RESUMEN
A structural, profile-based algorithm was used to identify interleukin 20 (IL-20), a novel IL-10 homolog. Chromosomal localization of IL-20 led to the discovery of an IL-10 family cytokine cluster. Overexpression of IL-20 in transgenic (TG) mice causes neonatal lethality with skin abnormalities including aberrant epidermal differentiation. Recombinant IL-20 protein stimulates a signal transduction pathway through STAT3 in a keratinocyte cell line, demonstrating a direct action of this ligand. An IL-20 receptor was identified as a heterodimer of two orphan class II cytokine receptor subunits. Both receptor subunits are expressed in skin and are dramatically upregulated in psoriatic skin. Taken together, these results demonstrate a role in epidermal function and psoriasis for IL-20, a novel cytokine identified solely by bioinformatics analysis.
Asunto(s)
Epidermis/inmunología , Interleucinas/genética , Receptores de Citocinas/genética , Receptores de Citocinas/inmunología , Animales , Línea Celular , Mapeo Cromosómico , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Dimerización , Epidermis/química , Epidermis/patología , Expresión Génica/inmunología , Humanos , Interleucina-10/genética , Interleucina-10/inmunología , Interleucinas/química , Interleucinas/inmunología , Queratinocitos/citología , Queratinocitos/inmunología , Queratinas/genética , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Psoriasis/inmunología , Psoriasis/patología , Receptores de Citocinas/química , Factor de Transcripción STAT3 , Homología de Secuencia de Aminoácido , Transactivadores/metabolismo , Regulación hacia Arriba/inmunologíaRESUMEN
A new member of the insulin gene superfamily (INSL5) was identified by searching EST databases for the presence of the conserved insulin B-chain cysteine motif. Human and murine INSL5 are both polypeptides of 135 amino acids, matching the classical signature of the insulin superfamily. Through the B- and A-chain regions, human INSL5 has 48% identity to shark relaxin, 40% identity to human relaxin, and 34% identity to human Leydig insulin-like factor. Northern blot analysis detected expression of human INSL5 in rectal, colon, and uterine tissue and of murine INSL5 only in thymic tissue. Using quantitative RT-PCR, expression of murine INSL5 was detected in the highest quantity in colon followed by thymus, and minimal expression was seen in testis. By radiation hybrid mapping and the use of surrounding markers, human INSL5 maps to chromosome 1 in the 1p31.1 to 1p22.3 region.
Asunto(s)
Hormonas/genética , Insulina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , Cromosomas Humanos Par 1/genética , Mapeo Contig , ADN Complementario/química , ADN Complementario/genética , Femenino , Expresión Génica , Humanos , Células Híbridas , Masculino , Ratones , Datos de Secuencia Molecular , Hormonas Peptídicas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
Two human cDNAs that encode novel vitamin K-dependent proteins have been cloned and sequenced. The predicted amino acid sequences suggest that both are single-pass transmembrane proteins with amino-terminal gamma-carboxyglutamic acid-containing domains preceded by the typical propeptide sequences required for posttranslational gamma-carboxylation of glutamic acid residues. The polypeptides, with deduced molecular masses of 23 and 17 kDa, are proline-rich within their putative cytoplasmic domains and contain several copies of the sequences PPXY and PXXP, motifs found in a variety of signaling and cytoskeletal proteins. Accordingly, these two proteins have been called proline-rich Gla proteins (PRGP1 and PRGP2). Unlike the gamma-carboxyglutamic acid domain-containing proteins of the blood coagulation cascade, the two PRGPs are expressed in a variety of extrahepatic tissues, with PRGP1 and PRGP2 most abundantly expressed in the spinal cord and thyroid, respectively, among those tissues tested. Thus, these observations suggest a novel physiological role for these two new members of the vitamin K-dependent family of proteins.
