RESUMEN
The orphan receptor ChemR23 is a G-protein coupled receptor (GPCR) with homology to neuropeptide and chemoattractant receptors. Tazarotene, a synthetic retinoid activating retinoic acid receptor (RAR), up-regulates tazarotene-induced gene-2 (TIG2). The function and molecular target of this protein are now described. By means of reverse pharmacology screening using a peptide library generated from human hemofiltrate, we have isolated and identified TIG2 as the natural ligand of ChemR23 and report the specific molecular form of the bioactive, circulating TIG2, representing the amino-acid residues 21 to 154 of the 163 amino acid-containing prepropeptide. Based on the expression pattern of ChemR23 and TIG2, the physiological role in bone development, immune and inflammatory responses and the maintenance of skin is now being investigated.
Asunto(s)
Ácidos Nicotínicos/genética , Receptores de Quimiocina/metabolismo , Receptores de Ácido Retinoico/sangre , Receptores de Ácido Retinoico/genética , Secuencia de Aminoácidos , Animales , Células CHO , Calcio/análisis , Calcio/metabolismo , Cricetinae , Fluorometría/métodos , Hemofiltración , Humanos , Ligandos , Datos de Secuencia Molecular , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/química , Receptores de Quimiocina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , TransfecciónRESUMEN
The apical sorting of the small intestinal membrane glycoprotein sucrase-isomaltase (SI) depends on the presence of O-linked glycans and the transmembrane domain. Here, we investigate the role of O-glycans carried by the Ser/Thr-rich stalk region of SI as an apical sorting signal and evaluate the spatial requirements for an efficient recognition of this signal. Several hybrid proteins are generated comprising the unsorted and unglycosylated protein, the rat growth hormone (rGH), fused to either the transmembrane domain of SI (GH-SI(TM)), or the transmembrane and the stalk domains (GH-SI(SR/TM)). Both constructs are randomly distributed over the apical and basolateral membranes of MDCK cells indicating that neither the transmembrane domain nor the O-glycans are sufficient per se for an apical delivery. Only when a polyglycine spacer is inserted between the stalk region of SI and the luminal part of rGH in the GH-SI(Gly/SR/TM) fusion protein does efficient apical sorting of an O-glycosylated protein as well as a time-dependent association with detergent-insoluble lipid microdomains occur. Obviously, the polyglycine spacer facilitates the accessibility of the O-glycans in GH-SI(Gly/SR/TM) to a putative sorting receptor, whereas these glycans are inadequately recognized in GH-SI(SR/TM). We conclude that the O-glycans in the stalk region of SI act as an apical sorting signal within a sorting machinery that comprises at least a carbohydrate-binding protein and fulfills specific spatial requirements provided, for example by a polyglycine spacer in the context of rGH or the P-domain within the SI enzyme complex.
Asunto(s)
Hormona del Crecimiento/metabolismo , Intestino Delgado/enzimología , Transporte de Proteínas , Complejo Sacarasa-Isomaltasa/metabolismo , Animales , Secuencia de Bases , Línea Celular , Membrana Celular/metabolismo , Cartilla de ADN , Perros , Glicosilación , Hormona del Crecimiento/biosíntesis , RatasRESUMEN
The impaired sorting profile to the apical membrane of human intestinal sucrase-isomaltase is the underlying cause in the pathogenesis of a novel phenotype of intestinal congenital sucrase-isomaltase deficiency. Molecular characterization of this novel phenotype reveals a point mutation in the coding region of the sucrase-isomaltase (SI) gene that results in an amino acid substitution of a glutamine by arginine at residue 117 of the isomaltase subunit. This substitution is located in a domain revealing features of a trefoil motif or a P-domain in immediate vicinity of the heavily O-glycosylated stalk domain. Expression of the mutant SI phenotype in epithelial Madin-Darby canine kidney cells reveals a randomly targeted SI protein to the apical and basolateral membranes confirming an exclusive role of the Q117R mutation in generating this phenotype. Unlike wild type SI, the mutant protein is completely extractable with Triton X-100 despite the presence of O-glycans that serve in the wild type protein as an apical sorting signal and are required for the association of SI with detergent-insoluble lipid microdomains. Obviously the O-glycans are not adequately recognized in the context of the mutant SI, most likely due to altered folding of the P-domain that ultimately affects the access of the O-glycans to a putative sorting element.
