RESUMEN
Notch and the m9/10 gene (groucho) of the Enhancer of split (E(spI)) complex are members of the "Notch group" of genes, which is required for a variety of cell fate choices in Drosophila. We have characterized human cDNA clones encoding a family of proteins, designated TLE, that are homologous to the E(spI) m9/10 gene product, as well as a novel Notch-related protein. The TLE genes are differentially expressed and encode nuclear proteins, consistent with the presence of sequence motifs associated with nuclear functions. The structural redundancy implied by the existence of more than one TLE and Notch-homologous gene may be a feature of the human counterparts of the developmentally important Drosophila Notch group genes.
Asunto(s)
Drosophila melanogaster/genética , Proteínas Nucleares/genética , Secuencia de Aminoácidos , Animales , Proteínas de Drosophila , Elementos de Facilitación Genéticos , Humanos , Inmunohistoquímica , Hormonas de Insectos/química , Hormonas de Insectos/genética , Hormonas de Insectos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Estructura Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , ARN Mensajero/genética , Receptores Notch , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Notch receptors are involved in cell-fate determination in organisms as diverse as flies, frogs and humans. In Drosophila melanogaster , loss-of-function mutations of Notch produce a 'neurogenic' phenotype in which cells destined to become epidermis switch fate and differentiate to neural cells. Upon ligand activation, the intracellular domain of Notch (ICN) translocates to the nucleus, and interacts directly with the DNA-binding protein Suppressor of hairless (Su(H)) in flies, or recombination signal binding protein Jkappa (RBP-Jkappa) in mammals, to activate gene transcription. But the precise mechanisms of Notch-induced gene expression are not completely understood. The gene mastermind has been identified in multiple genetic screens for modifiers of Notch mutations in Drosophila. Here we clone MAML1, a human homologue of the Drosophila gene Mastermind, and show that it encodes a protein of 130 kD localizing to nuclear bodies. MAML1 binds to the ankyrin repeat domain of all four mammalian NOTCH receptors, forms a DNA-binding complex with ICN and RBP-Jkappa, and amplifies NOTCH-induced transcription of HES1. These studies provide a molecular mechanism to explain the genetic links between mastermind and Notch in Drosophila and indicate that MAML1 functions as a transcriptional co-activator for NOTCH signalling.
Asunto(s)
Proteínas de Unión al ADN , Proteínas de Drosophila , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Células 3T3 , Animales , Células COS , Drosophila/genética , Genes de Insecto , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Receptores Notch , Especificidad de la Especie , Factores de Transcripción , TransfecciónRESUMEN
A fundamental cell-fate control mechanism regulating multicellular development is defined by the Notch-signalling pathway. Developmental and genetic studies of wild type and activated Notch-receptor expression in diverse organisms suggest that Notch plays a general role in development by governing the ability of undifferentiated precursor cells to respond to specific signals. Notch signalling has been conserved throughout evolution and controls the differentiation of a broad spectrum of cell types during development. Genetic studies in Drosophila have led to the identification of several components of the Notch pathway. Two of the positive regulators of the pathway are encoded by the suppressor of hairless [Su(H)] and deltex (dx) genes. Drosophila dx encodes a ubiquitous, novel cytoplasmic protein of unknown biochemical function. We have cloned a human deltex homologue and characterized it in parallel with its Drosophila counterpart in biochemical assays to assess deltex function. Both human and Drosophila deltex bind to Notch across species and carry putative SH3-binding domains. Using the yeast interaction trap system, we find that Drosophila and human deltex bind to the human SH3-domain containing protein Grb2 (ref. 10). Results from two different reporter assays allow us for the first time to associate deltex with Notch-dependent transcriptional events. We present evidence linking deltex to the modulation of basic helix-loop-helix (bHLH) transcription factor activity.