Asunto(s)
Ácido 1-Carboxiglutámico , Péptidos/genética , Prolina , Proteínas/genética , Vitamina K/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Humanos , Datos de Secuencia Molecular , Péptidos/metabolismo , Dominios Proteicos Ricos en Prolina , Proteínas/metabolismo , Alineación de Secuencia , Análisis de SecuenciaRESUMEN
An alternative spliced variant of metabotropic glutamate receptor subtype mGluR4a, termed mGluR4b was isolated from a rat cDNA library. Subtype mGluR4b was identical to the previously described mGluR4a, except for the last 64 amino acids in the C-terminal region in which were replaced by 135 new amino acids in mGluR4b. Recombinant baculoviruses coding for mGluR4a and mGluR4b were expressed in Spodoptera frugiperda, Sf-9, insect cells and characterized pharmacologically by measuring [3H]-L-2-amino-4-phosphonobutyrate ([3H]-L-AP4) binding and second messenger formation. [3H]-L-AP4 binding to membranes prepared from Sf-9 cells expressing mGluR4a and mGluR4b revealed respective affinities (Kd) of 480 and 360 nM and maximal binding densities (Bmax) of 4.2 and 0.8 pmol/mg protein. The ligand selectivity of mGluR4a and mGluR4b was similar: L-AP4 > L-serine-O-phosphate > L-glutamate > L-2-amino 2-methyl-4-phosphonobutyrate > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate > or = quisqualate. A decrease in the affinity of [3H]-L-AP4 was observed in the presence of 0.1 mM guanosine 5'-O-(3-thio)trisphosphate-gamma-S, indicating that mGluR4a and mGluR4b were functionally coupled to G-proteins in Sf-9 cells. Agonists of mGluR4 caused a minor decrease in forskolin-induced cAMP formation in Sf-9 cells expressing either mGluR4a or mGluR4b, suggesting that both receptors are coupled to adenylate cyclase in an inhibitory manner. Thus, mGluR4a and mGluR4b share a common signal transduction pathway and pharmacology when expressed in Sf-9 insect cells.
Asunto(s)
Receptores de Glutamato Metabotrópico/química , Secuencia de Aminoácidos , Aminobutiratos/metabolismo , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , AMP Cíclico/biosíntesis , Datos de Secuencia Molecular , Ratas , Receptores de Glutamato Metabotrópico/efectos de los fármacos , Receptores de Glutamato Metabotrópico/metabolismoRESUMEN
Metabotropic glutamate receptors mediate their intracellular response by coupling to G proteins and may be divided into three subfamilies: mGluR1 and mGluR5, which stimulate phosphatidylinositol hydrolysis; mGluR2 and mGluR3, which are negatively coupled to cyclic AMP formation; and mGluR4 and mGluR6, which also inhibit forskolin-stimulated cyclic AMP formation. The mGluR4 subtypes may represent L-2-amino-4-phosphonobutyrate-sensitive presynaptic autoreceptors, and two alternatively spliced variants of the mGluR4 coding for two receptors with different C termini have been identified. Using in situ hybridization, we measured the levels of mGluR1-mGluR5 mRNA in regions of the rat brain 24 h after transient global ischemia, a time point when no neuronal damage can yet be observed morphologically. In the hippocampus, the mRNA levels for mGluR1, mGluR2, and mGluR5 were decreased, mGluR3 mRNA levels were unchanged, and the mGluR4 mRNA levels were strongly increased. The strongest increase appeared to be in the mRNA encoding mGluR4b. The mGluR4 mRNA was also increased in the parietal cortex, whereas the ventral posteromedial thalamic nucleus showed a small decrease in its mRNA content. These results suggest that vulnerable neurons react to an increased extracellular glutamate concentration by differential regulation of the mRNA for pre- and postsynaptically located metabotropic glutamate receptors.
