Synaptic targeting of glutamate receptors.

The proper targeting and clustering of neurotransmitter receptors at appropriate postsynaptic sites are principal requirements for the formation of functional synapses. Recently, new studies have begun to elucidate the mechanisms underlying the targeting and clustering of glutamate receptors at excitatory synapses in the brain. Members of the SAP90/PSD-95 family of proteins have emerged as potential regulators of glutamate-receptor membrane distribution. Further, targeting motifs within glutamate receptor subunits have been identified. These findings provide important clues in the effort to understand the molecular features of synaptic organization.

Budding yeast Cdc20: a target of the spindle checkpoint.

The spindle checkpoint regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis. In the two-hybrid system, three proteins that are components of the checkpoint, Mad1, Mad2, and Mad3, were shown to interact with Cdc20, a protein required for exit from mitosis. Mad2 and Mad3 coprecipitated with Cdc20 at all stages of the cell cycle. The binding of Mad2 depended on Mad1 and that of Mad3 on Mad1 and Mad2. Overexpression of Cdc20 allowed cells with a depolymerized spindle or damaged DNA to leave mitosis but did not overcome the arrest caused by unreplicated DNA. Mutants in Cdc20 that were resistant to the spindle checkpoint no longer bound Mad proteins, suggesting that Cdc20 is the target of the spindle checkpoint.

SynGAP: a synaptic RasGAP that associates with the PSD-95/SAP90 protein family.

The PSD-95/SAP90 family of proteins has recently been implicated in the organization of synaptic structure. Here, we describe the isolation of a novel Ras-GTPase activating protein, SynGAP, that interacts with the PDZ domains of PSD-95 and SAP102 in vitro and in vivo. SynGAP is selectively expressed in brain and is highly enriched at excitatory synapses, where it is present in a large macromolecular complex with PSD-95 and the NMDA receptor. SynGAP stimulates the GTPase activity of Ras, suggesting that it negatively regulates Ras activity at excitatory synapses. Ras signaling at the postsynaptic membrane may be involved in the modulation of excitatory synaptic transmission by NMDA receptors and neurotrophins. These results indicate that SynGAP may play an important role in the modulation of synaptic plasticity.

SAP102, a novel postsynaptic protein that interacts with NMDA receptor complexes in vivo.

Synapse-associated proteins (SAPs) are constituents of the pre- and postsynaptic submembraneous cytomatrix. Here, we present SAP102, a novel 102kDa SAP detected in dendritic shafts and spines of asymmetric type 1 synapses. SAP102 is enriched in preparations of synaptic junctions, where it biochemically behaves as a component of the cortical cytoskeleton. Antibodies directed against NMDA receptors coimmunoprecipitate SAP102 from rat brain synaptosomes. Recombinant proteins containing the carboxy-terminal tail of NMDA receptor subunit NR2B interact with SAP102 from rat brain homogenates. All three PDZ domains in SAP102 bind the cytoplasmic tail of NR2B in vitro. These data represent direct evidence that in vivo SAP102 is involved in linking NMDA receptors to the submembraneous cytomatrix associated with postsynaptic densities at excitatory synapses.

Involvement of JunD in transcriptional activation of the orphan receptor gene nur77 by nerve growth factor and membrane depolarization in PC12 cells.

nur77, an immediate-early gene that encodes an orphan nuclear receptor, is rapidly and transiently induced by nerve growth factor (NGF) stimulation or membrane depolarization in the rat pheochromocytoma-derived cell line PC12. The Nur77 protein can act as a potent transcription activator and may function to regulate the expression of downstream genes in response to extracellular stimuli. We show here that activation of nur77 by NGF treatment and membrane depolarization is signalled through distinct pathways. These distinct signals appear to converge on the same transcription factors acting on the same promoter elements. We show that nur77 activation by both processes requires two cis-acting AP1-like elements, NAP1 and NAP2, which contain the core sequence TGCGTCA centered at 67 and 38 nucleotides upstream of the transcription start site. The NAP elements can confer inducibility by NGF and membrane depolarization on an otherwise unresponsive heterologous promoter. We identified JunD as a key mediator of nur77 activation by reason of the following observations. (i) JunD, but not CREB or other members of the Fos/Jun family, is a component of NAP binding activity in PC12 cell nuclear extracts. (ii) JunD, but not other Fos/Jun family members, specifically transactivates the nur77 promoter through the NAP elements (iii) A dominant-negative mutant of JunD effectively abolishes the activation of nur77 by either NGF treatment or membrane depolarization. These data draw a contrast between the regulation of nur77 with that of c-fos, in which the sequence requirements for activation by NGF treatment and membrane depolarization appear separable, and CREB appears to play a role in activation by both NGF and membrane depolarization.