Asunto(s)
Proteínas de Drosophila , Proteínas de Insectos/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Drosophila , Humanos , Proteínas de Insectos/química , Datos de Secuencia Molecular , Receptores Notch , Homología de Secuencia de AminoácidoRESUMEN
An intricate interplay of signalling pathways dictates the acquisition of specific cell fates during development. The NOTCH receptor is the central element in a cell-interaction mechanism that controls the fate of a very broad spectrum of precursor cells. Conservation across species implies that signalling through this receptor is a tool frequently used by metazoans to modulate the fate of precursor cells. This article describes recent advances in the genetic and molecular dissection of this developmentally fundamental pathway that have provided new insights into the mechanism by which extracellular signals act through the NOTCH receptor to determine or alter cellular fate.
RESUMEN
The warthog (wrt) gene, recovered as a modifier for Notch signaling, was found to encode the Drosophila homologue of rab6, Drab6. Vertebrate and yeast homologues of this protein have been shown to regulate Golgi network to TGN trafficking. To study the function of this protein in the development of a multicellular organism, we analyzed three different warthog mutants. The first was an R62C point mutation, the second a genomic null, and the third was an engineered GTP-bound form. Our studies show, contrary to yeast, that the Drosophila homologue of rab6 is an essential gene. However, it has limited effects on development beyond the larval stage. Only the mechanosensory bristles on the head, notum, and scutellum are affected by warthog mutations. We present models for the modifying effect of Drab6 on Notch signaling.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Proteínas de la Membrana/metabolismo , Proteínas de Unión al GTP rab , Proteínas ras/genética , Proteínas ras/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Clonación Molecular , Proteínas de Drosophila , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Expresión Génica , Genes Esenciales/genética , Genes de Insecto/genética , Prueba de Complementación Genética , Guanosina Trifosfato/metabolismo , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Larva/genética , Larva/crecimiento & desarrollo , Datos de Secuencia Molecular , Mosaicismo , Mutación , Fenotipo , Receptores Notch , Homología de Secuencia de Aminoácido , Transducción de Señal , Proteínas ras/químicaRESUMEN
The Drosophila cell cycle gene fizzy (fzy) is required for normal execution of the metaphase-anaphase transition. We have cloned fzy, and confirmed this by P-element mediated germline transformation rescue. Sequence analysis predicts that fzy encodes a protein of 526 amino acids, the carboxy half of which has significant homology to the Saccharomyces cerevisiae cell cycle gene CDC20. A monoclonal antibody against fzy detects a single protein of the expected size, 59 kD, in embryonic extracts. In early embryos fzy is expressed in all proliferating tissues; in late embryos fzy expression declines in a tissue-specific manner correlated with cessation of cell division. During interphase fzy protein is present in the cytoplasm; while in mitosis fzy becomes ubiquitously distributed throughout the cell except for the area occupied by the chromosomes. The metaphase arrest phenotype caused by fzy mutations is associated with failure to degrade both mitotic cyclins A and B, and an enrichment of spindle microtubules at the expense of astral microtubules. Our data suggest that fzy function is required for normal cell cycle-regulated proteolysis that is necessary for successful progress through mitosis.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Ciclinas/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insecto/genética , Mitosis/fisiología , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Cdc20 , Compartimento Celular , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/inmunología , Proteínas de Ciclo Celular/aislamiento & purificación , Clonación Molecular , Drosophila melanogaster/embriología , Proteínas Fúngicas/genética , Immunoblotting , Inmunohistoquímica , Microtúbulos/metabolismo , Mitosis/genética , Datos de Secuencia Molecular , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Huso Acromático/fisiologíaRESUMEN
The Notch locus of Drosophila melanogaster is one of a small number of zygotically acting "neurogenic" genes involved in the correct segregation of neural from epidermal lineages during embryogenesis as well as in other postembryonic developmental events. We have generated antibody probes against three regions of the Notch protein to study the expression of Notch and begin a biochemical characterization of the protein. Consistent with predictions based on DNA sequence data, here we gather evidence showing that Notch encodes a large, glycosylated surface protein with an apparent molecular mass of 300 kD: (a) all three antibodies detect Notch on Western blots as a high molecular mass, primarily full-length product; (b) immunoelectron microscopy localizes the Notch protein to the cell membrane; and (c) lentil lectin column binding demonstrates that the protein is glycosylated, indicative of its surface protein nature. In general, the distribution of the Notch protein coincides with that of the Notch transcript determined previously by in situ hybridizations. Notch is expressed in a much wider range of tissue types than those disrupted in the neurogenic mutant, as determined by antibody localization. Early labeling in the blastoderm appears ubiquitous except for the pole cells, but as development proceeds some distinctive features emerge: stronger staining is seen within the germ band layer where neuroblast delamination occurs, and the developing embryonic nervous system shows pronounced axonal staining. In third instar larvae, Notch is expressed in imaginal disks and in the central nervous system. Based on these results, certain models for how Notch controls the neuroblast cell fate choice are eliminated. We discuss how Notch may function in this choice as well as in other lineage fate determinations.
