcripción Genética/genética , Animales , Secuencia de Bases , Sitios de Unión , Northern Blotting , Células Cultivadas , Embrión de Pollo , Clonación Molecular , Proteínas de Unión al ADN/genética , Genes Reporteros , Datos de Secuencia Molecular , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Secuencia , ATPasa Intercambiadora de Sodio-Potasio/genética , Transfección/genéticaRESUMEN
It has long been suspected that proteolytic activity associated with advancing growth cones may be required for axon extension. We have isolated mutations in the kuzbanian (kuz) gene, which is expressed in the nervous system and encodes a putative zinc metalloprotease with a disintegrin domain. Drosophila embryos with loss-of-function mutations in kuz have dramatic defects in the development of central nervous system axon pathways, with many axons stalling and failing to extend through the nerve cord. This phenotype is rescued by panneural expression of kuz mRNA in the embryo. These results show that the Kuz metalloprotease is required for axon extension, suggesting a requirement for proteolytic activity at the growth cone surface.
Asunto(s)
Axones/fisiología , Desintegrinas/genética , Desintegrinas/metabolismo , Proteínas de Drosophila , Drosophila/embriología , Genes de Insecto , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Fenómenos Fisiológicos del Sistema Nervioso , Animales , Axones/enzimología , Drosophila/genética , Embrión no Mamífero , Femenino , Homocigoto , Hibridación in Situ , Masculino , Mutagénesis , Sistema Nervioso/citología , ARN Mensajero/biosíntesis , Transcripción GenéticaRESUMEN
The endoplasmic reticulum undergoes rapid, microscopic changes in its structure, including extension and anastomosis of tubular elements. Such dynamism is expected to manifest itself also as rapid intermixing of membrane components, at least within subdomains of the endoplasmic reticulum. Here we present evidence of a similar dynamism in the sarcoplasmic reticulum of developing skeletal muscle. The sarcoplasmic reticulum is sometimes considered a specialized type of endoplasmic reticulum, but it appears to be a rather static set of membrane-bound elements, repetitively arranged to enwrap each sarcomere of each myofibril. Both endoplasmic reticulum and sarcoplasmic reticulum contain P-type Ca(2+)-ATPases that transport calcium from the cytosol into their lumen. In the experiments reported here, chicken and mouse cells were fused by polyethylene glycol, natural myogenic cell fusion, or Sendai virus. The redistribution of Ca(2+)-ATPase molecules between chick and mouse endoplasmic reticulum/sarcoplasmic reticulum was followed by immunofluorescence microscopy in which species-specific monoclonal antibodies to chick and mouse Ca(2+)-ATPases were used. Redistribution was time- and temperature-dependent but independent of protein synthesis as well as the method of cell fusion. Intermixing occurred on a time scale of tens of minutes at 37 degrees C. These results verify the dynamic nature of the sarcoplasmic reticulum and illustrate an aspect of the special relationship between endoplasmic reticulum and sarcoplasmic reticulum.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Músculo Esquelético/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Fusión Celular/fisiología , Línea Celular , Embrión de Pollo , Cicloheximida/farmacología , Fibroblastos , Técnica del Anticuerpo Fluorescente Directa , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Ratones , Músculo Esquelético/efectos de los fármacos , Retículo Sarcoplasmático/química , Temperatura , Factores de TiempoRESUMEN
Motor axons make synaptic connections with specific muscles, and this specificity unfolds during development as motoneuron growth cones make specific pathway choices and ultimately recognize and synapse on their specific muscle targets. The Drosophila clueless mutation was identified previously in a genetic screen for mutations that disrupt motoneuron guidance and connectivity. We show here that clueless is allelic to abrupt. The abrupt gene is required for the embryonic formation of specific synaptic connections between a subset of motoneurons and a subset of muscles. Mutations in abrupt also reveal its role in establishing and maintaining muscle attachments, adult sensory cell formation, and morphogenesis of adult appendages. The abrupt gene encodes a zinc finger protein with a conserved BTB domain. Abrupt is expressed in muscle nuclei but not motoneurons, suggesting that abrupt controls the muscle expression of molecules required for correct motoneuron targeting, as well as molecules required for correct muscle attachments.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila , Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto , Músculos/embriología , Factores de Transcripción/genética , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Núcleo Celular/genética , Núcleo Celular/metabolismo , Clonación Molecular , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/química , Datos de Secuencia Molecular , Neuronas Motoras/metabolismo , Desarrollo de Músculos , Músculos/metabolismo , Mutación , Sistema Nervioso/embriología , Sistema Nervioso/metabolismo , Alineación de Secuencia , Factores de Transcripción/biosíntesis , Factores de Transcripción/química , Dedos de Zinc/genéticaRESUMEN
We have cloned a Na,K-ATPase alpha-subunit gene from Caenorhabditis elegans and discovered that it is identical to the gene eat-6, eat-6 mutations cause feeble contractions and slow, delayed relaxations of pharyngeal muscle. The resting membrane potential of eat-6 mutant pharynxes is consistently depolarized compared to wild-type. The action potentials are smaller, and the return to resting potential is slower. To explain these abnormalities, we propose that a reduction of Na,K-ATPase activity in eat-6 mutants leads to a reduction of the ion concentration gradients that power membrane potential changes.