Asunto(s)
Encéfalo/metabolismo , Ataque Isquémico Transitorio/metabolismo , Neuronas/metabolismo , ARN Mensajero/metabolismo , Receptores de Glutamato/biosíntesis , Animales , Autorradiografía , Encéfalo/patología , Corteza Cerebral/metabolismo , Clonación Molecular , Hipocampo/metabolismo , Hibridación in Situ , Ataque Isquémico Transitorio/patología , Masculino , Neuronas/patología , Especificidad de Órganos , Lóbulo Parietal/metabolismo , Sondas ARN , Ratas , Ratas Wistar , Radioisótopos de Azufre , Sinapsis/metabolismo , Factores de TiempoRESUMEN
The pharmacological specificity of the mGluR1 alpha subtype of the metabotropic glutamate receptor (mGluR) was examined in a cloned baby hamster kidney cell line (BHK-ts13) measuring [3H]glutamate binding and inositol phosphate (PI) hydrolysis. PI-hydrolysis was maximally stimulated by quisqualate (1112 +/- 105% of basal), glutamate (1061 +/- 70% of basal), ibotenate (1097 +/- 115% of basal) and beta-N-methylamino-L-alanine (BMAA) (1010 +/- 104% of basal). In contrast, the maximal stimulation of PI-hydrolysis by (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (t-ACPD) was only 673 +/- 78% of the basal level. The relative order of potency was quisqualate > glutamate > ibotenate > t-ACPD > BMAA. Agonist-stimulated PI-hydrolysis was attenuated (25 +/- 4% inhibition) by L-2-amino-3-phosphonopropionic acid and partially blocked (44 +/- 7%) by pertussis toxin treatment. Saturation binding studies with [3H]glutamate on membranes prepared from BHK-ts13 cells expressing the mGluR1 alpha subtype showed that glutamate binds to a single affinity state of this receptor with a limited capacity (Kd = 296 nM, Bmax = 0.8 pmol/mg protein). In competition experiments, [3H]glutamate was displaced by quisqualate, glutamate, ibotenate, t-ACPD and BMAA with a rank order of potency similar to that found for stimulation of PI-hydrolysis.
Asunto(s)
Glutamatos/metabolismo , Fosfatos de Inositol/metabolismo , Receptores de Glutamato/metabolismo , Aminoácidos Diaminos/farmacología , Aminobutiratos/farmacología , Animales , Unión Competitiva , Línea Celular , Membrana Celular/metabolismo , Células Clonales , Cricetinae , Toxinas de Cianobacterias , Maleato de Dizocilpina/farmacología , Glutamatos/farmacología , Ácido Glutámico , Ácido Iboténico/análogos & derivados , Ácido Iboténico/farmacología , Riñón , Cinética , Neurotoxinas/farmacología , Toxina del Pertussis , Quinoxalinas/farmacología , Ácido Quiscuálico/farmacología , Receptores de Glutamato/biosíntesis , Receptores de Glutamato/clasificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Transfección , Factores de Virulencia de Bordetella/farmacología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol PropiónicoRESUMEN
Receptors for the major excitatory neurotransmitter glutamate include metabotropic (G protein-coupled) and ionotropic (glutamate-gated ion channel) types. These receptors have large, presumably extracellular, amino-terminal domains. Sensitive sequence analysis techniques indicate that the metabotropic receptor extracellular domain is similar to bacterial periplasmic amino acid binding proteins. A structural model built using the observed similarity predicts a ligand-binding site, and mutants with conservative amino acid substitutions at this site are shown to have reduced ligand affinity. The metabotropic receptor extracellular domain is a member of a family of structural domains linked to a variety of receptor types, including ionotropic glutamate receptors.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bases de Datos Factuales , Predicción , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Two new complementary DNAs overlapping cDNA clones that encode the G-protein coupled metabotropic glutamate receptor mGluR1 from rat brain have been isolated and sequenced in their entirety. These new clones represent mRNA with 3' untranslated regions approximately 2.5 kilobases longer than the previously isolated cDNA clones. These results indicate that the previously observed two size classes of approximately 4 kb and approximately 7 kb which hybridize to sequences that encode this receptor use different polyadenylation signals and differ in the extent of their 3' untranslated sequence. There is a striking asymmetry in the distribution of a sequence involved in mRNA instability between the two mRNA species.