Asunto(s)
Drosophila melanogaster/genética , Genes , Glicoproteínas de Membrana/genética , Proteínas del Tejido Nervioso/genética , Animales , Western Blotting , Membrana Celular/metabolismo , Clonación Molecular , ADN/genética , Drosophila melanogaster/crecimiento & desarrollo , Células Epidérmicas , Epidermis/metabolismo , Larva , Neuronas/citología , Neuronas/metabolismo , Pupa , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo RestrictivoRESUMEN
The Notch gene in Drosophila encodes a transmembrane protein with homology to EGF that appears to mediate cell-cell interactions necessary for proper epidermal vs. neural fate decisions. In this study, we examine Notch expression in detail throughout embryonic and imaginal development using confocal laser-scanning microscopy and specific mAb probes. We find that Notch is expressed in a tissue-specific manner as early as the cellular blastoderm stage, when cells of the presumptive mesoderm clearly express less Notch than adjacent ectodermal precursors. Notch is abundantly expressed during the initial determination of neuronal lineages, such as the embryonic neuroblasts and the precursors of sensory neurons in the imaginal disc epithelia, but expression quickly decreases during subsequent differentiation. These changing patterns of Notch expression do not correlate well with cell movements, and thus do not appear to support the notion that the major function of Notch is to maintain epithelial integrity via adhesive mechanisms. Our data suggest instead that Notch may act as a cell-surface receptor, perhaps functioning in the lateral inhibition mechanism that is necessary for proper spacing of neuronal precursors.
Asunto(s)
Drosophila/crecimiento & desarrollo , Hormonas de Insectos/biosíntesis , Proteínas de la Membrana/biosíntesis , Animales , Blastodermo/metabolismo , Drosophila/embriología , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Hormonas de Insectos/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Proteínas de la Membrana/genética , Mesodermo/metabolismo , Metamorfosis Biológica , Mitosis , Neuronas/citología , Neuronas/metabolismo , Pupa/crecimiento & desarrollo , Pupa/metabolismo , Receptores NotchRESUMEN
The exuberant growth of neurites during development becomes markedly reduced as cortical neurons mature. In vitro studies of neurons from mouse cerebral cortex revealed that contact-mediated Notch signaling regulates the capacity of neurons to extend and elaborate neurites. Up-regulation of Notch activity was concomitant with an increase in the number of interneuronal contacts and cessation of neurite growth. In neurons with low Notch activity, which readily extend neurites, up-regulation of Notch activity either inhibited extension or caused retraction of neurites. Conversely, in more mature neurons that had ceased their growth after establishing numerous connections and displayed high Notch activity, inhibition of Notch signaling promoted neurite extension. Thus, the formation of neuronal contacts results in activation of Notch receptors, leading to restriction of neuronal growth and a subsequent arrest in maturity.
