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Results for “Receptors, Notch”

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At least 19 recordsLinked to original sources

PubMed2000

Expression of notch receptors, notch ligands, and fringe genes in hematopoiesis.

OBJECTIVE: Hematopoiesis is the process by which mature blood cell types are generated from a small population of pluripotent hematopoietic stem cells. How these cells undergo fate selection, however, is not fully understood. The Notch signaling system is known to mediate cell fate decisions of multipotent precursors in a wide range of complex animals throughout development. As Notch signaling involves cell-cell interactions, we sought to determine the expression of Notch receptors, ligands, and regulators in individual cell populations along the hematopoietic differentiation pathway. MATERIALS: Described here is a single cell RT-PCR analysis of Notch1, Notch3, Notch4, Notch ligands (Dll1 and Jagged1), and Fringe gene expression in cells of the blood system. As previously described, single cell globally amplified cDNA was generated by RT-PCR from various hematopoietic precursor cells whose potential was known from sibling analysis. A precursor hierarchy slot blot was created containing these cDNAs as well as samples from maturing blood cell populations and two fibroblast cell lines. The precursor slot blot was screened with probes for each of the candidate genes. RESULTS: Macrophage precursors expressed high levels of Notch1 transcript, while maturing macrophages expressed high levels of both Notch1 and Notch4. The Jagged 1 ligand transcript was highly expressed in terminally maturing cells including mast cells and megakaryocytes. In contrast, the Manic Fringe gene was highly expressed in uncommitted bi- and tri-potential precursors as well as in committed neutrophil and macrophage precursors. CONCLUSIONS: Distinct expression patterns of Jagged1 and Manic Fringe suggest that their corresponding proteins could regulate cell fate choices during hematopoiesis and may be responsible for regulating communication between lineage compartments during hematopoietic development.

Animals↗

PubMed2000

Calcium depletion dissociates and activates heterodimeric notch receptors.

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.

3T3 Cells↗

PubMed2000

MAML1, a human homologue of Drosophila mastermind, is a transcriptional co-activator for NOTCH receptors.

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.

3T3 Cells↗

PubMed2004

The Notch amino terminus regulates protein levels and Delta-induced clustering of Drosophila Notch receptors.

Notch signaling is required for the development of almost all animal tissues. It is a cell surface receptor that generates intracellular signals in response to Delta binding its extracellular domain. Notch response to Delta is affected by mutations in its extracellular domain outside of the Delta binding region. One such region is the Notch amino terminus. Mutations in this region are associated with developmental defects. How a mutation in the Notch amino terminus affects Notch function is unknown. We explored this issue in Drosophila melanogaster. We report that Notch receptors mutated in the amino terminus accumulate to abnormal levels, are deficient in Delta induced receptor clustering, and exhibit reduced rate of internalization and signaling. Notch receptors lacking the whole or the carboxy-terminal half of the intracellular domain are defective in internalization but not in clustering or accumulation. None of the other mutated Notch receptors showed defects in clustering, accumulation, or internalization. These observations suggest that the Notch amino terminus regulates Notch levels and clustering, which could affect the rate of Notch signaling and down-regulation.

Animals↗

PubMed2004

Nuclear betaII-tubulin associates with the activated notch receptor to modulate notch signaling.

The Notch signal pathway plays important roles in proliferation, apoptosis, and differentiation. Abnormalities in Notch signaling are linked to many human diseases. After ligand binding, Notch signaling is activated through the cleavage of Notch receptors to release and translocate the Notch intracellular domain into the nucleus. The Notch1 receptor intracellular domain (N1IC), the activated form of the Notch1 receptor, can modulate downstream target genes via C promoter-binding factor 1-dependent and -independent pathways. To further dissect the Notch1 signaling pathway, we screened the N1IC-associated proteins using a yeast two-hybrid system and identified nuclear beta(II)-tubulin as a candidate for the N1IC-associated proteins. It was suggested that the presence of beta(II)-tubulin in nuclei might be correlated with the cancerous state of cells. However, the function of beta(II)-tubulin locating in the nucleus still is unknown. Herein, we show that the complex of alpha- and beta(II)-tubulin is associated with N1IC in cancer cells by a coimmunoprecipitation analysis. The ankyrin domain of the Notch1 receptor alone was sufficient to associate with beta(II)-tubulin. Furthermore, alpha- and beta(II)-tubulin were localized in the nucleus and formed a complex with N1IC. Treatment with Taxol increased the amounts of nuclear alpha- and beta(II)-tubulin in K562 and HeLa cells and promoted the C promoter-binding factor 1-dependent transactivation activity of N1IC. We also show that nuclear beta(II)-tubulin was bound on the C promoter-binding factor 1 response elements via the association with N1IC. These results suggest that nuclear beta(II)-tubulin can modulate Notch signaling through interaction with N1IC in cancer cells.