Asunto(s)
Errores Innatos del Metabolismo de los Carbohidratos/enzimología , Errores Innatos del Metabolismo de los Carbohidratos/genética , Polaridad Celular , Complejo Sacarasa-Isomaltasa/deficiencia , Complejo Sacarasa-Isomaltasa/genética , Animales , Línea Celular , Membrana Celular/metabolismo , Preescolar , Perros , Humanos , Microdominios de Membrana/metabolismo , Fenotipo , Mutación Puntual , Transporte de Proteínas , Complejo Sacarasa-Isomaltasa/metabolismoRESUMEN
Lactase-phlorizin hydrolase is a brush-border enzyme which is specifically expressed in the small intestine where it hydrolyses lactose, the main carbohydrate found in milk. We have previously demonstrated in transgenic mice that the tissue-specific and developmental expression of lactase is controlled by a 1 kb upstream region of the pig lactase gene. Two homeodomain transcription factors, caudal-related homeodomain protein (Cdx2) and hepatic nuclear factor 1alpha (HNF1alpha), are known to bind to regulatory cis elements in the promoters for several intestine-specific genes, including lactase, and are present in mammalian intestinal epithelia from an early stage in development. In the present study, we examined whether Cdx2 and HNF1alpha physically interact and co-operatively activate transcription from the lactase-phlorizin hydrolase promoter. We show that the presence of both factors leads to a much higher level of transcription than the sum of the activation by either factor alone. The N-terminal activation domain of Cdx2 is required for maximal synergy with HNF1alpha. With the use of pull-down assays, we demonstrate a direct protein-protein interaction between Cdx2 and HNF1alpha. The interaction domain includes the homeodomain region of both proteins. This is the first demonstration of a functional interaction between two transcription factors involved in the activation of a number of intestine-specific genes. Synergistic interaction between tissue-restricted factors is likely to be an important mechanism for reinforcing developmental and tissue-specific gene expression within the intestine.
Asunto(s)
Proteínas de Unión al ADN , Regulación Enzimológica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Lactasa-Florizina Hidrolasa/biosíntesis , Lactasa-Florizina Hidrolasa/genética , Proteínas Nucleares , Factores de Transcripción/metabolismo , Animales , Factor de Transcripción CDX2 , Células CACO-2 , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Proteínas de Homeodominio/genética , Humanos , Ratones , Regiones Promotoras Genéticas , Transactivadores , Factores de Transcripción/genéticaRESUMEN
BACKGROUND & AIMS: One-kilobase sequence of the upstream fragment of the pig lactase-phlorizin hydrolase gene has been shown to control small intestinal-specific expression and postweaning decline of lactase-phlorizin hydrolase in transgenic mice. The aim of this study was to identify the regulatory DNA elements and transcription factors controlling lactase-phlorizin hydrolase expression. METHODS: The activity of different lactase-phlorizin hydrolase promoter fragments was investigated by transfection experiments using Caco-2 cells. Electrophoretic mobility shift assays and supershift analyses were used to characterize the interaction between intestinal transcription factors and the identified regulatory elements. RESULTS: Functional analysis revealed three previously undescribed regulatory regions in the lactase-phlorizin hydrolase promoter: a putative enhancer between -894 and -798 binding hepatocyte nuclear factor (HNF)-1 at position -894 to -880; a repressor-binding element between -278 to -264 to which an HNF-3-like factor is able to bind; and an element between -178 to -164 that binds an activating transcription factor. CONCLUSIONS: Identification of three new regulatory regions and HNF-1 and HNF-3-like transcription factor as players in the regulation of lactase-phlorizin hydrolase gene transcription has an impact on the understanding of the molecular mechanisms behind age-dependent, tissue-specific, differentiation-dependent, and regional regulation of expression in the intestine.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Regulación Enzimológica de la Expresión Génica , Lactasa-Florizina Hidrolasa/genética , Transactivadores/fisiología , Factores de Transcripción/fisiología , Animales , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT , Células CACO-2 , Diferenciación Celular , Factores de Transcripción Forkhead , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Humanos , Proteínas Nucleares/fisiología , Regiones Promotoras Genéticas , Porcinos , Transcripción GenéticaRESUMEN
Lactase-phlorizin hydrolase is exclusively expressed in the small intestine and is often used as a marker for the differentiation of enterocytes. The cis-element CE-LPH1 found in the lactase-phlorizin hydrolase promoter has previously been shown to bind an intestinal-specific nuclear factor. By electrophoretic mobility-shift assay it was shown that the factor Cdx-2 (a homoeodomain-protein related to caudal) binds to a TTTAC sequence in the CE-LPH1. Furthermore it was demonstrated that Cdx-2 is able to activate reporter gene transcription by binding to CE-LPH1. A mutation in CE-LPH1, which does not affect Cdx-2 binding, results in a higher transcriptional activity, indicating that the CE-LPH1 site contains other binding site(s) in addition to the Cdx-2-binding site.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Proteínas de Homeodominio/genética , Lactasa-Florizina Hidrolasa/genética , Secuencia de Bases , Factor de Transcripción CDX2 , Células CACO-2 , Células HeLa , Humanos , Lactasa-Florizina Hidrolasa/biosíntesis , Datos de Secuencia Molecular , TransactivadoresRESUMEN
Adult-type hypolactasia is a genetic condition making approximately one half of the human population intolerant to milk because of abdominal symptoms. The cause is a post-weaning down-regulation of the intestinal-specific enzyme lactase-phlorizin hydrolase (LPH) reducing the intestinal capacity to hydrolyze lactose. We here demonstrate that the stretch -17 to -994 in the pig LPH-promoter carries cis-elements which direct a small intestinal-specific expression and a post-weaning decline of a linked rabbit beta-globin gene. These data demonstrate that the post-weaning decline of LPH is mainly due to a transcriptional down-regulation.