Asunto(s)
Comunicación Celular , Corteza Cerebral/citología , Proteínas de la Membrana/metabolismo , Neuritas/fisiología , Neuronas/citología , Receptores de Superficie Celular/metabolismo , Factores de Transcripción , Animales , Recuento de Células , Diferenciación Celular , Movimiento Celular , Núcleo Celular/metabolismo , Tamaño de la Célula , Células Cultivadas , Corteza Cerebral/embriología , Inhibición de Contacto , Humanos , Ligandos , Ratones , Mitosis , Neuritas/química , Neuronas/metabolismo , Estructura Terciaria de Proteína , Receptor Notch1 , Receptor Notch2 , Transducción de Señal , Activación Transcripcional , Regulación hacia ArribaRESUMEN
The Notch/Lin-12/Glp-1 receptor family mediates the specification of numerous cell fates during development in Drosophila and Caenorhabditis elegans. Studies on the expression, mutant phenotypes, and developmental consequences of unregulated receptor activation have implicated these proteins in a general mechanism of local cell signaling, which includes interactions between equivalent cells and between different cell types. Genetic approaches in flies and worms have identified putative components of the signaling cascade, including a conserved family of extracellular ligands and two cellular factors that may associate with the Notch Intracellular domain. One factor, the Drosophila Suppressor of Hairless protein, is a DNA-binding protein, which suggests that Notch signaling may involve relatively direct signal transmission from the cell surface to the nucleus. Several vertebrate Notch receptors have also been discovered recently and play important roles in normal development and tumorigenesis.
Asunto(s)
Proteínas de Caenorhabditis elegans , Diferenciación Celular , Proteínas de la Membrana/fisiología , Receptores de Superficie Celular/fisiología , Transducción de Señal , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Proteínas de Drosophila , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Proteínas del Helminto/fisiología , Humanos , Ligandos , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/genética , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Superficie Celular/genética , Receptores NotchRESUMEN
In Drosophila, the Wingless and Notch signaling pathways function in m any of the same developmental patterning events. Genetic analysis demonstrates that the dishevelled gene, which encodes a molecule previously implicated in implementation of the Winglass signal, interacts antagonistically with Notch and one of its known ligands, Delta. A direct physical interaction between Dishevelled and the Notch carboxyl terminus, distal to the cdc10/ankyrin repeats, suggests a mechanism for this interaction. It is proposed that Dishevelled, in addition to transducing the Wingless signal, blocks Notch signaling directly, thus providing a molecular mechanism for the inhibitory cross talk observed between these pathways.
Asunto(s)
Proteínas de Drosophila , Drosophila/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Células Clonales , Proteínas Dishevelled , Drosophila/genética , Drosophila/crecimiento & desarrollo , Genes de Insecto , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación , Fenotipo , Proteínas/genética , Proteínas Proto-Oncogénicas/genética , Pupa/metabolismo , Receptores Notch , Alas de Animales/citología , Alas de Animales/crecimiento & desarrollo , Proteína Wnt1RESUMEN
Notch signaling defines an evolutionarily ancient cell interaction mechanism, which plays a fundamental role in metazoan development. Signals exchanged between neighboring cells through the Notch receptor can amplify and consolidate molecular differences, which eventually dictate cell fates. Thus, Notch signals control how cells respond to intrinsic or extrinsic developmental cues that are necessary to unfold specific developmental programs. Notch activity affects the implementation of differentiation, proliferation, and apoptotic programs, providing a general developmental tool to influence organ formation and morphogenesis.
Asunto(s)
Proteínas de la Membrana/fisiología , Receptores de Superficie Celular/fisiología , Transducción de Señal , Animales , Apoptosis , Comunicación Celular , División Celular , Núcleo Celular/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ligandos , Receptores Notch , Transcripción GenéticaRESUMEN
Signaling by the Notch surface receptor controls cell fate determination in a broad spectrum of tissues. This signaling is triggered by the interaction of the Notch protein with what, so far, have been thought to be transmembrane ligands expressed on adjacent cells. Here biochemical and genetic analyses show that the ligand Delta is cleaved on the surface, releasing an extracellular fragment capable of binding to Notch and acting as an agonist of Notch activity. The ADAM disintegrin metalloprotease Kuzbanian is required for this processing event. These observations raise the possibility that Notch signaling in vivo is modulated by soluble forms of the Notch ligands.
