RESUMEN
Trafficking of receptors to and from the cell surface is a powerful mechanism for regulating neuronal excitability. To date, the majority of studies concerning glutamate receptor trafficking have been performed in neuronal cultures in which surface expression can be readily assayed by immunofluorescence techniques. Results from such studies have had important implications in the field of synaptic plasticity. However, cultured neurons are, by necessity, prepared from very young animals. Moreover, although an enhancement of excitatory neurotransmission can be induced in such systems, classic long-term potentiation (LTP) can be produced only in acute slices or in vivo. To study trafficking in adult tissues, we have adapted two biochemical techniques, proteolysis and cross-linking. These techniques help define surface-expressed and intracellular pools of native receptors in acute hippocampal slices.
Asunto(s)
Antígenos de Superficie/biosíntesis , Hipocampo/metabolismo , Receptores de Glutamato/biosíntesis , Animales , Antígenos de Superficie/metabolismo , Western Blotting , Líquido Cefalorraquídeo/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Electrofisiología , Potenciales Postsinápticos Excitadores , Técnicas In Vitro , Péptido Hidrolasas/metabolismo , Ratas , Receptores de Glutamato/metabolismoRESUMEN
In the CA1 region of the rat hippocampus, long-term potentiation (LTP) requires the activation of NMDA receptors (NMDARs) and leads to an enhancement of AMPA receptor (AMPAR) function. In neonatal hippocampus, this increase in synaptic strength seems to be mediated by delivery of AMPARs to the synapse. Here we studied changes in surface expression of native AMPA and NMDA receptors following induction of LTP in the adult rat brain. In contrast to early postnatal rats, we find that LTP in the adult rat does not alter membrane association of AMPARs. Instead, LTP leads to rapid surface expression of NMDARs in a PKC- and Src-family-dependent manner. The present study suggests a developmental shift in the LTP-dependent trafficking of AMPA receptors. Moreover, our results indicate that insertion of NMDA receptors may be a key step in regulating synaptic plasticity.
Asunto(s)
Hipocampo/fisiología , Potenciación a Largo Plazo/fisiología , Neuronas/metabolismo , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Fraccionamiento Celular , Membrana Celular/química , Membrana Celular/metabolismo , Quimotripsina/farmacología , Reactivos de Enlaces Cruzados/farmacología , Electrofisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Hipocampo/citología , Técnicas In Vitro , Neuronas/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Ratas , Transducción de Señal/fisiología , Familia-src Quinasas/metabolismoRESUMEN
The N-methyl-D-aspartate receptor (NMDAR) is an ionotropic glutamate receptor, which plays crucial roles in synaptic plasticity and development. We have recently shown that potentiation of NMDA receptor function by protein kinase C (PKC) appears to be mediated via activation of non-receptor tyrosine kinases. The aim of this study was to test whether this effect could be mediated by direct tyrosine phosphorylation of the NR2A or NR2B subunits of the receptor. Following treatment of rat hippocampal CA1 mini-slices with 500 nM phorbol 12-myristate 13-acetate (PMA) for 15 min, samples were homogenized, immunoprecipitated with anti-NR2A or NR2B antibodies and the resulting pellets subjected to Western blotting with antiphosphotyrosine antibody. An increase in tyrosine phosphorylation of both NR2A (76 +/- 11% above control) and NR2B (41 +/- 11%) was observed. This increase was blocked by pretreatment with the selective PKC inhibitor chelerythrine, with the tyrosine kinase inhibitor Lavendustin A or with the Src family tyrosine kinase inhibitor PP2. PMA treatment also produced an increase in the phosphorylation of serine 890 on the NR1 subunit, a known PKC site, at 5 min with phosphorylation returning to near basal levels by 10 min while tyrosine phosphorylation of NR2A and NR2B was sustained for up to 15 min. These results suggest that the modulation of NMDA receptor function seen with PKC activation may be the result of tyrosine phosphorylation of NR2A and/or NR2B.