Cell Line, Tumor↗

PubMed2001

Role of Deltex-1 as a transcriptional regulator downstream of the Notch receptor.

Intercellular signaling through the cell-surface receptor Notch plays important roles in a variety of developmental processes as well as in pathogenesis of some human cancers and genetic disorders. However, the mechanisms by which Notch signals are transduced into cells still remain elusive. Here we investigated the signaling mechanisms for Notch in the cell fate control of neural progenitor cells. We show that Deltex-1 (DTX1), a mammalian homolog of Drosophila Deltex, mediates a Notch signal to block differentiation of neural progenitor cells. We found that a significant fraction of DTX1 proteins were localized in the nucleus and physically interacted with the transcriptional coactivator p300. Through its binding to p300, DTX1 inhibited transcriptional activation by the neural-specific helix-loop-helix-type transcription factor MASH1, and this mechanism is likely responsible for the differentiation inhibition of neural progenitor cells. Our results further suggest that DTX1 regulates transcription independently of the previously characterized Notch signaling pathway involving RBP-J and HES1/HES5. Thus, DTX1 serves as an important signaling component downstream of Notch that regulates transcription in the nucleus.

Animals↗

PubMed2003

Structure and stability of the ankyrin domain of the Drosophila Notch receptor.

The Notch receptor contains a conserved ankyrin repeat domain that is required for Notch-mediated signal transduction. The ankyrin domain of Drosophila Notch contains six ankyrin sequence repeats previously identified as closely matching the ankyrin repeat consensus sequence, and a putative seventh C-terminal sequence repeat that exhibits lower similarity to the consensus sequence. To better understand the role of the Notch ankyrin domain in Notch-mediated signaling and to examine how structure is distributed among the seven ankyrin sequence repeats, we have determined the crystal structure of this domain to 2.0 angstroms resolution. The seventh, C-terminal, ankyrin sequence repeat adopts a regular ankyrin fold, but the first, N-terminal ankyrin repeat, which contains a 15-residue insertion, appears to be largely disordered. The structure reveals a substantial interface between ankyrin polypeptides, showing a high degree of shape and charge complementarity, which may be related to homotypic interactions suggested from indirect studies. However, the Notch ankyrin domain remains largely monomeric in solution, demonstrating that this interface alone is not sufficient to promote tight association. Using the structure, we have classified reported mutations within the Notch ankyrin domain that are known to disrupt signaling into those that affect buried residues and those restricted to surface residues. We show that the buried substitutions greatly decrease protein stability, whereas the surface substitutions have only a marginal affect on stability. The surface substitutions are thus likely to interfere with Notch signaling by disrupting specific Notch-effector interactions and map the sites of these interactions.

Amino Acid Sequence↗

PubMed2006

[Role of Cre-loxP-mediated conditional gene targeting in understanding the function of Notch receptors].

The Notch family of cell surface receptors and their ligands constitute an evolutionarily conserved signaling pathway that is used by invertebrates and vertebrates to regulate a broad spectrum of cell specification events through local cell interactions. After ligand binding Notch receptor undergoes proteolytic processing ultimately liberating the cytoplasmic domain of the Notch receptor which translocates to the nucleus and activates target genes. In all animal models tested, mutations in Notch genes invariably resulted in developmental abnormalities. In mammals, Notch signaling controls key stages of lymphocyte differentiation as well as activation and several abnormalities in Notch pathway have been suggested to cause human leukemias. Cre-loxP mediated conditional gene targeting significantly contributed to our current understanding of the physiological roles of different Notch family members in hematopoietic compartment. This technique helped to overcome embryonic lethality of Notch mutants providing the opportunity to inactivate specific Notch gene in adulthood.