Asunto(s)
Desintegrinas/metabolismo , Proteínas de Drosophila , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Células Cultivadas , Desintegrinas/genética , Drosophila/embriología , Drosophila/genética , Drosophila/metabolismo , Femenino , Péptidos y Proteínas de Señalización Intracelular , Ligandos , Masculino , Proteínas de la Membrana/genética , Metaloendopeptidasas/genética , Datos de Secuencia Molecular , Mutación , Neuronas/citología , Procesamiento Proteico-Postraduccional , Receptores Notch , Transducción de Señal , TransfecciónAsunto(s)
Síndrome de Alagille/genética , Proteínas de la Membrana/genética , Receptores de Superficie Celular/genética , Animales , Proteínas de Unión al Calcio , Diferenciación Celular , Drosophila/genética , Proteínas de Drosophila , Genes Dominantes , Humanos , Péptidos y Proteínas de Señalización Intercelular , Péptidos y Proteínas de Señalización Intracelular , Proteína Jagged-1 , Ligandos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Mutación , Receptores de Superficie Celular/metabolismo , Receptores Notch , Proteínas Serrate-Jagged , Transducción de SeñalRESUMEN
During the development of Drosophila melanogaster, individual cells must make choices between a restricted set of possible fates in order to give rise to spatial patterns composed of different types of differentiated cells. The Notch locus appears to play a central and general role in the regulative events that control the local architecture of the final cellular pattern in several tissues, among them being the central and peripheral nervous systems.
Asunto(s)
Drosophila melanogaster/embriología , Hormonas de Insectos/fisiología , Proteínas de la Membrana/fisiología , Animales , Diferenciación Celular , Proteínas de Drosophila , Drosophila melanogaster/genética , Ectodermo , Desarrollo Embrionario , Desarrollo Embrionario y Fetal , Epistasis Genética , Retroalimentación , Regulación de la Expresión Génica , Genes , Hormonas de Insectos/genética , Proteínas de la Membrana/genética , Mosaicismo , Sistema Nervioso/embriología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/fisiología , Receptores NotchRESUMEN
The Notch genes of Drosophila melanogaster and vertebrates encode transmembrane receptors that help determine cell fate during development. Although ligands for Notch proteins have been identified, the signaling cascade downstream of the receptors remains poorly understood. In human acute lymphoblastic T-cell leukemia, a chromosomal translocation damages the NOTCH1 gene. The damage apparently gives rise to a constitutively activated version of NOTCH protein. Here we show that a truncated version of NOTCH1 protein resembling that found in the leukemic cells can transform rat kidney cells in vitro. The transformation required cooperation with the E1A oncogene of adenovirus. The transforming version of NOTCH protein was located in the nucleus. In contrast, neither wild-type NOTCH protein nor a form of the truncated protein permanently anchored to the plasma membrane produced transformation in vitro. We conclude that constitutive activation of NOTCH similar to that found in human leukemia can contribute to neoplastic transformation. Transformation may require that the NOTCH protein be translocated to the nucleus. These results sustain a current view of how Notch transduces a signal from the surface of the cell to the nucleus.