Activation of the inducible orphan receptor gene nur77 by serum growth factors: dissociation of immediate-early and delayed-early responses.

We have characterized the genetic elements that mediate the transcriptional activation of nur77, a growth factor-inducible gene encoding a member of the steroid/thyroid hormone receptor superfamily. Although initially identified as a serum-inducible immediate-early gene with expression kinetics similar to those of c-fos, we found that transcriptional activation of nur77 by serum growth factors in fibroblasts is in fact composed of two components: an immediate-early component, which can occur in the absence of de novo protein synthesis, and a delayed-early component, which is dependent on de novo protein synthesis. The expression of nur77 following serum stimulation reflects the superimposition of immediate-early and delayed-early expression. Immediate-early and delayed-early expression can be dissociated from one another by deletion or base substitution mutations of the nur77 promoter. Immediate-early expression of nur77 is mediated primarily by sequences located between nucleotides -86 and -126 upstream of the transcription start site. This region includes a sequence that resembles but differs from the CArG element found in other serum-inducible promoters. Upstream of the CArG-like element is a potential binding site for a transcription factor of the Ets family; the presence of this site is required for significant transcriptional induction. Delayed-early expression of nur77 is mediated by multiple AP-1-like and GC-rich elements, which can interact with products of immediate-early genes such as Fos/Jun and Zif268, respectively. Furthermore, we show that Zif268 can activate transcription of the nur77 promoter, suggesting that it may play a role in the delayed-early expression of nur77.

Endocrine and neurogenic regulation of the orphan nuclear receptors Nur77 and Nurr-1 in the adrenal glands.

nurr77 and nurr-1 are growth factor-inducible members of the steroid/thyroid hormone receptor gene superfamily. In order to gain insight into the potential roles of nur77 in the living organism, we used pharmacologic treatments to examine the expression of nur77 in the mouse adrenal gland. We found that nur77 and nurr-1 are induced in the adrenal gland upon treatment with pentylene tetrazole (Ptz; Metrazole). This induction is separable into distinct endocrine and neurogenic mechanisms. In situ hybridization analysis demonstrates that nur77 expression upon Ptz treatment in the adrenal cortex is localized primarily to the inner cortical region, the zona fasciculata-reticularis, with minimal induction in the zona glomerulosa. This induction is inhibitable by pretreatment with dexamethasone, indicating involvement of the hypothalamic-pituitary-adrenal axis in the activation of adrenal cortical expression. When mice were injected with adrenocorticotrophic hormone (ACTH), nur77 expression in the adrenal gland spanned all cortical layers including the zona glomerulosa, but medullary expression was not induced. Ptz also induces expression of both nur77 and nurr-1 in the adrenal medulla. Medullary induction is likely to have a neurogenic origin, as nur77 expression was not inhibitable by dexamethasone pretreatment and induction was seen after treatment with the cholinergic neurotransmitter nicotine. nur77 is also inducible by ACTH, forskolin, and the second messenger analog dibutyryl cyclic AMP in the ACTH-responsive adrenal cortical cell line Y-1. Significantly, Nur77 isolated from ACTH-stimulated Y-1 cells bound to its response element whereas Nur77 present in unstimulated cells did not. Moreover, Nur77 in ACTH-treated Y-1 cells was hypophosphorylated at serine 354 compared with that in untreated cells. These results, taken together with the previous observation that dephosphorylation of serine 354 affects DNA binding affinity in vitro, show for the first time that phosphorylation of Nur77 at serine 354 is under hormonal regulation, modulating its DNA binding affinity. Thus, ACTH regulates Nur77 in two ways: activation of its gene and posttranslational modification. A promoter analysis of nur77 induction in Y-1 cells indicates that the regulatory elements mediating ACTH induction differ from those required for induction in the adrenal medullary tumor cell line PC12 and in 3T3 fibroblasts.

