RESUMEN
In the rat, the alpha2B-adrenergic receptor (alpha2B-AR) is encoded by the rat non-glycosylated (RNG) gene and is primarily expressed in the kidney, brain and liver of adult animals. High levels of alpha2B-AR are also found during fetal life in the placenta, liver and blood, where it is borne by cells of the erythropoietic lineage. As a first step to define the mechanisms responsible for the spatio-temporal pattern of alpha2B-AR expression, a genomic fragment containing 2.8 kb of the 5'-flanking region, the ORF and approximately 20 kb of the 3'-flanking region of the RNG gene was isolated. RNase protection assays performed on RNA from placenta or kidney using a series of riboprobes permitted to locate the transcription start site 372 bases upstream from the start codon. Transient transfection of various cells, including rat proximal tubule in primary culture, with constructs containing luciferase as a reporter gene demonstrated that: (i) the 5'-flanking region exhibited a strong and sense-dependent transcriptional activity and (ii) the 332 bp fragment (-732/-401 relative to the start codon), which lacks a TATA box but contains Sp1 sites, is sufficient to drive expression. Analysis of chromatin susceptibility to DNaseI digestion identified two hypersensitive sites (HS1 and HS2) located 1.7 and 1.0 kb, respectively, upstream from ATG and containing recognition sequences for erythroid transcription factors. EMSA showed specific binding of GATA1 and NF-E2 to these elements. Taken together, the results suggest that the chromatin environment in the vicinity of these boxes plays a critical role for alpha2B-AR expression during fetal life.
Asunto(s)
Regiones Promotoras Genéticas , Receptores Adrenérgicos beta 2/genética , Animales , Secuencia de Bases , Sitios de Unión , Clonación Molecular , ADN , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Factores de Unión al ADN Específico de las Células Eritroides , Factor de Transcripción GATA1 , Datos de Secuencia Molecular , Factor de Transcripción NF-E2 , Ratas , Ratas Sprague-Dawley , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The engulfment of dying cells is a specialized form of phagocytosis that is extremely conserved across evolution. In the worm, it is genetically controlled by two parallel pathways, which are only partially reconstituted in mammals. We focused on the recapitulation of the CED-1 defined pathway in mammalian systems. We first explored and validated MEGF10, a novel receptor bearing striking structural similarities to CED-1, as a bona fide functional ortholog in mammals and hence progressed toward the analysis of molecular interactions along the corresponding pathway. We ascertained that, in a system of forced expression by transfection, MEGF10 function can be modulated by the ATP binding cassette transporter ABCA1, ortholog to CED-7. Indeed, the coexpression of either a functional or a mutant ABCA1 exerted a transdominant positive or negative modulation on the MEGF10-dependent engulfment. The combined use of biochemical and biophysical approaches indicated that this functional cooperation relies on the alternate association of these receptors with a common partner, endogenously expressed in our cell system. We provide the first working model structuring in mammals the CED-1 dependent pathway.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de la Membrana/metabolismo , Fagocitosis/fisiología , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sustitución de Aminoácidos , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Muerte Celular , Transferencia Resonante de Energía de Fluorescencia , Células HeLa , Humanos , Proteínas de la Membrana/genética , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Fagocitosis/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Especificidad de la Especie , TransfecciónRESUMEN
The EMI domain, first named after its presence in proteins of the EMILIN family, was identified here in several metazoan proteins with various domain architectures, among which the mammalian NEU1/NG3 proteins and Caenorhabditis elegans CED-1, identified as a transmembrane receptor that mediates cell corpse engulfment. Functional data available for EMILIN proteins suggest that the EMI domain could be a protein-protein interaction module. Sequence profiles specific of the EMI family of domains led to identify the probable orthologs of the C. elegans CED-1 protein in mammals and insects, which were yet uncovered.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Proteínas de la Membrana/genética , Homología de Secuencia de Aminoácido , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans , Bases de Datos de Proteínas/estadística & datos numéricos , Insectos , Mamíferos , Datos de Secuencia Molecular , Neuraminidasa/genética , Estructura Terciaria de Proteína/genética , Análisis de Secuencia de ProteínaRESUMEN
By using a novel time- and space-correlated single-photon counting detector, we show that fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to herpes simplex virus thymidine kinase (TK) monomers can be used to reveal homodimerization of TK in the nucleus and cytoplasm of live cells. However, the quantification of energy transfer was limited by the intrinsic biexponential fluorescence decay of the donor CFP (lifetimes of 1.3 +/- 0.2 ns and 3.8 +/- 0.4 ns) and by the possibility of homodimer formation between two TK-CFP. In contrast, the heterodimerization of the transcriptional factor NF-E2 in the nucleus of live cells was quantified from the analysis of the fluorescence decays of GFP in terms of 1) FRET efficiency between GFP and DsRed chromophores fused to p45 and MafG, respectively, the two subunits of NF-E2 (which corresponds to an interchromophoric distance of 39 +/- 1 A); and 2) fractions of GFP-p45 bound to DsRed-MafG (constant in the nucleus, varying in the range of 20% to 70% from cell to cell). The picosecond resolution of the fluorescence kinetics allowed us to discriminate between very short lifetimes of immature green species of DsRed-MafG and that of GFP-p45 involved in FRET with DsRed-MafG.