Role of the fourth membrane domain of the NR2B subunit in the assembly of the NMDA receptor

N-methyl-D-aspartate (NMDA) receptors play crucial roles in excitatory synaptic transmission as well as in excitotoxicity. A growing body of evidence suggests that the regulation of both subunit composition and the number of NMDA receptors reaching the surface membrane are tightly regulated. Recently, we have shown that the third membrane domains (M3) of both NR1 and NR2B subunits contain endoplasmic reticulum (ER) retention signals that prevent the unassembled subunits from leaving the ER. Furthermore, these membrane domains together with NR1 M4 are necessary for negating the ER retention signals found in M3 of NR1 and NR2B. In this addendum, we present new electrophysiological data showing that mutation of the HLFY motif, located immediately after M4 of the NR2B subunit, abolishes the surface trafficking of full-length NR1/NR2B complexes (supporting previous immunofluorescent experiments from our lab); however, the deletion of the NR2B C-terminus including the HLFY motif did not affect the formation of functional receptors when two pieces of the NR2B subunit, NR2B truncated before M4 and NR2B M4, were co-expressed together with the NR1 subunit. These observations will help to uncover the processes involved in the assembly of NR1 and NR2 subunits into functional NMDA receptors.     

Role of the fourth membrane domain of the NR2B subunit in the assembly of the NMDA receptor

N-methyl-D-aspartate (NMDA) receptors play crucial roles in excitatory synaptic transmission as well as in excitotoxicity. A growing body of evidence suggests that the regulation of both subunit composition and the number of NMDA receptors reaching the surface membrane are tightly regulated. Recently, we have shown that the third membrane domains (M3) of both NR1 and NR2B subunits contain endoplasmic reticulum (ER) retention signals that prevent the unassembled subunits from leaving the ER. Furthermore, these membrane domains together with NR1 M4 are necessary for negating the ER retention signals found in M3 of NR1 and NR2B. In this addendum, we present new electrophysiological data showing that mutation of the HLFY motif, located immediately after M4 of the NR2B subunit, abolishes the surface trafficking of full-length NR1/NR2B complexes (supporting previous immunofluorescent experiments from our lab); however, the deletion of the NR2B C-terminus including the HLFY motif did not affect the formation of functional receptors when two pieces of the NR2B subunit, NR2B truncated before M4 and NR2B M4, were co-expressed together with the NR1 subunit. These observations will help to uncover the processes involved in the assembly of NR1 and NR2 subunits into functional NMDA receptors.     

NMDA受体在焦虑症发病机制的研究进展

伴随社会生活和工作压力的增大,常见精神类疾病焦虑症的发病率逐年攀升。焦虑症的发病机制非常复杂,迄今尚未完全阐明。本文概述了焦虑症发病机制与NMDA受体不同亚型的关系。NMDA受体主要广泛分布于脑、脊髓和周围神经系统。NR1广泛分布于中枢神经,在NR2D亚基敲除小鼠中,NR1和NR2D的相互影响可能参与了焦虑样行为。NR2A与NR2B是NMDA受体的两个重要亚基,NR2B的高选择性拮抗剂艾芬地尔在小鼠的高架十字迷宫实验中发挥了抗焦虑功效。将小鼠全脑的NR2C基因用NR2B替代之后,1月龄变异小鼠的高架十字迷宫实验显示有明显的非条件性焦虑行为,表明NR2B和NR2C均可能参与焦虑的发生。因此,深入阐明调控NMDA受体亚基组成的确切作用机制,将有助于探索焦虑症潜在治疗靶点的发现,并针对性地开展新药的研发。     

Isolation and Initial Characterization of Constitutive Nitrate Reductase-Deficient Mutants NR328 and NR345 of Soybean (Glycine max)

Two nitrate reductase deficient mutants of soybean (Glycine max [L.] Merr. cv Bragg) were isolated from approximately 10,000 M₂ seedlings, using a direct enzymic assay in microtiter plates. Stable inheritance of NR345 and NR328 phenotypes has been demonstrated through to the M₅ generation. Both mutants were affected in constitutive nitrate reductase activity. Assayable activities of cNR in nitrate-free grown seedlings was about 3 to 4% of the control for NR345 and 14 to 16% of the control for NR328. Both mutants expressed inducible NR during early plant development and were sensitive to nitrate and urea inhibition of nodulation. These new mutants will allow an extension of the characterization of nitrate reductases and their function in soybean. Preliminary evidence indicates that NR345 is similar to the previously isolated mutant nr₁, while NR328 is different.     