Bop1 is a mouse WD40 repeat nucleolar protein involved in 28S and 5. 8S RRNA processing and 60S ribosome biogenesis.

We have identified and characterized a novel mouse protein, Bop1, which contains WD40 repeats and is highly conserved through evolution. bop1 is ubiquitously expressed in all mouse tissues examined and is upregulated during mid-G(1) in serum-stimulated fibroblasts. Immunofluorescence analysis shows that Bop1 is localized predominantly to the nucleolus. In sucrose density gradients, Bop1 from nuclear extracts cosediments with the 50S-80S ribonucleoprotein particles that contain the 32S rRNA precursor. RNase A treatment disrupts these particles and releases Bop1 into a low-molecular-weight fraction. A mutant form of Bop1, Bop1Delta, which lacks 231 amino acids in the N- terminus, is colocalized with wild-type Bop1 in the nucleolus and in ribonucleoprotein complexes. Expression of Bop1Delta leads to cell growth arrest in the G(1) phase and results in a specific inhibition of the synthesis of the 28S and 5.8S rRNAs without affecting 18S rRNA formation. Pulse-chase analyses show that Bop1Delta expression results in a partial inhibition in the conversion of the 36S to the 32S pre-rRNA and a complete inhibition of the processing of the 32S pre-rRNA to form the mature 28S and 5.8S rRNAs. Concomitant with these defects in rRNA processing, expression of Bop1Delta in mouse cells leads to a deficit in the cytosolic 60S ribosomal subunits. These studies thus identify Bop1 as a novel, nonribosomal mammalian protein that plays a key role in the formation of the mature 28S and 5.8S rRNAs and in the biogenesis of the 60S ribosomal subunit.

Fisp12/mouse connective tissue growth factor mediates endothelial cell adhesion and migration through integrin alphavbeta3, promotes endothelial cell survival, and induces angiogenesis in vivo.

Fisp12 was first identified as a secreted protein encoded by a growth factor-inducible immediate-early gene in mouse fibroblasts, whereas its human ortholog, CTGF (connective tissue growth factor), was identified as a mitogenic activity in conditioned media of human umbilical vein endothelial cells. Fisp12/CTGF is a member of a family of secreted proteins that includes CYR61, Nov, Elm-1, Cop-1/WISP-2, and WISP-3. Fisp12/CTGF has been shown to promote cell adhesion and mitogenesis in both fibroblasts and endothelial cells and to stimulate cell migration in fibroblasts. These findings, together with the localization of Fisp12/CTGF in angiogenic tissues, as well as in atherosclerotic plaques, suggest a possible role for Fisp12/CTGF in the regulation of vessel growth during development, wound healing, and vascular disease. In this study, we show that purified Fisp12 (mCTGF) protein promotes the adhesion of microvascular endothelial cells through the integrin receptor alphavbeta3. Furthermore, Fisp12 stimulates the migration of microvascular endothelial cells in culture, also through an integrin-alphavbeta3-dependent mechanism. In addition, the presence of Fisp12 promotes endothelial cell survival when cells are plated on laminin and deprived of growth factors, a condition that otherwise induces apoptosis. In vivo, Fisp12 induces neovascularization in rat corneal micropocket implants. These results demonstrate that Fisp12 is a novel angiogenic inducer and suggest a direct role for Fisp12 in the adhesion, migration, and survival of endothelial cells during blood vessel growth. Taken together with the recent finding that the related protein CYR61 also induces angiogenesis, we suggest that Fisp12/mCTGF and CYR61 comprise prototypes of a new family of angiogenic regulators that function, at least in part, through integrin-alphavbeta3-dependent pathways.

Evidence of p53-dependent cross-talk between ribosome biogenesis and the cell cycle: effects of nucleolar protein Bop1 on G(1)/S transition.

