We reported previously that nerve growth factor (NGF) up-regulates activity of the N-methyl-D-aspartate receptor 1 (NR1) promoter. We have explored the pathways and nuclear targets of NGF signaling in regulating the NR1 promoter. PD98059 and wortmannin, but not rapamycin, significantly attenuated NGF-induced transcriptional activity from an NR1 promoter-luciferase construct. Coexpressing constitutively active forms of Ras, Raf, or MAPK/ERK kinase 1 (MEK1) increased promoter activity dramatically. The MEK1-induced increase was largely prevented by mutations of the tandem GC boxes in the promoter. Promoter activity was also increased significantly by coexpressed GC boxbinding proteins (Sp1, 3, or 4) in nonstimulated PC12 cells. Either an extracellular signal-regulated kinase-1 (ERK1)-or Sp1-specific antibody coprecipitated Sp1 with ERKs, and the coprecipitation was enhanced significantly by NGF treatment of PC12 cells. ERK2 also incorporated radioactivity of [␥ 32 P]ATP into recombinant Sp1. However, ERK2-treated Sp1 and PC12 nuclear extracts or nuclear extracts from NGF-treated cells exhibited reduced binding to the promoter or a consensus GC box. Our results suggest that NGF utilizes both the Ras/ERK and phosphatidylinositol 3-kinase pathways to up-regulate NR1 promoter activity and that Sp1 is a novel substrate of NGF-activated ERKs. NGF-increased NR1 promoter activity may involve a complicated mechanism of Sp1 phosphorylation and possible transcription factor exchange.

Our previous studies revealed that a proximal region of the N-methyl-D-aspartate receptor 1 (NMDAR1) promoter is important for cell-type-specific expression. We have now explored the contributions of several regulatory elements to this specificity. Deletion of the neuronrestrictive silencer element partially relieved the suppression of promoter activity in C6 glioma and HeLa cells. An overlapping G(C/G)G/tandem Sp1-containing region crucial for both basal and nerve growth factor (NGF)-regulated promoter activity specifically bound nuclear proteins on its purine-rich sense strand. A faster migrating complex, single-stranded binding protein complex 1 (SBPC1), was highly enriched in HeLa cells, whereas a slower migrating complex, SBPC2, was enriched in PC12 cells. A high ratio of 2/1 complex correlated with a high level of promoter activity. NGF treatment of PC12 cells reduced SBPC1 but increased SBPC2. Competition experiments showed that the SBPC1 binding required a dG 4 sequence and the SBPC2 needed a core of TG 3 A plus a 5-flanking sequence. Singlestranded DNA encompassing TG 3 A and/or dG 4 specifically suppressed cotransfected NMDAR1 promoter activity. UV cross-linking studies indicated that a 31.5-kDa protein mainly formed SBPC1, whereas SBPC2 contained several larger proteins. Our results suggest that neuron-restrictive silencer factor and single-stranded DNA-binding proteins may both play a role in cell-type specificity of the NMDAR1 gene, and the latter may also be involved in basal and NGF-regulated activity.The NMDA 1 subtype of glutamate receptor plays important roles in voltage-dependent Ca 2ϩ influx, synaptic plasticity, and excitotoxic neuronal death in the mammalian central nervous system (1-3). Functional NMDA receptors contain a key NMDAR1 subunit in combination with one or more members of the NMDAR2(A-D) subunits (4 -7). The expression pattern of the NMDAR1 gene is widespread in the brain and restricted to neuronal cells (8 -10). A great deal of data suggest that interactions of cis elements in a gene with tissue-specific transacting factors play crucial roles in determining tissue-specific expression of the gene (11-14). We previously isolated a 3-kilobase pair promoter of the rat NMDAR1 gene and observed that a proximal region with 356 bp is sufficient to confer to the promoter a cell-type specificity in neuronal-like PC12 cells compared with C6 glioma and HeLa cervical cancer cells (15)(16)(17). This proximal promoter region includes a 5Ј-untranslated region that contains a neuron-restrictive silencer element (NRSE)/RE1-like element. Several neuronal genes contain this element, and a neuron-restrictive silencer factor/REST, expressed in nonneuronal tissues, may recognize this element and restrict gene expression in nonneuronal cells (11,18).Recently, several factors were identified that preferentially bind transcriptional elements on single-stranded DNA and function as suppressors or activators (19 -31). For example, heterogeneous nuclear ribonucleoprotein K specifically binds to a cytosine-r...