Transcription of the NR1 subunit of the N-methyl-D-aspartate receptor is down-regulated by excitotoxic stimulation and cerebral ischemia

The N-methyl-D-aspartate (NMDA) type of glutamate receptor (NMDAR) plays central roles in normal and pathological neuronal functioning. We have examined the regulation of the NR1 subunit of the NMDAR in response to excessive activation of this receptor in in vitro and in vivo models of excitotoxicity. NR1 protein expression in cultured cortical neurons was specifically reduced by stimulation with 100 microM NMDA or glutamate. NMDA decreased NR1 protein amounts by 71% after 8 h. Low NMDA concentrations (< or = 10 microM) had no effect. NR1 down-regulation was inhibited by the general NMDAR antagonist DL-AP5 and also by ifenprodil, which specifically antagonizes NMDARs containing NR2B subunits. Arrest of NMDAR signaling with DL-AP5 after brief exposure to NMDA did not prevent subsequent NR1 decrease. Down-regulation of NR1 did not involve calpain cleavage but resulted from a decrease in de novo synthesis consequence of reduced mRNA amounts. In contrast, NMDA did not alter the expression of NR2A mRNA or newly synthesized protein. In neurons transiently transfected with an NR1 promoter/luciferase reporter construct, promoter activity was reduced by 68% after 2 h of stimulation with NMDA, and its inhibition required extracellular calcium. A similar mechanism of autoregulation of the receptor probably operates during cerebral ischemia, because NR1 mRNA and protein were strongly decreased at early stages of blood reperfusion in the infarcted brains of rats subjected to occlusion of the middle cerebral artery. Because NR1 is the obligatory subunit of NMDARs, this regulatory mechanism will be fundamental to NMDAR functioning.     

Conformational adaptation of nuclear receptor ligand binding domains to agonists: potential for novel approaches to ligand design

Ligands occupy the core of nuclear receptor (NR) ligand binding domains (LBDs) and modulate NR function. X-ray structures of NR LBDs reveal most NR agonists fill the enclosed pocket and promote packing of C-terminal helix 12 (H12), whereas the pockets of unliganded NR LBDs differ. Here, we review evidence that NR pockets rearrange to accommodate different agonists. Some thyroid hormone receptor (TR) ligands with 5′ extensions designed to perturb H12 act as antagonists, but many are agonists. One mode of adaptation is seen in a TR/thyroxine complex; the pocket expands to accommodate a 5′ iodine extension. Crystals of other NR LBDs reveal that the pocket can expand or contract and some agonists do not fill the pocket. A TRβ structure in complex with an isoform selective drug (GC-24) reveals another mode of adaptation; the LBD hydrophobic interior opens to accommodate a bulky 3′ benzyl extension. We suggest that placement of extensions on NR agonists will highlight unexpected areas of flexibility within LBDs that could accommodate extensions; thereby enhancing the selectivity of agonist binding to particular NRs. Finally, agonists that induce similar LBD structures differ in their activities and we discuss strategies to reveal subtle structural differences responsible for these effects.     

Light control of extractable nitrate reductase activity in higher plants

Light regulation of extractable nitrate reductase (NR) activity of higher plants is complicated by: 1) involvement of several photoreceptors, 2) differences in the relative importance of the several photoreceptors among species and among developmental stages of the same species, 3) two types of effects – alteration of activity of existing NR and influences on de novo synthesis of NR, and 4) differing forms of NR within the same species. The interrelationships of all of these factors are not clear. It may be that each system will have to be understood separately before a general model can be developed. Immunochemical quantification of NR from systems exposed to varied light regimes may enhance our understanding of this area. Currently few general conclusions can be made; however, we think that the following statements are true or are usually true: (1) Phytochrome influences extractable NR activity by the low irradiance response and high irradiance response in etiolated tissues. (2) In de-etiolated tissues phytochrome can influence NR activity decay at the end of a light period by the low irradiance response. (3) The phytochrome equilibrium or the absolute level of P_fr influences extractable NR activity in green tissues under white light. (4) Blue light influences extractable NR activity through phytochrome and another, unknown, blue light-absorbing pigment. Flavins may be involved in vitro in reactivation of inactivated NR. (5) Photosynthesis does not directly influence the induction of the forms of NR that require substrate and light for induction. (6) In some tissues there appears to be a close link between nitrite-reducing and nitrate-reducing capabilities. (7) Much circumstantial evidence from kinetic and protein-synthesis-inhibitor studies and the only available immunochemical data indicate that light induces de novo synthesis of NR, resulting in increased extractable activity.     

