Hideo Taniura

Necdin, A Postmitotic Neuron-specific Growth Suppressor, Interacts with Viral Transforming Proteins and Cellular Transcription Factor E2F1

Necdin is a nuclear protein expressed in virtually all postmitotic neurons, and ectopic expression of this protein strongly suppresses the proliferation of NIH3T3 cells. Simian virus 40 large T antigen targets both p53 and the retinoblastoma protein (Rb) for cellular transformation. By analogy with the interactions of the large T antigen with these nuclear growth suppressors, we examined the ability of necdin to bind to the large T antigen. Necdin was co-immunoprecipitated with the large T antigen from the nuclear extract of necdin cDNA-transfected COS-1 cells. Yeast two-hybrid and in vitro binding analyses revealed that necdin bound to an amino-terminal region of the large T antigen, which encompasses the Rb-binding domain. Moreover, necdin bound to adenovirus E1A, another viral oncoprotein that forms a specific complex with Rb. We then examined the ability of necdin to bind to the transcription factor E2F1, a cellular Rb-binding factor involved in cell-cycle progression. Intriguingly, necdin, like Rb, bound to a carboxyl-terminal domain of E2F1, and repressed E2F-dependent transactivation in vivo. In addition, necdin suppressed the colony formation of Rb-deficient SAOS-2 osteosarcoma cells. These results suggest that necdin is a postmitotic neuron-specific growth suppressor that is functionally similar to Rb.

Physical and Functional Interactions of Neuronal Growth Suppressor Necdin with p53

Necdin is expressed in virtually all postmitotic neurons, and ectopic expression of this protein suppresses cell proliferation. Necdin, like the retinoblastoma protein, interacts with cell cycle promoting proteins such as simian virus 40 large T antigen, adenovirus E1A, and the transcription factor E2F1. Here we demonstrate that necdin interacts with the tumor suppressor protein p53 as well. The yeast two-hybrid and in vitro binding analyses revealed that necdin bound to a narrow region (amino acids 35–62) located between the MDM2-binding site and the proline-rich region in the amino-terminal domain of p53. The electrophoretic mobility shift assay showed that necdin supershifted a complex between p53 and its binding DNA, implying that the p53-necdin complex is competent for DNA binding. In p53-deficient osteosarcoma SAOS-2 cells, necdin markedly suppressed p53-dependent activation of the p21/WAF promoter. Necdin and p53 inhibited cell growth in an additive manner as assessed by the colony formation of SAOS-2 cells, suggesting that necdin does not affect p53-mediated growth suppression. On the other hand, necdin inhibited p53-induced apoptosis of osteosarcoma U2OS cells. Thus, necdin can be a growth suppressor that targets p53 and modulates its biological functions in postmitotic neurons.

Necdin-related MAGE proteins differentially interact with the E2F1 transcription factor and the p75 neurotrophin receptor

Necdin is a growth suppressor expressed predominantly in postmitotic neurons and implicated in their terminal differentiation. Necdin shows a moderate homology to the MAGE family proteins, the functional roles of which are largely unknown. Human genes encoding necdin, MAGEL2 (necdin-like 1), and MAGE-G1 (necdin-like 2) are located in proximal chromosome 15q, a region associated with neurodevelopmental disorders such as Prader-Willi syndrome, Angelman syndrome, and autistic disorder. The necdin and MAGEL2 genes are subjected to genomic imprinting and suggested to be involved in the etiology of Prader-Willi syndrome. In this study, we compared biochemical and functional characteristics of murine orthologs of these necdin-related MAGE proteins. The colony formation and bromodeoxyuridine incorporation analyses revealed that necdin and MAGE-G1, but not MAGEL2, induced growth arrest. Necdin and MAGE-G1 interacted with the transcription factor E2F1 via its transactivation domain, repressed E2F1-dependent transcription, and antagonized E2F1-induced apoptosis of N1E-115 neuroblastoma cells. In addition, necdin and MAGE-G1 interacted with the p75 neurotrophin receptor via its distinct intracellular domains. In contrast, MAGEL2 failed to bind to these necdin interactors, suggesting that MAGEL2 has no necdin-like function in developing brain. Overexpression of p75 translocated necdin and MAGE-G1 in the proximity of the plasma membrane and reduced their association with E2F1 to facilitate E2F1-induced death of neuroblastoma cells. These results suggest that necdin and MAGE-G1 target both E2F1 and p75 to regulate cell viability during brain development.