Animals↗

PubMed2004

Distribution of presenilin 1 and 2 and their relation to Notch receptors and ligands in human embryonic/foetal central nervous system.

Notch signaling in vertebrates is mediated by four Notch receptors (Notch-1, -2, -3, and -4) that are activated by interacting with at least five different Notch ligands, Jagged-1, Jagged-2, Delta-1, -2, and -3. Recent studies have shown that the gamma-secretase-like intramembranous cleavage of Notch receptors to release their cytoplasmic signaling domains requires the presenilin (PS) proteins 1 and 2 (PS1 and PS2). Here, we used immunohistochemistry to compare the distribution of all four Notch receptor proteins and three ligands in the context of co-localization with PS1 and PS2 in first trimester human central nervous system (CNS). In addition, we investigated Notch receptors and ligands expression by Western blotting. The study was performed on the forebrain and spinal cord of human embryonic/foetal CNS (5-11 gestational weeks). Results showed a divergent distribution of the different Notch receptor proteins with only Notch-1 being co-localized with PS1 and PS2. Notch-2 was only seen occasionally within the developing cortex and spinal cord. Notch-3 expression was restricted to neuroepithelial cells of the spinal cord and endothelial cells in blood vessels of both developing cerebral cortex and spinal cord. The weak, punctate staining of Notch-4 in the neuroepithelium of the spinal cord could not be confirmed with Western blotting. Neither Notch-2, nor -3 showed overlap with either PS1 or PS2 immunoreactivity. The ligand Jagged-1 was found sporadically in the neuroepithelial cell layer in cerebral cortex of the earlier stages of development and of the spinal cord during the first trimester while Jagged-2 was not detected. Jagged-1 and Jagged-2 immunoreactivities were not found in the 9-11-week cortex. No co-distribution of Jagged-1 and PS1 or PS2 was found. Delta-1 ligand expression was detected in neuroepithelial cells of the ventricular zone of the cerebral cortex, and also in maturating neurons in the cortical plate and ventral horns of the developing spinal cord. The presence of Notch-1, Delta-1 and Jagged-1 in the neuroepithelium of developing CNS indicates that Notch signaling in proliferating human progenitor cells only involves these two receptor ligands and that cleavage of Notch-1 is mediated both by PS1 and PS2. The strong immunoreactivity of Notch-1, Delta-1 and PS1 in the cortical plate and in maturating neurons of the spinal cord also suggests that these proteins may regulate the maturation processes of post-mitotic neurons. The pronounced PS1 immunoreactivity in neurites in the hindbrain and spinal cord without detectable expression of any Notch receptor or ligand suggests that a possible role for PS1 in neurite growth involves either gamma-secretase-mediated cleavage of other substrates or gamma-secretase-independent mechanisms.

Blotting, Western↗

PubMed2005

Propensity of adult lymphoid progenitors to progress to DN2/3 stage thymocytes with Notch receptor ligation.

Notch family receptors control critical events in the production and replenishment of specialized cells in the immune system. However, it is unclear whether Notch signaling regulates abrupt binary lineage choices in homogeneous progenitors or has more gradual influence over multiple aspects of the process. A recently developed coculture system with Delta 1-transduced stromal cells is being extensively used to address such fundamental questions. Different from fetal progenitors, multiple types of adult marrow cells expanded indefinitely in murine Delta-like 1-transduced OP9 cell cocultures, progressed to a DN2/DN3 thymocyte stage, and slowly produced TCR(+) and NK cells. Long-term cultured cells of this kind retained some potential for T lymphopoiesis in vivo. Adult marrow progressed through double-positive and single-positive stages only when IL-7 concentrations were low and passages were infrequent. Lin(-)c-Kit(low)GFP(+)IL-7Ralpha(+/-) prolymphocytes were the most efficient of adult bone marrow cells in short-term cultures, but the assay does not necessarily reflect cells normally responsible for replenishing the adult thymus. Although marrow-derived progenitors with Ig D(H)-J(H) rearrangements acquired T lineage characteristics in this model, that was not the case for more B committed cells with V(H)-D(H)J(H) rearrangement products.

Aging↗

PubMed2001

Notch receptors and hematopoiesis.

