RESUMEN
AID (activation-induced cytidine deaminase) catalyzes transcription-dependent deamination of C --> U in immunoglobulin variable (IgV) regions to initiate somatic hypermutation (SHM) in germinal center B-cells. SHM is essential in generating high affinity antibodies. Here we show that when coexpressed with GANP (germinal center-associated nuclear protein) in COS-7 cells, AID is transported from the cytoplasm and concentrated in the nucleus. GANP forms a complex with AID in cotransfected cells in vivo and in vitro. We have isolated AID mutants that bind with reduced affinity to GANP compared with wild type AID. One of these mutants, AID (D143A) binds GANP with a 10-fold lower affinity compared with wild type AID yet retains substantial C-deamination activity in vitro. Mutant AID (D143A) remains localized predominantly in the cytoplasm when coexpressed with GANP. Exogenous expression of GANP in Ramos B-cells promotes binding of AID to IgV DNA and mRNA and increases SHM frequency. These data suggest that GANP may serve as an essential link required to transport AID to B-cell nuclei and to target AID to actively transcribed IgV regions.
Asunto(s)
Acetiltransferasas/genética , Núcleo Celular/metabolismo , Citidina Desaminasa/genética , Región Variable de Inmunoglobulina/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Acetiltransferasas/metabolismo , Animales , Células COS , Chlorocebus aethiops , Citidina Desaminasa/química , Citoplasma/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Mapeo de Interacción de ProteínasRESUMEN
Recent studies indicate that the balance between cell survival and proapoptotic signals determines which cells commit to life or death. We have shown that the balance between follicle-stimulating hormone and prolactin determines differentiation or apoptosis in 7th generation spermatogonia during newt spermatogenesis; however, the molecular mechanisms specifying their fate are poorly understood. Here we show that the newt RNA-binding protein (nRBP) plays a critical role in determining their fate. nRBP was identified as a clone whose mRNA is decreased by prolactin, resulting in the reduction of the protein, which is otherwise expressed predominantly in the spermatogonia. nRBP protein associated with the mRNA for newt programmed cell death protein 4 (nPdcd4) at the 3'-untranslated region. nRBP reduction increased nPdcd4 mRNA but decreased its protein. In a cell-free system, cytoplasmic extracts containing reduced amounts of nRBP and nPdcd4 protein induced apoptosis, whereas adding nRBP protein to the extracts blocked apoptosis. Furthermore, overexpression of nRBP protected cells from apoptosis, stabilized the chimeric transcript containing the nPdcd4 3'-untranslated region, and accelerated its translation. These data suggest that, in the absence of nRBP, nPdcd4 mRNA is not stabilized and its translation is suppressed, leading to apoptosis in the spermatogonia.
Asunto(s)
Proteínas Anfibias/genética , Proteínas Reguladoras de la Apoptosis/genética , Apoptosis , Prolactina/metabolismo , Interferencia de ARN , Salamandridae/metabolismo , Espermatogonias/metabolismo , Secuencia de Aminoácidos , Proteínas Anfibias/química , Proteínas Anfibias/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Femenino , Masculino , Datos de Secuencia Molecular , Prolactina/química , Prolactina/genética , Unión Proteica , Biosíntesis de Proteínas , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Salamandridae/genética , Alineación de Secuencia , Espermatogonias/química , Espermatogonias/citologíaRESUMEN
Neuregulin1 (NRG1) is a growth factor that signals through the interaction of the epidermal growth factor (EGF)-like domain with ErbB receptors. An immunoglobulin (Ig)-like domain is contained together with EGF-like domain in the ectodomain of some isoforms generated by alternative splicing, but its role in NRG1 signaling remained unclear. In the present study, we identified a novel isoform of NRG1 containing an Ig-like domain conserved among species from adult Xenopus laevis, which is predominantly expressed in the testis and brain. We generated recombinant proteins for the whole ectodomain and EGF-like domain alone of the isoform to compare their effects on cell proliferation, and phosphorylation of and their association with ErbB receptor, demonstrating that the ectodomain had approximately 10(3)-fold higher abilities than the EGF-like domain. Therefore, the Ig-like domain is probably essential for efficient interaction of an EGF-like domain with ErbB receptors.