Asunto(s)
Hipocampo/metabolismo , Proteína Quinasa C/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Tirosina/metabolismo , Secuencia de Aminoácidos/genética , Animales , Sinergismo Farmacológico , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Ácido Ocadaico/farmacología , Fosforilación , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Acetato de Tetradecanoilforbol/farmacología , Factores de TiempoRESUMEN
Amplification and resulting overexpression of the HER-2/ neu proto-oncogene is found in approximately 30% of human breast and 20% of human ovarian cancers. To better understand the molecular events associated with overexpression of this gene in human breast cancer cells, differential hybridization was used to identify genes whose expression levels are altered in cells overexpressing this receptor. Of 16 000 clones screened from an overexpression cell cDNA library, a total of 19 non-redundant clones were isolated including seven whose expression decreases (C clones) and 12 which increase (H clones) in association with HER-2/ neu overexpression. Of these, five C clones and 11 H clones have been confirmed to be differentially expressed by northern blot analysis. This group includes nine genes of known function, three previously sequenced genes of relatively uncharacterized function and four novel genes without a match in GenBank. Examination of the previously characterized genes indicates that they represent sequences known to be frequently associated with the malignant phenotype, suggesting that the subtraction cloning strategy used identified appropriate target genes. In addition, differential expression of 12 of 16 (75%) cDNAs identified in the breast cancer cell lines are also seen in HER-2/ neu -overexpressing ovarian cancer cells, indicating that they represent generic associations with HER-2/ neu overexpression. Finally, up-regulation of two of the identified cDNAs, one novel and one identified but as yet uncharacterized gene, was confirmed in human breast cancer specimens in association with HER-2/ neu overexpression. Further characterization of these genes may yield insight into the fundamental biology and pathogenetic effects of HER-2/ neu overexpression in human breast and ovarian cancer cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Receptor ErbB-2/biosíntesis , Secuencia de Aminoácidos , Northern Blotting , Neoplasias de la Mama/genética , Femenino , Amplificación de Genes , Humanos , Datos de Secuencia Molecular , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Fenotipo , Proto-Oncogenes Mas , Receptor ErbB-2/genética , Células Tumorales CultivadasRESUMEN
Platelet-activating factor acetylhydrolases (PAF-AHs) play an important role in the metabolism of PAF, a potent phospholipid mediator affecting various physiological processes. The heterotrimeric form of intracellular PAF-AH consists of two catalytic subunits (PAF-AH Ib beta and PAF-AH Ib gamma) and a potential regulatory subunit (PAF-AH Ib alpha). Hemizygous deletion of the gene encoding the alpha subunit has been implicated in two related neurological disorders: isolated lissencephaly sequence and Miller-Dieker syndrome. Here we report the isolation and characterization of mouse Pafaha/Lis1 cDNAs and the corresponding Pafaha/Lis1 gene. We have cloned five cDNAs representing alternatively polyadenylated messages. Northern blot analysis revealed that the various Pafaha/Lis1 mRNAs are differentially expressed in mouse tissues. The Pafaha/Lis1 gene spans a genomic region of more than 50 kb and consists of 12 exons, the first 2 of which are embedded in CpG islands. We have identified two sites of alternative splicing of Pafaha/Lis1: one affecting the length of the 5' untranslated region, the other potentially resulting in a truncated form of the encoded protein.
Asunto(s)
Clonación Molecular , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Proteínas Asociadas a Microtúbulos , Fosfolipasas A/genética , Factor de Activación Plaquetaria/metabolismo , Proteínas/genética , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Empalme Alternativo , Animales , Encéfalo/metabolismo , Exones , Intrones , Ratones , Datos de Secuencia Molecular , Fosfolipasas A/química , Reacción en Cadena de la Polimerasa , Proteínas/química , Análisis de Secuencia de ADNRESUMEN
A novel human chemokine STCP-1 (stimulated T cell chemotactic protein) was isolated from an activated macrophage cDNA library. The chemokine has four cysteines positioned in a manner that identifies STCP-1 as a member of the CC chemokine family. The amino acid sequence shows 34% identity with RANTES. The gene consists of 3 exons and 2 introns with the position of intron/exon boundaries similar to that of RANTES. The gene is expressed as a 3.4-kilobase transcript on lymph node, thymus, and Appendix. STCP-1 induces Ca2+ mobilization in a small percentage of primary activated T lymphocytes, but on repeated stimulation the percentage of T lymphocytes that respond to STCP-1 increases. The chemokine STCP-1 does not induce Ca2+ mobilization in monocytes, dendritic cells, neutrophils, eosinophils, lipopolysaccharide-activated B lymphocytes, and freshly isolated resting T lymphocytes. Similarly, STCP-1, while acting as a mild chemoattractant for primary activated T lymphocytes, is a potent chemoattractant for chronically activated T lymphocytes but has no chemoattractant activity for monocytes, neutrophils, eosinophils, and resting T lymphocytes. As STCP-1 acts specifically on activated T lymphocytes, it may play a role in the trafficking of activated/effector T lymphocytes to inflammatory sites and other aspects of activated T lymphocyte physiology.
Asunto(s)
Quimiocinas/biosíntesis , Quimiocinas/farmacología , Quimiotaxis de Leucocito/efectos de los fármacos , Macrófagos/fisiología , Linfocitos T/fisiología , Secuencia de Aminoácidos , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/fisiología , Secuencia de Bases , Calcio/metabolismo , Quimiocinas/química , Factores Quimiotácticos/farmacología , Clonación Molecular , ADN Complementario , Femenino , Expresión Génica , Humanos , Leucocitos/efectos de los fármacos , Leucocitos/fisiología , Activación de Linfocitos , Activación de Macrófagos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Masculino , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Linfocitos T/efectos de los fármacosRESUMEN
The mechanism of BRCA1 tumor suppression in human breast and ovarian cells is the focus of intense investigation. In this report, full length BRCA1 (230 kDa) introduced into cells with CMV promoter constructs was nuclear when transgene expression was low whereas high expression resulted in cytoplasmic accumulation, aberrant nuclear and cell morphology. A nuclear localization signal (NLS) was mapped to BRCA1 amino acid positions 262-570. We describe a splice variant, BRCA1-delta11b, missing the majority of exon 11 including the NLS. Exogenous BRCA1-delta11b (110 kDa) was cytoplasmic and, unlike the full-length protein, overexpression of the protein encoded by the variant did not appear to be toxic. RNA probe titrations and RT-PCR demonstrated that BRCA1 and delta11b transcripts are coexpressed in a wide variety of cells and tissues. Interestingly, BRCA1-delta11b message was greatly reduced or absent in several breast and ovarian tumor lines relative to exon 11 transcripts and a delta9,10 splice variant. Taken together our results suggest that full-length BRCA1 and BRCA1-delta11b may have distinct roles in cell growth regulation and tumorigenesis.