Bop1 is a novel nucleolar protein involved in rRNA processing and ribosome assembly. We have previously shown that expression of Bop1Delta, an amino-terminally truncated Bop1 that acts as a dominant negative mutant in mouse cells, results in inhibition of 28S and 5.8S rRNA formation and deficiency of newly synthesized 60S ribosomal subunits (Z. Strezoska, D. G. Pestov, and L. F. Lau, Mol. Cell. Biol. 20:5516-5528, 2000). Perturbation of Bop1 activities by Bop1Delta also induces a powerful yet reversible cell cycle arrest in 3T3 fibroblasts. In the present study, we show that asynchronously growing cells are arrested by Bop1Delta in a highly concerted fashion in the G(1) phase. Kinase activities of the G(1)-specific Cdk2 and Cdk4 complexes were downregulated in cells expressing Bop1Delta, whereas levels of the Cdk inhibitors p21 and p27 were concomitantly increased. The cells also displayed lack of hyperphosphorylation of retinoblastoma protein (pRb) and decreased expression of cyclin A, indicating their inability to progress through the restriction point. Inactivation of functional p53 abrogated this Bop1Delta-induced cell cycle arrest but did not restore normal rRNA processing. These findings show that deficiencies in ribosome synthesis can be uncoupled from cell cycle arrest and reveal a new role for the p53 pathway as a mediator of the signaling link between ribosome biogenesis and the cell cycle. We propose that aberrant rRNA processing and/or ribosome biogenesis may cause "nucleolar stress," leading to cell cycle arrest in a p53-dependent manner.

Rapid and preferential activation of the c-jun gene during the mammalian UV response.

Exposure of mammalian cells to DNA-damaging agents leads to activation of a genetic response known as the UV response. Because several previously identified UV-inducible genes contain AP-1 binding sites within their promoters, we investigated the induction of AP-1 activity by DNA-damaging agents. We found that expression of both c-jun and c-fos, which encode proteins that participate in formation of the AP-1 complex, is rapidly induced by two different DNA-damaging agents: UV and H2O2. Interestingly, the c-jun gene is far more responsive to UV than any other immediate-early gene that was examined, including c-fos. Other jun and fos genes were only marginally affected by UV or H2O2. Furthermore, UV is a much more efficient inducer of c-jun than phorbol esters, the standard inducers of c-jun expression. This preferential response of the c-jun gene is mediated by its 5' control region and requires the TPA response element, suggesting that this element also serves as an early target for the signal transduction pathway elicited by DNA damage. Both UV and H2O2 lead to a long-lasting increase in AP-1 binding activity, suggesting that AP-1 may mediate the induction of other damage-inducible genes such as human collagenase.

c-myc can induce expression of G0/G1 transition genes.

The human c-myc oncogene was linked to the heat shock-inducible Drosophila hsp70 promoter and used to stably transfect mouse BALB/c 3T3 cells. Heat shock of the transfectants at 42 degrees C followed by recovery at 37 degrees C resulted in the appearance of the human c-myc protein which was appropriately localized to the nuclear fraction. Two-dimensional analysis of the proteins of density-arrested cells which had been heat shock treated revealed the induction of eight protein species and the repression of five protein species. All of the induced and repressed proteins were nonabundant. cDNA clones corresponding to genes induced during the G0/G1 transition were used as probes to assay for c-myc inducibility of these genes. Two anonymous sequences previously identified as serum inducible (3CH77 and 3CH92) were induced when c-myc was expressed. In response to serum stimulation, 3CH77 and 3CH92 were expressed before c-myc mRNA levels increased. However, in response to specific induction of c-myc by heat shock of serum arrested cells, 3CH77 and 3CH92 mRNA levels increased after the rise in c-myc mRNA. Therefore, we hypothesize that abnormal expression of c-myc can induce genes involved in the proliferative response.

Cyr61, a product of a growth factor-inducible immediate-early gene, promotes cell proliferation, migration, and adhesion.