Notch receptors are involved in a variety of cell-fate decisions that affect the development and function of many organs, including hematopoiesis and the immune system. There are four mammalian Notch receptors that have only partially overlapping functions despite sharing similar structures and ligands. The ligands for Notch are transmembrane proteins expressed on adjacent cells, including Jagged and Delta, and it is quite possible that signaling is bidirectional. A large Notch precursor protein is proteolytically cleaved to form the mature cell-surface receptor. Ligand binding induces additional proteolytic events followed by translocation of the intracellular domain to the nucleus. There, Notch interacts with transcription factors such as RBPJ kappa, activating transcription of basic helix-loop-helix genes such as HES1. These in turn regulate expression of tissue-specific transcription factors that influence lineage commitment and other events. In this review, the details of Notch signaling will be discussed, with a focus on what is known about the role of Notch in hematopoiesis.

Animals↗

PubMed2005

Chaperone activity of protein O-fucosyltransferase 1 promotes notch receptor folding.

Notch proteins are receptors for a conserved signaling pathway that affects numerous cell fate decisions. We found that in Drosophila, Protein O-fucosyltransferase 1 (OFUT1), an enzyme that glycosylates epidermal growth factor-like domains of Notch, also has a distinct Notch chaperone activity. OFUT1 is an endoplasmic reticulum protein, and its localization was essential for function in vivo. OFUT1 could bind to Notch, was required for the trafficking of wild-type Notch out of the endoplasmic reticulum, and could partially rescue defects in secretion and ligand binding associated with Notch point mutations. This ability of OFUT1 to facilitate folding of Notch did not require its fucosyltransferase activity. Thus, a glycosyltransferase can bind its substrate in the endoplasmic reticulum to facilitate normal folding.

Amino Acid Motifs↗

PubMed2004

Monoubiquitination and endocytosis direct gamma-secretase cleavage of activated Notch receptor.

Activation of mammalian Notch receptor by its ligands induces TNFalpha-converting enzyme-dependent ectodomain shedding, followed by intramembrane proteolysis due to presenilin (PS)-dependent gamma-secretase activity. Here, we demonstrate that a new modification, a monoubiquitination, as well as clathrin-dependent endocytosis, is required for gamma-secretase processing of a constitutively active Notch derivative, DeltaE, which mimics the TNFalpha-converting enzyme-processing product. PS interacts with this modified form of DeltaE, DeltaEu. We identified the lysine residue targeted by the monoubiquitination event and confirmed its importance for activation of Notch receptor by its ligand, Delta-like 1. We propose a new model where monoubiquitination and endocytosis of Notch are a prerequisite for its PS-dependent cleavage, and discuss its relevance for other gamma-secretase substrates.

Amino Acid Sequence↗

PubMed2002

Identification of a family of mastermind-like transcriptional coactivators for mammalian notch receptors.

The molecular mechanisms by which Notch receptors induce diverse biological responses are not fully understood. We recently cloned a mammalian homologue of the Mastermind gene of Drosophila melanogaster, MAML1 (Mastermind-like-1 molecule) and determined that it functions as a transcriptional coactivator for Notch receptors. In this report, we characterize two additional genes in this Mastermind-like gene family: MAML2 and MAML3. The three MAML genes are widely expressed in adult tissues but exhibit distinct expression patterns in mouse early spinal cord development. All MAML proteins localize to nuclear bodies, share a conserved basic domain in their N termini that binds to the ankyrin repeat domain of Notch, and contain a transcriptional activation domain in their C termini. Moreover, as determined by using coimmunoprecipitation assays, each MAML protein was found to be capable of forming a multiprotein complex with the intracellular domain of each Notch receptor (ICN1 to -4) and CSL in vivo. However, MAML3 bound less efficiently to the ankyrin repeat domain of Notch1. Also, in U20S cells, whereas MAML1 and MAML2 functioned efficiently as coactivators with each of the Notch receptors to transactivate a Notch target HES1 promoter construct, MAML3 functioned more efficiently with ICN4 than with other forms of ICN. Similarly, MAML1 and MAML2 amplified Notch ligand (both Jagged2 and Delta1)-induced transcription of the HES-1 gene, whereas MAML3 displayed little effect. Thus, MAML proteins may modify Notch signaling in different cell types based on their own expression levels and differential activities and thereby contribute to the diversity of the biological effects resulting from Notch activation.