植物中硝酸还原酶和亚硝酸还原酶的作用

植物通过硝酸盐同化途径以硝酸盐和氨的形式吸收氮元素。硝酸盐的同化是一个受到严格控制的过程,其中两个先后参加反应的酶——硝酸还原酶(NR)和亚硝酸还原酶(NiR)对初级氮的同化起主要调控。在高等植物中,NR和NiR基因的转录及转录后加工受到各种内在和外在因素的影响,翻译后调控是消除亚硝酸盐积累的重要机制。随着分子生物学技术的发展,可以更容易地通过突变体和转基因方式来研究NR和NiR基因的调控。     

Tungstate: is it really a specific nitrate reductase inhibitor in plant nitric oxide research?

Nitrate reductase (NR) is an enzymatic source of nitric oxide (NO) in plants, and it needs Mo for the Mo-cofactor to be activated. Because NR-deficient mutants are not always available in some species, a cheap and simple pharmacological application of tungstate, which substitutes for Mo in the Mo-cofactor as a competitive antagonist, is widely used as a NR inhibitor in plant NO research. However, evidence indicates that tungstate not only inactivates NR but also inhibits other molybdate-dependent enzymes in plants. In addition, a number of investigations have shown that tungstate also inhibits root growth, affects cortical microtubule formation, and induces programmed cell death (PCD) in plants, just like other heavy metals do. Therefore, tungstate has been shown to exert many other effects that are not connected with the inhibition of NR activity. The origin and mechanism of using tungstate as a NR inhibitor in plants is reviewed here and the progress regarding tungstate toxicity to plants and the possible problems involved in using tungstate as a NR inhibitor in plant NO research are analysed. In summary, the use of tungstate as a NR inhibitor in plant NO research must be treated with caution, keeping in mind that it is not completely specific. It is necessary to search for more NR-deficient mutants and new, specific NR inhibitors. A combination of pharmacological and biochemical analysis with a genetic approach will be necessary in order to investigate the roles of NO in plants.     

Minireview: nuclear receptors and breast cancer

Until recently, the study of nuclear receptor (NR) function in breast cancer biology has been largely limited to estrogen and progesterone receptors. The development of reliable gene expression arrays, real-time quantitative RT-PCR, and immunohistochemical techniques for studying NR superfamily members in primary human breast cancers has now revealed the presence and potential importance of several additional NRs in the biology of breast cancer. These include receptors for steroid hormones (including androgens and corticosteroids), fat-soluble vitamins A and D, fatty acids, and xenobiotic lipids derived from diet. It is now clear that after NR activation, both genomic and nongenomic NR pathways can coordinately activate growth factor signaling pathways. Advances in our understanding of both NR functional networks and epithelial cell growth factor signaling pathways have revealed a frequent interplay between NR and epithelial cell growth factor family signaling that is clinically relevant to breast cancer. Understanding how growth factor receptors and their downstream kinases are activated by NRs (and vice-versa) is a central goal for maximizing treatment opportunities in breast cancer. In addition to the estrogen receptor, it is predicted that modulating the activity of other NRs will soon provide novel prevention and treatment approaches for breast cancer patients.     

The N-Methyl-d-Aspartate Receptor Subunits NR2A and NR2B Bind to the SH2 Domains of Phospholipase C-γ

Abstract: The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-γ (PLC-γ). A glutathione S -transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-γ was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-γ and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.     

NMDA receptor expression and C terminus structure in the rotifer Brachionus plicatilis and long-term potentiation across the Metazoa

The C termini of N-methyl-d-aspartate (NMDA) receptor NR2 subunits are thought to play a major role in the molecular establishment of memory across the Bilateria, via the phenomenon known as long-term potentiation (LTP). Despite their long history of use as models in the study of memory, the expression and structure of the NR2 subunit in the Lophotrochozoa has remained uncategorized. Here, we report the phylogenic relationships of NR subunits across the Bilateria, and the cloning and in situ analysis of expression of NMDA NR1 and NR2 subunits in the monogont rotifer Brachionus plicatilis. RNA in situ hybridization suggests expression of NMDA receptor subunits in B. plicatilis is neural, consistent with expression observed in other species, and ours is the first report confirming NR2 expression in the lophotrochozoan clade. However, the single NR2 subunit identified in B. plicatilis was found to lack the long C terminal domain found in vertebrates, which is believed to modulate LTP. Further investigation revealed that mollusc and annelid NR2 subunits possess long intracellular C terminal domains. As data from molluscs (and particularly Aplysia californica) are the basis for much of our understanding of LTP, understanding how these diverse lophotrochozoan C termini function in vivo will have many implications for how we consider the evolution of the molecular control of learning and memory across the Metazoa as a whole and interpret the results of experiments into this vital component of cognition.     

