The NR1 gene undergoes induction in neurogenesis mainly via promoter de-repression, and up-regulation during neuronal differentiation by undefined mechanism(s). Here, we show that in the distal region the NR1 promoter has an active NF-B site sharing the consensus with the immunoglobulin (Ig)/human immunodeficiency virus NF-B site. Mutation of this site significantly reduced NR1 promoter up-regulation during neuronal differentiation of P19 cells. Electrophoretic mobility shift assays revealed that P19 nuclei constitutively contained p50 and that neuronal differentiation not only increased nuclear p50 but also induced p65 nuclear translocation. Responding to this change was an up-regulation of NF-B-dependent promoter activity. The N-methyl-D-aspartate (NMDA) 1 subtype of glutamate receptors plays an important role in neuronal differentiation and plasticity. The essential component of the NMDA receptor complexes is encoded by the NR1 gene (1, 2). This gene is induced during neurogenesis and up-regulated in neurons undergoing differentiation (3, 4). In studies of NR1 gene upregulation, a negative mechanism has been proposed: repressor element 1 (RE1) silencing transcription factor (REST)/neuronrestriction silencer factor (NRSF) acts on the RE1 site in the NR1 promoter and suppresses transcription in neural progenitors. After neurogenesis, REST/NRSF is down-regulated, and the suppression of the NR1 gene is removed to let de-repression occur (4). In studies of neuronal differentiation of P19 cells, we observed that there was a time gap between the down-regulation of the REST/NRSF protein and the gradually robust upregulation of the NR1 mRNA/promoter activity during neuronal differentiation (4), suggesting that a positive mechanism is required for NR1 gene transcription after de-repression. The NR1 promoter contains in the proximal region GC-boxes and MEF2 sites that interact with transcription activators sensitive to neuronal differentiation and thus could contribute to this positive mechanism (5-7). However, our recent observation suggests that the distal region of this promoter is required for fully up-regulated transcription (4) and very likely contains one or more important enhancers that positively respond to the signals of neuronal differentiation.Sequence analysis of the NR1 promoter uncovered a putative nuclear factor-B (NF-B) site in the region distal to the transcription start points (TSPs) (4). NF-B sites interact with dimerized NF-B factors and positively regulates cis-promoter. Five different NF-B factors have been found in mammalian tissues, p65 (Rel-A), p50, p52, C-Rel, and RelB. Homo-or heterodimers of NF-B factors are associated with IB factor in the cytoplasm and remain inactive. Activating signals triggered by environmental cues induce IB dissociation, thus releasing NF-B dimers that then become activated and nuclearly translocated to regulate transcription of target genes (8 -10). In the nervous system, NF-B activity is differentially distributed in the developing brain at different stages (11). Fo...

To understand the genetic mechanism controlling the expression of the NMDA subtype of glutamate receptors during neuronal differentiation, we studied activation of the N-methyl-D-aspartate receptor subunit 1 (NR1) gene and the role of the repressor element-1 (RE1) element in NR1 promoter activation. Following neuronal differentiation of P19 embryonic carcinoma cells, the NR1 transcription rate and mRNA level were significantly increased, while the nuclear level of the repressor RE1 silencing transcription factor (REST)/neuron-restriction silencer factor (NRSF) was reduced. Nuclear REST/NRSF from undifferentiated cells formed a large complex with the NR1 RE1 element. While this complex was significantly reduced after the differentiation, REST/NRSF from differentiated cells formed a new, faster migrating complex. In transient transfections, deletion of the RE1 element increased activity of the 5.4-kb NR1 promoter sixfold in undifferentiated cells, but only induced approximately 1.4-fold increase in differentiated cells. Forced expression of REST/NRSF in differentiated cells suppressed the promoter, while forced expression of a dominant-negative REST/NRSF induced promoter activity as well as the mRNA of the NR1 gene in undifferentiated cells. In stable transfectants, the wild-type promoter showed a robust increase in activity following differentiation in a pattern similar to the NR1 mRNA increase. Conversely, the promoter lacking the RE1 element showed only a moderate increase. Our data suggest that the NR1 gene up-regulation during neuronal differentiation is controlled by its promoter activation, which is largely determined by the interaction between the RE1 element and the repressor REST/NRSF.