cyr61 was first identified as a growth factor-inducible immediate-early gene in mouse fibroblasts. The encoded Cyr61 protein is a secreted, cystein-rich heparin-binding protein that associates with the cell surface and the extracellular matrix, and in these aspects it resembles the Wnt-1 protein and a number of known growth factors. During embryogenesis, cyr61 is expressed most notably in mesenchymal cells that are differentiating into chondrocytes and in the vessel walls of the developing circulatory system. cyr61 is a member of an emerging gene family that encodes growth regulators, including the connective tissue growth factor and an avian proto-oncoprotein, Nov cyr61 also shares sequence similarities with two Drosophila genes, twisted gastrulation and short gastrulation, which interact with decapentaplegic to regulate dorsal-ventral patterning. In this report we describe the purification of the Cyr61 protein in a biologically active form, and we show that purified Cyr61 has the following activities: (i) it promotes the attachment and spreading of endothelial cells in a manner similar to that of fibronectin; (ii) it enhances the effects of basic fibroblast growth factor and platelet-derived growth factor on the rate of DNA synthesis of fibroblasts and vascular endothelial cells, although it has no detectable mitogenic activity by itself; and (iii) it acts as a chemotactic factor for fibroblasts. Taken together, these activities indicate that Cyr61 is likely to function as an extracellular matrix signaling molecule rather than as a classical growth factor and may regulate processes of cell proliferation, migration, adhesion, and differentiation during development.

Raf and fibroblast growth factor phosphorylate Elk1 and activate the serum response element of the immediate early gene pip92 by mitogen-activated protein kinase-independent as well as -dependent signaling pathways.

Previous studies have shown that a mitogen activated protein (MAP) kinase (MEK)-independent signaling pathway is required by activated Raf or fibroblast-derived growth factor (FGF) for the differentiation of rat hippocampal neuronal H19-7 cells. We now demonstrate that both Raf and FGF similarly induce prolonged transcription and translation of the immediate early gene pip92 in the absence of activation of the MAP kinases (MAPKs) ERK1 and ERK2. To determine the mechanism by which this occurs and to identify novel Raf-activated signaling pathways, we investigated the induction of the pip92 promoter by both FGF and an estradiol-activated Raf-1-estrogen receptor fusion protein (deltaRaf-1:ER) in H19-7 cells. Deletion analysis of the pip92 promoter indicated that activation by the MAPK-independent pathway occurs primarily within the region containing a serum response element (SRE). Further analysis of the SRE by using a heterologous thymidine kinase promoter showed that both an Ets and CArG-like site are required. Elk1, which binds to the Ets site, was phosphorylated both in vitro and in vivo by the MAPK-independent pathway, and phosphorylation of an Elk1-GAL4 fusion protein by this pathway was sufficient for transactivation. Finally, at least two Elk1 kinases were fractionated by gel filtration, and analysis by an in-gel kinase assay revealed at least three novel Raf-activated Elk1 kinases. These results indicate that both FGF and Raf activate MAPK-independent kinases that can stimulate Elk1 phosphorylation and immediate early gene transcription.

Pip92: a short-lived, growth factor-inducible protein in BALB/c 3T3 and PC12 cells.

pip92 is a cellular immediate-early gene inducible by serum growth factors in fibroblasts. It is also induced in the rat pheochromocytoma cell line PC12 by agents that cause proliferation, neuronal differentiation, and membrane depolarization. We show that the pip92-encoded polypeptide is a proline-rich protein of 221 amino acids, has an extremely short half-life, and is localized in the cytoplasm. We hypothesize that Pip92 plays a role in mediating the cellular responses to a variety of extracellular signals.

Expression of cyr61, a growth factor-inducible immediate-early gene.

A set of immediate-early genes that are rapidly activated by serum or purified platelet-derived growth factor in mouse 3T3 fibroblasts has been previously identified. Among these genes, several are related to known or putative transcription factors and growth factors, supporting the notion that some of these genes encode regulatory molecules important to cell growth. We show here that a member of this set of genes, cyr61 (originally identified by its cDNA 3CH61), encodes a 379-amino-acid polypeptide rich in cysteine residues. cyr61 can be induced through protein kinase C-dependent and -independent pathways. Unlike many immediate-early genes that are transiently expressed, the cyr61 mRNA is accumulated from the G0/G1 transition through mid-G1. This expression pattern is due to persistent transcription, while the mRNA is rapidly turned over during the G0/G1 transition and in mid-G1 at the same rate. In logarithmically growing cells, the cyr61 mRNA level is constant throughout the cell cycle. Cyr61 contains an N-terminal secretory signal sequence; however, it is not detected in the culture medium by immunoprecipitation. Cyr61 is synthesized maximally at 1 to 2 h after serum stimulation and has a short half-life within the cell.

