RESUMO
CTLA-4, expressed by activated T cells, transduces an inhibitory signal. We show here that PCR amplification of the coding sequence of CTLA-4 in nonstimulated human T lymphocytes results in the amplification of two transcripts of 650 and 550 bp. Sequencing shows that the larger form codes for membrane CTLA-4 and the 550-bp transcript is a spliced variant in which exon 2 coding for the transmembrane region is deleted. This spliced cDNA has been named CTLA-4delTM. The splicing induces a frame shift which results in the addition of 22 extra amino acids before a translational termination. Activation of T cells with phorbol 12-myristate 13-acetate plus ionomycin or anti-CD3 plus anti-CD28 monoclonal antibodies induces a suppression of CTLA-4delTM mRNA expression associated with a preferential expression of the membrane CTLA-4 mRNA, showing that CTLA-4delTM mRNA expression is restricted to nonactivated T cells. A soluble immunoreactive form of CTLA-4 was detected in the serum of 14 / 64 healthy subjects. These results suggest that nonstimulated T cells may constitutively produce a soluble form of CTLA-4 which may have an important role in the regulation of immune homeostasis.
Assuntos
Processamento Alternativo , Antígenos de Diferenciação/biossíntese , Imunoconjugados , Linfócitos T/metabolismo , Abatacepte , Sequência de Aminoácidos , Animais , Antígenos CD , Antígenos de Diferenciação/genética , Sequência de Bases , Células COS , Antígeno CTLA-4 , Células Cultivadas , Expressão Gênica , Humanos , Ativação Linfocitária , Dados de Sequência Molecular , RNA Mensageiro , Proteínas Recombinantes de Fusão/genética , Solubilidade , Linfócitos T/imunologiaRESUMO
The 2-5A/RNase L system is one of the pathways induced by interferon (IFN). It plays a major role in the antiviral and antiproliferative activities of IFNs. Recently, we have shown that the activity of the RNase L could be inhibited by a proteic inhibitor, the RNase L Inhibitor (RLI). Human RLI (Hu-RLI) was cloned and characterized. We describe here the isolation and characterization of the cDNA encoding the murine RLI (Mu-RLI). Hu-RLI and Mu-RLI protein have 98% amino acid identity. Mu-RLI is functionally homologous to Hu-RLI, and all the structural features and amino acid sequence motifs of Hu-RLI are conserved in Mu-RLI. Moreover, reticulocyte lysate translated Mu-RLI protein is also able to inhibit 2-5A binding on 2-5A-dependent RNAse-L. Northern blot analysis revealed that Mu-RLI cDNA hybridizes with one mRNA of 3.5 kb except for the testis where two mRNA of 3.5 and 2.1 kb, respectively, are detected, suggesting a tissue-specific regulation.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Chaperoninas , Inibidores Enzimáticos/química , Biossíntese de Proteínas , Proteínas/química , Nucleotídeos de Adenina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Competitiva , Clonagem Molecular , Sequência Conservada , DNA Complementar , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Mathanococcus/genética , Camundongos , Dados de Sequência Molecular , Oligorribonucleotídeos/metabolismo , Especificidade de Órgãos , Proteínas/farmacologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
This study demonstrates that alpha1-adrenergic receptors previously identified in the pregnant rat myometrium are heterogeneous. They can be subtyped alpha1A- and alpha1B-adrenergic receptors on the basis of their affinity for the antagonists WB4101 (alpha1A > alpha1B) and chloroethylclonidine (alpha1B selective). Between Day 21 of pregnancy and term, the proportion of [3H]prazosin binding sites with low affinity for WB4101 and sensitive to inactivation by 10(-5) M chloroethylclonidine under hypotonic conditions (alpha1B subtype) remained constant. In contrast, the number of [3H]prazosin binding sites with a high affinity for WB4101 and insensitive to chloroethylclonidine (alpha1A subtype) increased by 88% at term. The effect of 5'-guanylylimidodiphosphate (Gpp[NH]p) on competition of the agonist phenylephrine for [3H]prazosin binding in the presence of WB4101 or after chloroethylclonidine pretreatment indicates that the alpha1A-adrenergic receptor underwent uncoupling whereas the alpha1B-adrenergic receptor-G protein coupled state was increased (+ 63%). Phenylephrine consistently stimulated phospholipase C activity on membrane fractions prepared from term myometria. This stimulation was completely inhibited after 10(-5) M chloroethylclonidine but was not consistently decreased with 5-methylurapidyl, a selective alpha1A-antagonist. Furthermore QL antibody (anti-G alpha(q)/G alpha11) also specifically blocked the phenylephrine-stimulated phospholipase C activity. Altogether these results strongly suggest that activation of the alpha1B-adrenergic receptor subtype in the pregnant myometrium at term may contribute to the stimulation of the G alpha(q)/G alpha11/phospholipase C signaling pathway.