The nicotinic acetylcholine receptor (AChR) is a pentameric complex made up of four types of subunits in the stoichiometry alpha 2 beta gamma delta. These subunits have been shown to be differentially phosphorylated by cAMP-dependent protein kinase (PKA) protein kinase C, and a protein tyrosine kinase. A variety of studies have suggested that phosphorylation of the AChR in vitro and in vivo regulates the rate of desensitization of the receptor. In this study we have used site-specific mutagenesis and patch-clamp techniques to examine the role of phosphorylation in the regulation of desensitization of the AChR expressed in Xenopus oocytes Expression of wild-type AChR in Xenopus oocytes results in the constitutive phosphorylation of the AChR on the gamma and delta subunits. This phosphorylation is apparently due to the high basal level of PKA in oocytes since a specific peptide inhibitor of PKA completely eliminated phosphorylation of the AChR by oocyte extracts in vitro. The phosphorylation of the AChR in oocytes was not significantly enhanced by forskolin or cAMP analogs or by coexpression with the catalytic subunit of PKA, suggesting that the basal activity of PKA in oocytes is sufficient to phosphorylate the receptor to a high stoichiometry. Using site-specific mutagenesis, the sites of phosphorylation were determined to be serines 353 and 354 on the gamma subunit and serines 361 and 362 on the delta subunit. To examine the functional properties of wild-type and mutant receptors lacking phosphorylation sites, we used patch-clamp techniques to measure the responses of out-side-out patches to repetitive pulses of ACh using a rapid perfusion system. Wild-type and mutant receptors showed rapid concentration-dependent activation and desensitization to applied agonist. The time constant of desensitization of ensemble mean currents ranged from several hundred milliseconds at low ACh concentrations to 100-200 msec at saturating concentrations. The desensitization time constants for mutant receptors lacking all phosphorylation sites were significantly slower than wild-type phosphorylated receptors at all concentrations of ACh tested. In addition, mutant receptors that had the serine residues changed to glutamate residues in order to mimic the negative charge of the phosphorylated serine residue produced receptors that had desensitization rates approaching those of the wild-type phosphorylated receptor. These results provide further support that phosphorylation of the nicotinic ACh receptor regulates rate of desensitization.

Two DNA elements which we have termed SAA and GAG have been shown to control expression of the rat amyloid precursor protein (APP) gene, and the region containing the SAA element has been shown to interact with nuclear proteins [Hoffman and Chernak (1994) Biochem. Biophys. Res. Commun. 201, 610-617]. In this report we study DNA sequences and proteins which influence the activity of the SAA element. An oligonucleotide containing the SAA element is specifically bound by nuclear proteins derived from rat PC12 cells, consistently forming four complexes designated C25, C30, C35 and C40 in electrophoretic mobility shift assays (EMSAs). We demonstrate that the C25, C30 and C40 complexes involve the binding of nuclear proteins to an SP1 consensus sequence located within the SAA element and that the C25 complex contains a protein antigenically related to the human SP1 protein. We establish further that the C35 complex requires a USF recognition site located within the SAA element and contains a protein antigenically related to the human upstream stimulatory factor (USF) protein. Using APP promoter/luciferase reporter gene constructs, we demonstrate that both the SP1 and the USF sites can play a role in the transcriptional activity of the SAA element. Finally, we show that complexes similar to the C25, C30 and C35 complexes are formed by rat cortex nuclear extracts and the SAA element in EMSA experiments, suggesting the relevance of our in vitro observations to the in vivo functioning of the rat APP promoter.