A growth factor-responsive gene of murine BALB/c 3T3 cells encodes a protein homologous to human tissue factor.

Polypeptide growth factors rapidly induce the transcription of a set of genes that appear to mediate cell growth. We report that one of the genes induced in BALB/c mouse 3T3 cells encodes a transmembrane protein (mTF) homologous to human tissue factor, which is involved in the proteolytic activation of blood clotting. mTF mRNA is present in many murine tissues and cell lines. Our results raise the possibility that mTF may also play a role in cell growth.

Nur77 is differentially modified in PC12 cells upon membrane depolarization and growth factor treatment.

The rat pheochromocytoma cell line PC12 can be induced by growth factors to undergo proliferation and neuronal differentiation. These cells also have excitable membranes that can be depolarized by neurotransmitters or elevated levels of extracellular KCl. Treatment of PC12 cells with growth factors or membrane-depolarizing agents rapidly activates the expression of specific genes whose products are thought to mediate the subsequent biological responses. One such gene, nur77, is a member of the steroid and thyroid hormone receptor gene superfamily. We have identified the Nur77 protein and shown that it is synthesized rapidly and transiently in PC12 cells following stimulation, has a short half-life of 30 to 40 min, and is located in both the nucleus and the cytoplasm. Nur77 is posttranslationally modified, primarily by phosphorylation on serine residues. Phosphopeptide analysis reveals that Nur77 is modified differently upon membrane depolarization than after treatment with growth factors. We hypothesize that the activity of Nur77 is regulated by both differential gene expression and posttranslational modification and that these modes of regulation contribute to distinct downstream responses specific to membrane depolarization and growth factor treatment.

p27Kip1 induces an accumulation of the repressor complexes of E2F and inhibits expression of the E2F-regulated genes.

p27Kip1 is an inhibitor of the cyclin-dependent kinases and it plays an inhibitory role in the progression of cell cycle through G1 phase. To investigate the mechanism of cell cycle inhibition by p27Kip1, we constructed a cell line that inducibly expresses p27Kip1 upon addition of isopropyl-1-thio-beta-D-galactopyranoside in the culture medium. Isopropyl-1-thio-beta-D-galactopyranoside-induced expression of p27Kip1 in these cells causes a specific reduction in the expression of the E2F-regulated genes such as cyclin E, cyclin A, and dihydrofolate reductase. The reduction in the expression of these genes correlates with the p27Kip1-induced accumulation of the repressor complexes of the E2F family of factors (E2Fs). Our previous studies indicated that p21WAF1 could disrupt the interaction between cyclin/cyclin-dependent kinase 2 (cdk2) and the E2F repressor complexes E2F-p130 and E2F-p107. We show that p27Kip1, like p21WAF1, disrupts cyclin/cdk2-containing complexes of E2F-p130 leading to the accumulation of the E2F-p130 complexes, which is found in growth-arrested cells. In transient transfection assays, expression of p27Kip1 specifically inhibits transcription of a promoter containing E2F-binding sites. Mutants of p27Kip1 harboring changes in the cyclin- and cdk2-binding motifs are deficient in inhibiting transcription from the E2F sites containing reporter gene. Moreover, these mutants of p27Kip1 are also impaired in disrupting the interaction between cyclin/cdk2 and the repressor complexes of E2Fs. Taken together, these observations suggest that p27Kip1 reduces expression of the E2F-regulated genes by generating repressor complexes of E2Fs. Furthermore, the results also demonstrate that p27Kip1 inhibits expression of cyclin A and cyclin E, which are critical for progression through the G1-S phases.

Molecular mechanisms of glutamate receptor clustering at excitatory synapses.

The targeting of AMPA- and NMDA-type glutamate receptors to synapses in the central nervous system is essential for efficient excitatory synaptic transmission. Recent studies have indicated that protein-protein interactions of these receptors with synaptic proteins that contain PDZ domains are crucial for receptor targeting. NMDA receptors have been found to bind to the PSD-95 family of proteins, whereas AMPA receptors interact with the PDZ-domain-containing protein GRIP (glutamate receptor interacting protein). PSD-95 and GRIP contain multiple PDZ domains as well as other protein-protein interaction motifs that help to form large macromolecular complexes that may be important for the formation and plasticity of synapses.