Amino Acid Sequence↗

PubMed2006

Roles of O-fucosyltransferase 1 and O-linked fucose in notch receptor function.

O-fucosyltransferase 1 (Ofut1) is a soluble endoplasmic reticulum protein that directly transfers fucose onto serine or threonine residues of EGF domain-containing proteins such as Notch receptors. Genetic analysis indicates that Ofut1 is essential for Notch receptor activation. To explore the molecular basis for the absolute requirement of Ofut1 for Notch function, biochemical and cell biological approaches were used. Ligand-binding assay revealed that Ofut1 is essential for Notch receptors to physically interact with their ligands. In addition, secretion assay and cell surface staining showed that secretion of Notch receptors is impaired in OFUT1-depleted cells, indicating that the structure of Notch receptors is altered. Interestingly, promotion of Notch secretion by OFUT1 does not require its enzyme activity. Together with the fact that OFUT1 physically associates with Notch and that OFUT1 prevents misfolding of Notch mutants, it is proposed that OFUT1 acts as a chaperone that promotes the folding of the EGF repeat of Notch receptors. This chapter focuses on the methods used to analyze the roles of Ofut1 in Notch receptor structure and function.

Animals↗

PubMed1996

[Role of the Notch receptors in intercellular communication].

The Notch gene was discovered in Drosophila at the beginning of the century and is currently the subject of intensive investigation, not only in invertebrates but also in vertebrates where remarkably well conserved homologues have been recently found. Notch encodes a new kind of cellular receptor whose functioning is still unclear and plays a role in a large number of cell interactions throughout development and in tissue renewal in the adult. Detailed study in invertebrates of some of these interactions has led to the identification of other genes required for transduction of the signal initiated by the receptor. Notch is always involved in processes where cells have the potential to choose between several different programmes of differentiation. Cells adopt a specific developmental pathway as a result of the inhibition of some programmes through Notch signalling. In this review we discuss the contribution of different experimental models to an understanding of the role of Notch in intercellular signalling.

Animals↗

PubMed2004

Expression of notch receptors and ligands in the adult gut.

The Notch signaling pathway has become recognized as a vitally important pathway in regulating proliferative/differentiative decisions and cell fate. To explore the involvement of the Notch pathway in adult gut, we investigated the expression of Notch receptors and their ligands by Northern blotting and in situ hybridization. Notch receptors and ligands were expressed in both proliferative and post-mitotic cells throughout adult rat gut, variously in epithelial, immune, and endothelial cells. Expression of Notch1, Jagged1, and Jagged2 frequently overlapped, whereas Notch2 expression was restricted to specific crypt cells, the lamina propria of the large intestine, and Peyer's patch lymphocytes. We propose that the expression of multiple Notch receptors and ligands in a range of different intestinal cell types indicates that this signaling pathway underpins many of the processes involved in the maintenance and function of the adult gut.

Animals↗

PubMed2004

Axoglial interaction via the notch receptor in oligodendrocyte differentiation.

INTRODUCTION: Increasing evidence has revealed that the Notch signalling pathway is one of the pivotal systems that mediate oligodendrocyte development. The Notch receptor is a type I transmembrane molecule that represents a novel cellular signalling paradigm, namely, regulated intramembrane proteolysis (RIP). METHOD: The typical Notch ligands, such as Delta, Serrate/Jagged and Lag2 (DSL), promote the formation of oligodendocyte precursor cells (OPCs) and maintain them in an uncommitted stage, thus retarding oligodendrocyte appearance in the central nervous system (CNS). RESULTS: In contrast, our recent studies have revealed that F3/contactin, a GPI-linked neural adhesion molecule, interacts with Notch and speeds up the generation and maturation of oligodendrocytes. CONCLUSIONS: Considering the distinct, albeit somewhat overlapping expression patterns of F3 and DSL in the CNS, the Notch receptor appears to function ligand-dependently during oligodendrocyte development. This multipotentiality may well designate the Notch receptor as one of the therapeutic targets that one can manoeuvre to treat demyelinating diseases, such as multiple sclerosis, that is characterised by chronic myelin degeneration.

Animals↗