Molecular cloning and functional expression of gicerin, a novel cell adhesion molecule that binds to neurite outgrowth factor

Gicerin is an integral membrane glycoprotein of about 82 kd that is transiently expressed in the developing CNS. Gicerin was first identified as a binding protein for neurite outgrowth factor (NOF), a member of the laminin family of extracellular matrix proteins. By isolating and sequencing a gicerin cDNA, we have found that this protein is a novel member of the immunoglobulin superfamily. The deduced protein (584 amino acids) consists of five immunoglobulin-like loop structures in an extracellular domain, a single transmembrane region, and a short cytoplasmic tail. Cells transfected stably with gicerin cDNA adhered to NOF and aggregated with each other, indicating that gicerin exhibits both heterophilic and homophilic adhesion activities.

The human chromosomal gene for necdin, a neuronal growth suppressor, in the Prader–Willi syndrome deletion region

Necdin is a growth suppressor expressed in virtually all postmitotic neurons in the brain. The human necdin gene, NDN, is maternally imprinted and deleted in the Prader-Willi syndrome, a neurobehavioral contiguous gene disorder. Here, we isolated and characterized the human chromosomal necdin gene and its promoter region. The necdin gene is intronless, and it encodes a protein of 321 amino acid residues, four residues shorter than mouse Necdin. By fluorescence in-situ hybridization analysis, the necdin gene was localized to chromosome 15q11.2-q12 within the Prader-Willi syndrome deletion region. CpG islands were found in a region extending from the proximal 5-flanking sequence to the protein coding region. The 5-flanking sequence, which lacks canonical TATA and CAAT boxes, possessed a promoter activity in postmitotic neurons derived from murine embryonal carcinoma P19 cells. Methylation in vitro of HhaI CpG sites in the promoter region reduced the transcriptional activity. These results suggest that the necdin gene is silenced through methylation of the CpG island encompassing its promoter region.

Ectopic expression of necdin induces differentiation of mouse neuroblastoma cells.

Necdin is expressed predominantly in postmitotic neurons, and ectopic expression of this protein strongly suppresses cell growth. Necdin has been implicated in the pathogenesis of Prader-Willi syndrome, a human neurodevelopmental disorder associated with genomic imprinting. Here we demonstrate that ectopic expression of necdin induces a neuronal phenotype in neuroblastoma cells. Necdin was undetectable in mouse neuroblastoma N1E-115 cells under undifferentiated and differentiated conditions. N1E-115 cells transfected with necdin cDNA showed morphological differentiation such as neurite outgrowth and expression of the synaptic marker proteins synaptotagmin and synaptophysin. In addition, Western blot analysis of the retinoblastoma protein (Rb) family members Rb, p130, and p107 revealed that necdin cDNA transfectants contained an increased level of p130 and a reduced level of p107, a pattern seen in differentiated G0 cells. The transcription factors E2F1 and E2F4 physically interacted with necdin via their carboxyl-terminal transactivation domains, but only E2F1 abrogated necdin-induced growth arrest and neurite outgrowth of neuroblastoma cells. Overexpression of E2F1 in differentiated N1E-115 cells induced apoptosis, which was antagonized by co-expression of necdin. These results suggest that necdin promotes the differentiation and survival of neurons through its antagonistic interactions with E2F1.

Expression and Functional Analysis of a Novel Isoform of Gicerin, an Immunoglobulin Superfamily Cell Adhesion Molecule

We have cloned a novel cDNA of gicerin, a cell adhesion molecule belonging to the immunoglobulin superfamily. Both gicerin isoforms share the same extracellular domain, which has five immunoglobulin-like loop structures and a transmembrane domain as s-gicerin, but differ in the cytoplasmic tail domain. As the newly identified form has a larger cytoplasmic domain than the previously reported form, we refer to them as l-gicerin and s-gicerin, respectively. l-gicerin is transcribed from a distinct mRNA containing an inserted sequence not found in s-gicerin mRNA which caused a frameshift for the coding region for a cytoplasmic domain. Previous studies demonstrated that gicerin showed a doublet band of 82 and 90 kDa in chicken gizzard smooth muscle. We report that the 82-kDa protein corresponds to s-gicerin and the 90-kDa protein to l-gicerin. We also found that the two gicerin isoforms are expressed differentially in the developing nervous system. Functional analysis of these gicerin isoforms in stable transfectants revealed that they had differ in their homophilic adhesion properties, as well as in heterophilic cell adhesion assayed with neurite outgrowth factor. In addition, these isoforms have neurite-promoting activity by their homophilic adhesion, but differ in their ability to promote neurite outgrowth.