Asunto(s)
Poli A/metabolismo , ARN Mensajero/metabolismo , Receptores de Glutamato/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN , Datos de Secuencia Molecular , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato/clasificación , Mapeo RestrictivoRESUMEN
Glutamate and L-AP4 inhibited forskolin-stimulated cyclic AMP (cAMP) production in baby hamster kidney (BHK) cells transfected with the type IV metabotropic receptor (mGluR4). In situ hybridization revealed a high level of mRNA for the mGluR4 in the entorhinal cortex, but not in the dentate gyrus. These data demonstrate that mGluR4 receptors are negatively coupled to the cAMP cascade, and suggest that the mGluR4 receptor may be the previously described presynaptic L-AP4 receptor.
Asunto(s)
Adenilil Ciclasas/metabolismo , Aminobutiratos/farmacología , Receptores de Glutamato/efectos de los fármacos , Animales , Células Cultivadas , Colforsina/farmacología , Cricetinae , AMP Cíclico/metabolismo , Glutamatos/farmacología , Ácido Glutámico , Riñón/citología , Riñón/metabolismoRESUMEN
The Drosophila pecanex locus contains a maternal-effect neurogenic gene. A homologue of this gene has not yet been described in mammals or other organisms. We report here a partial complementary DNA clone from rat brain mRNA that encodes sequences which are very similar (83% over 189 amino acids) to a portion of sequence encoded by a transcript from the Drosophila pecanex locus [LaBonne, S.G., Sunitha, I and Mahowald, A.P. (1989) Dev. Biology 136: 1-161].
Asunto(s)
Mapeo Cromosómico/métodos , ADN/genética , Drosophila/genética , Código Genético/genética , Biblioteca Genómica , Transcripción Genética/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Ratas , Homología de Secuencia de Ácido Nucleico , Programas InformáticosRESUMEN
High affinity binding of platelet-derived growth factor (PDGF) has been proposed to involve the interaction of the dimeric PDGF ligand with two receptor subunits, designated alpha and beta. We have cloned and expressed a human PDGF receptor cDNA which differs in sequence from the beta-subunit and which has the PDGF binding properties and monoclonal antibody recognition, predicted for the alpha-subunit. Scatchard analysis indicated that PDGF-AA and PDGF-AB bound to transfected alpha-subunits with affinities of Kd = 0.06 and 0.05 nM, respectively. PDGF-BB bound with a significantly lower affinity (Kd = 0.4 nM). Nevertheless, this affinity is still great enough to mediate substantial PDGF-BB binding at physiological concentrations and would be considered to be "high affinity." We have used wild-type and kinase-inactive human beta-subunits to show that PDGF binding promotes receptor subunit dimerization in intact cells. In addition, we found that PDGF stimulates tyrosine phosphorylation of the kinase-inactive beta-subunit when it is expressed with alpha-subunits. The kinase-inactive beta-subunits were phosphorylated at tyrosine 857 and 751, the major phosphorylation sites of the wild-type beta-subunit, indicating either that intra- and intermolecular phosphorylation occurs on the same sites, or that a significant fraction of receptor tyrosine phosphorylation is intermolecular.
Asunto(s)
Factor de Crecimiento Derivado de Plaquetas/farmacología , Receptores de Superficie Celular/metabolismo , Animales , Clonación Molecular , Cricetinae , ADN/genética , Técnicas In Vitro , Sustancias Macromoleculares , Fosforilación , Fosfotirosina , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Agregación de Receptores , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Receptores del Factor de Crecimiento Derivado de Plaquetas , Proteínas Recombinantes/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
A complementary DNA encoding a G protein-coupled glutamate receptor from rat brain, GluGR, was cloned by functional expression in Xenopus oocytes. The complementary DNA encodes a protein of 1199 amino acids containing a seven-transmembrane motif, flanked by large amino- and carboxyl-terminal domains. This receptor lacks any amino acid sequence similarity with other G protein-coupled receptors, suggesting that it may be a member of a new subfamily. The presence of two introns flanking the central core suggests that GluGR may have evolved by exon shuffling. Expressed in oocytes, GluGR is activated by quisqualate greater than glutamate greater than ibotenate greater than trans-1-aminocyclopentyl-1,3-dicarboxylate, and it is inhibited by 2-amino-3-phosphonopropionate. Activation is blocked by Bordella pertussis toxin. These properties are typical of some metabotropic glutamate receptors.