The thalamic intergeniculate leaflet mediates locomotor activity-induced reversal of phenotype in photoperiod nonresponsive Siberian hamsters

The role of the intergeniculate leaflet of the thalamus (IGL) in photoperiod responsiveness was examined in a laboratory-selected line of photoperiod nonresponsive (NR) Siberian hamsters. NR hamsters fail to exhibit typical winter-type responses (i.e., gonadal regression and development of winter-type pelage) when exposed to short day lengths (e.g., 10 h of light/day). Earlier studies revealed that NR hamsters will exhibit winter-type responses when exposed to short photoperiod if they are given free access to a running wheel. The present study tested the hypothesis that this locomotor activity-induced reversal of phenotype is dependent on the IGL. Male NR hamsters underwent destruction of the IGL prior to being housed in short day lengths in cages equipped with running wheels. Activity rhythms were monitored for 8 weeks, after which time pelage response and paired testes weights were obtained. In contrast to sham-operated NR animals given access to running wheels, IGL-ablated animals showed no increase in the duration of nocturnal running wheel activity and became active later in the night than sham-lesioned animals. Lesioned animals also failed to exhibit the typical short photoperiod-induced gonadal regression and pelage molt. The results implicate the IGL in the mechanism by which running wheel activity can influence photoperiodic responses.     

Nutrient restriction in combinatory therapy of tumors

The main objective of anticancer treatment is the elimination of degenerated cells by the induction of programmed cell death. Various chemotherapy drugs and radiation are able to activate cell death mechanisms in tumors. However, unfortunately, monotherapy will always be insufficiently effective because of the variety and virulence of tumors, as well as their ability to develop resistance to drugs. Moreover, monotherapy might constrain many negative side effects. Therefore, the combination of different approaches and/or drugs will increase the efficiency of treatment. One such promising approach is the combination of nutrient restriction (NR) and various chemotherapeutic drugs. This approach may not only affect the autophagy but also influence apoptotic cell death. This review is focused on the potential of NR use in anticancer therapy, as well as the molecular mechanisms underlying this approach.     

Triheteromeric NR1/NR2A/NR2B receptors constitute the major N-methyl-D-aspartate receptor population in adult hippocampal synapses

NMDA receptors (NMDARs), fundamental to learning and memory and implicated in certain neurological disorders, are heterotetrameric complexes composed of two NR1 and two NR2 subunits. The function of synaptic NMDARs in postnatal principal forebrain neurons is typically attributed to diheteromeric NR1/NR2A and NR1/NR2B receptors, despite compelling evidence for triheteromeric NR1/NR2A/NR2B receptors. In synapses, the properties of triheteromeric NMDARs could thus far not be distinguished from those of mixtures of diheteromeric NMDARs. To find a signature of NR1/NR2A/NR2B receptors, we have employed two gene-targeted mouse lines, expressing either NR1/NR2A or NR1/NR2B receptors without NR1/NR2A/NR2B receptors, and compared their synaptic properties with those of wild type. In acute hippocampal slices of mutants older than 4 weeks we found a distinct voltage dependence of NMDA R-mediated excitatory postsynaptic current (NMDA EPSC) decay time for the two diheteromeric NMDARs. In wild-type mice, NMDA EPSCs unveiled the NR1/NR2A characteristic for this voltage-dependent deactivation exclusively, indicating that the contribution of NR1/NR2B receptors to evoked NMDA EPSCs is negligible in adult CA3-to-CA1 synapses. The presence of NR1/NR2A/NR2B receptors was obvious from properties that could not be explained by a mixture of diheteromeric NR1/NR2A and NR1/NR2B receptors or by the presence of NR1/NR2A receptors alone. The decay time for NMDA EPSCs in wild type was slower than that for NR1/NR2A receptors, and the sensitivity of NMDA EPSCs to NR2B-directed NMDAR antagonists was 50%. Thus, NR2B is prominent in adult hippocampal synapses as an integral part of NR1/NR2A/NR2B receptors.