Necdin acts as a transcriptional repressor that interacts with multiple guanosine clusters

Necdin is a growth suppressor expressed predominantly in postmitotic neurons, and ectopic expression of this protein suppresses cell growth. Here we report that Necdin directly binds to specific DNA sequences and serves as a transcriptional repressor. Polyhistidine-tagged Necdin was used for selection of random-sequence oligonucleotides by polymerase chain reaction-based amplification. Necdin recognized guanosine (G)-rich sequences that encompass multiple G clusters and intervening mono- or di-nucleotides of A, T and C. These sequences, termed GN boxes, resemble multiply aligned forms of the canonical GC box which is recognized by Sp family members. Necdin directly bound to a GN box consisting of contiguous two GC boxes with four G clusters, but not to a single GC box with two G clusters, whereas Sp1 bound to both. In a reporter system using Drosophila Schneider Line 2 cells, Necdin repressed Sp1-dependent activity of mouse c-myc P1 promoter that contains a typical GN box. Deletion of the GN box from the c-myc P1 promoter or its conversion to the single GC box abolished the Necdin-dependent repression. These results suggest that Necdin modulates gene transcription via the GN box that is potentially recognized by GC box-targeting Sp family members.

Necdin interacts with the ribonucleoprotein hnRNP U in the nuclear matrix

Necdin is expressed predominantly in terminally differentiated neurons, and its ectopic expression suppresses cell proliferation. We screened a cDNA library from neurally differentiated embryonal carcinoma P19 cells for necdin‐binding proteins by the yeast two‐hybrid assay. One of the positive clones contained cDNA encoding a carboxyl‐terminal portion of heterogeneous nuclear ribonucleoprotein U (hnRNP U), a nuclear matrix‐associated protein that interacts with chromosomal DNA. We isolated cDNA encoding full‐length mouse hnRNP U to analyze its physical and functional interactions with necdin. The necdin‐binding site of hnRNP U was located near a carboxyl‐terminal region that mediated the association between hnRNP U and the nuclear matrix. In postmitotic neurons, endogenously expressed necdin and hnRNP U were detected in the nuclear matrix and formed a stable complex. Ectopically expressed necdin was concentrated in the nucleoli, but coexpressed hnRNP U recruited necdin to the nucleoplasmic compartment of the nuclear matrix. Furthermore, under the same conditions necdin and hnRNP U cooperatively suppressed the colony formation of transfected SAOS‐2 cells. These results suggest that necdin suppresses cell proliferation through its interaction with hnRNP U in the specific subnuclear structure. J. Cell. Biochem. 84: 545–555, 2002.

A Metabotropic Glutamate Receptor Family Gene in Dictyostelium discoideum

Metabotropic glutamate receptors (mGluRs) are a class of G-protein-coupled receptors that possess a seven transmembrane region involved in the modulation of excitatory synaptic transmission in the nervous system. mGluR orthologs have been identified in Drosophila, Caenorhabditis elegans, and higher organisms. Drosophila possesses two mGluR genes, DmGluRA and DmXR. We screened the Dictyostelium genome data base using the ligand binding domain of rat mGluR1 as bait, and identified a new receptor, DdmGluPR, belonging to the mGluR family. Similar to Drosophila DmXR, the residues of mGluRs involved in the binding of the α-carboxylic and α-amino groups of glutamate were well conserved in DdmGluPR, but the residues interacting with the γ-carboxylic group of glutamate were not. The phylogenetic analysis suggests that DdmGluPR diverged after the mGluR family-GABAB receptors split but before mGluR family divergence. DdmGluPR mRNA was expressed in vegetative cells and throughout starvation-induced development, but the level of the expression was relatively high until 4 h after starvation. DdmGluPR was localized to the plasma membrane of axenically grown Ax-2 cells expressed as a green fluorescent protein fusion protein. DdmGluPR-null cells grew faster at high cell density and reached higher densities than wild-type cells. DdmGluPR-null cells exhibited delayed aggregates formation upon starvation and impaired chemotaxis toward cAMP. Although expressions of cAR1 and aca, cAMP-signaling components, were rapidly induced and peaked at 2–4 h in wild-type cells, DdmGluPR-null cells displayed sustained and peaked at 8 h of the expressions of these genes. Our findings suggest the involvement of DdmGluPR in the early development of Dictyostelium discoideum.