Asunto(s)
Química Encefálica , Clonación Molecular , Proteínas de Unión al GTP/metabolismo , Receptores de Neurotransmisores/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/genética , Exones , Humanos , Intrones , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , ARN Mensajero/genética , Ratas , Receptores de Glutamato , Receptores de Neurotransmisores/química , Receptores de Neurotransmisores/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
The complete nucleotide sequence of a cDNA encoding the human platelet-derived growth factor (PDGF) receptor is presented. The cDNA contains an open reading frame that codes for a protein of 1106 amino acids. Comparison to the mouse PDGF receptor reveals an overall amino acid sequence identity of 86%. This sequence identity rises to 98% in the cytoplasmic split tyrosine kinase domain. RNA blot hybridization analysis of poly(A)+ RNA from human dermal fibroblasts detects a major (approximately 5.7 kb) and a minor (approximately 4.8 kb) transcript using the cDNA as a probe. Baby hamster kidney cells, transfected with an expression vector containing the receptor cDNA, express an approximately equal to 190-kDa cell surface protein that is recognized by an anti-human PDGF receptor antibody. The recombinant PDGF receptor is functional in the transfected baby hamster kidney cells as demonstrated by ligand-induced phosphorylation of the receptor. Binding properties of the recombinant PDGF receptor were also assessed with pure preparations of BB and AB isoforms of PDGF (i.e., PDGF dimers composed of two B chains or an A and a B chain). Unlike human dermal fibroblasts, which bind both isoforms with high affinity, the transfected baby hamster kidney cells bind only the BB isoform of PDGF with high affinity. This observation is consistent with the existence of more than one PDGF receptor class.
Asunto(s)
Clonación Molecular , ADN/genética , Genes , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores de Superficie Celular/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Enzimas de Restricción del ADN , Humanos , Ratones , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Receptores de Superficie Celular/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas , TransfecciónAsunto(s)
ADN Satélite/genética , Factor VII/genética , Genes , Secuencia de Bases , Exones , Humanos , Intrones , Datos de Secuencia MolecularRESUMEN
Activated factor VII (factor VIIa) is a vitamin K-dependent plasma serine protease that participates in a cascade of reactions leading to the coagulation of blood. Two overlapping genomic clones containing sequences encoding human factor VII were isolated and characterized. The complete sequence of the gene was determined and found to span about 12.8 kilobases. The mRNA for factor VII as demonstrated by cDNA cloning is polyadenylylated at multiple sites but contains only one AAUAAA poly(A) signal sequence. The mRNA can undergo alternative splicing, forming one transcript containing eight segments as exons and another with an additional exon that encodes a larger prepro leader sequence. The latter transcript has no known counterpart in the other vitamin K-dependent proteins. The positions of the introns with respect to the amino acid sequence encoded by the eight essential exons of factor VII are the same as those present in factor IX, factor X, protein C, and the first three exons of prothrombin. These exons code for domains generally conserved among members of this gene family. The comparable introns in these genes, however, are dissimilar with respect to size and sequence, with the exception of intron C in factor VII and protein C. The gene for factor VII also contains five regions made up of tandem repeats of oligonucleotide monomer elements. More than a quarter of the intron sequences and more than a third of the 3' untranslated portion of the mRNA transcript consist of these minisatellite tandem repeats.