Asunto(s)
Transformación Celular Neoplásica/genética , Leucemia Experimental/genética , Leucemia-Linfoma de Células T del Adulto/genética , Proteínas de la Membrana/genética , Receptores de Superficie Celular/genética , Factores de Transcripción , Proteínas E1A de Adenovirus/genética , Alelos , Animales , Transporte Biológico , Compartimento Celular , Núcleo Celular/metabolismo , Humanos , Ratas , Receptor Notch1 , Receptor Notch2 , Eliminación de SecuenciaRESUMEN
E47 is a widely expressed transcription factor that activates B-cell-specific immunoglobulin gene transcription and is required for early B-cell development. In an effort to identify processes that regulate E47, and potentially B-cell development, we found that activated Notch1 and Notch2 effectively inhibit E47 activity. Only the intact E47 protein was inhibited by Notch-fusion proteins containing isolated DNA binding and activation domains were unaffected-suggesting that Notch targets an atypical E47 cofactor. Although overexpression of the coactivator p300 partially reversed E47 inhibition, results of several assays indicated that p300/CBP is not a general target of Notch. Notch inhibition of E47 did not correlate with its ability to activate CBF1/RBP-Jkappa, the mammalian homolog of Suppressor of Hairless, a protein that associates physically with Notch and defines the only known Notch signaling pathway in drosophila. Importantly, E47 was inhibited independently of CBF1/RPB-Jkappa by Deltex, a second Notch-interacting protein. We provide evidence that Notch and Deltex may act on E47 by inhibiting signaling through Ras because (i) full E47 activity was found to be dependent on Ras and (ii) both Notch and Deltex inhibited GAL4-Jun, a hybrid transcription factor whose activity is dependent on signaling from Ras to SAPK/JNK.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Drosophila , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Transactivadores , Células 3T3 , Animales , Proteína de Unión a CREB , Proteínas de Unión al ADN/metabolismo , Proteína p300 Asociada a E1A , Proteínas de Insectos/metabolismo , Ratones , Proteínas Nucleares/metabolismo , Receptor Notch1 , Receptor Notch2 , Proteínas Represoras/metabolismo , Factores de Transcripción TCF , Proteína 1 Similar al Factor de Transcripción 7 , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas ras/metabolismoRESUMEN
Notch receptors participate in a highly conserved signaling pathway that regulates morphogenesis in multicellular animals. Maturation of Notch receptors requires the proteolytic cleavage of a single precursor polypeptide to produce a heterodimer composed of a ligand-binding extracellular domain (N(EC)) and a single-pass transmembrane signaling domain (N(TM)). Notch signaling has been correlated with additional ligand-induced proteolytic cleavages, as well as with nuclear translocation of the intracellular portion of N(TM) (N(ICD)). In the current work, we show that the N(EC) and N(TM) subunits of Drosophila Notch and human Notch1 (hN1) interact noncovalently. N(EC)-N(TM) interaction was disrupted by 0.1% sodium dodecyl sulfate or divalent cation chelators such as EDTA, and stabilized by millimolar Ca(2+). Deletion of the Ca(2+)-binding Lin12-Notch (LN) repeats from the N(EC) subunit resulted in spontaneous shedding of N(EC) into conditioned medium, implying that the LN repeats are important in maintaining the interaction of N(EC) and N(TM). The functional consequences of EDTA-induced N(EC) dissociation were studied by using hN1-expressing NIH 3T3 cells. Treatment of these cells for 10 to 15 min with 0.5 to 10 mM EDTA resulted in the rapid shedding of N(EC), the transient appearance of a polypeptide of the expected size of N(ICD), increased intranuclear anti-Notch1 staining, and the transient activation of an Notch-sensitive reporter gene. EDTA treatment of HeLa cells expressing endogenous Notch1 also stimulated reporter gene activity to a degree equivalent to that resulting from exposure of the cells to the ligand Delta1. These findings indicate that receptor activation can occur as a consequence of N(EC) dissociation, which relieves inhibition of the intrinsically active N(TM) subunit.
Asunto(s)
Calcio/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal , Células 3T3 , Animales , Dimerización , Drosophila , Proteínas de Drosophila , Humanos , Transporte Iónico , Proteínas de la Membrana/genética , Ratones , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores NotchRESUMEN
Since our characterization of the slit cDNA sequence, encoding a protein secreted by glial cells and involved in the formation of axonal pathways in Drosophila, we have discovered that the protein contains two additional sequence motifs that are highly conserved in a variety of proteins. A search of the GenPept database with the 73 amino acids at the carboxy terminus of slit revealed that this region contains significant similarity to a carboxy-terminal domain found in six other exported proteins. This observation has allowed us to define a new carboxy-terminal protein motif. In addition, comparisons with a 202 amino acid domain residing between epidermal growth factor (EGF) repeats in slit shows this region to be conserved in laminin, agrin and perlecan and, strikingly, also to lie between EGF repeats in both agrin and perlecan. Our analysis suggests this motif is involved in mediating interactions among extracellular proteins. Consistent with our previous characterization of the slit protein, both new motifs are found only in extracellular proteins. The identification of these two conserved motifs in slit reveals that the entire 1469 amino acids of the protein are made up of modular regions similar to those conserved in other extracellular proteins.