Molecular Dissection of Native Mammalian Forebrain NMDA Receptors Containing the NR1 C2 Exon: Direct Demonstration of NMDA Receptors Comprising NR1, NR2A, and NR2B Subunits Within the Same Complex

Abstract: The subunit compositions of the NR1 C2 exon-containing N -methyl- d -aspartate (NMDA) receptors of adult mammalian forebrain were determined by using a combination of immunoaffinity chromatography and immunoprecipitation studies with NMDA receptor subunit-specific antibodies. NMDA receptors were solubilised by sodium deoxycholate, pH 9, and purified by anti-NR1 C2 antibody affinity chromatography. The purified receptor subpopulation showed immunoreactivity with anti-NR1 C2, anti-NR1 N1, anti-NR1 C2', anti-NR2A, and anti-NR2B NMDA receptor antibodies. The NR1 C2-receptor subpopulation was subjected to immunoprecipitation using anti-NR2B antibodies and the resultant immune pellets analysed by immunoblotting where anti-NR1 C2, anti-NR1 C2', anti-NR2A, and anti-NR2B immunoreactivities were all found. Quantification of the immunoblots showed that 46% of the NR1 C2 immunoreactivity was associated with the NR2B subunit. Of this, 87% (i.e., 40% of total) were NR1 C2/NR2B receptors and 13% (6% of total) were NR1 C2/NR2A/NR2B, thus identifying the triple combination as a minor receptor subset. These results demonstrate directly, for the first time, the coexistence of the NR2A and NR2B subunits in native NMDA receptors. They show the coexistence of two splice forms of the NR1 subunit, i.e., NR1 C2 and NR1 C2', in native receptors and, in addition, they imply an NMDA receptor subpopulation containing four types of NMDA receptor subunit, NR1 C2, NR1 C2', NR2A, and NR2B, which, in accord with molecular size determinations, predicts that the NMDA receptor is at least tetrameric. These results are the first quantitative study of NMDA receptor subtypes and demonstrate molecular heterogeneity for both the NR1 and the NR2 subunits in native forebrain NMDA receptors.     

Appropriate NR1-NR1 disulfide-linked homodimer formation is requisite for efficient expression of functional, cell surface N-methyl-D-aspartate NR1/NR2 receptors

A c-Myc epitope-tagged N-methyl-D-aspartate receptor NR1-2a subunit was generated, NR1-2a(c-Myc), where the tag was inserted after amino acid 81. NR1-2a(c-Myc) /NR2A receptors when expressed in mammalian cells are not trafficked to the cell surface nor do they yield cell cytotoxicity post-transfection. NR1-2a(c-Myc) was, however, shown to assemble with NR2A subunits by immunoprecipitation and [(3)H]MK801 radioligand binding assays. Immunoblots of cells co-transfected with wild-type NR1-2a/NR2A subunits yielded two NR1-2a immunoreactive species with molecular masses of 115 and 226 kDa. Two-dimensional electrophoresis under non-reducing and reducing conditions revealed that the 226-kDa band contained disulfide-linked NR1-2a subunits. Only the 115-kDa NR1-2a species was detected for NR1-2a(c-Myc)/NR2A. The c-Myc epitope is inserted adjacent to cysteine 79 of the NR1-2a subunit; therefore, it is possible that the tag may prevent the formation of NR1 disulfide bridges. A series of cysteine --> alanine NR1-2a mutants was generated, and the NR1-2a mutants were co-expressed with NR2A or NR2B subunits in mammalian cells and characterized with respect to cell surface expression, cell cytotoxicity post-transfection, co-association by immunoprecipitation, and immunoblotting following SDS-PAGE under both reducing and non-reducing conditions. When co-expressed with NR2A in mammalian cells, NR1-2a(C79A)/NR2A displayed similar properties to NR1-2a(c-Myc)/NR2A in that the 226-kDa NR1 immunoreactive species was not detectable, and trafficking to the cell surface was impaired compared with wild-type NR1/NR2 receptors. These results provide the first biochemical evidence for the formation of NR1-NR1 intersubunit disulfide-linked homodimers involving cysteine 79. They suggest that disulfide bridging and structural integrity within the NR1 N-terminal domain is requisite for cell surface N-methyl-D-aspartate receptor expression.     

Biochemical Evidence for the Existence of a Pool of Unassembled C2 Exon-Containing NR1 Subunits of the Mammalian Forebrain NMDA Receptor

Abstract: Optimum conditions were determined for the solubilisation of native NMDA receptors of adult mammalian brain with the retention of [³H]MK-801 radioligand binding activity. The most efficient conditions were 1% Triton X-100/1 M NaCl. The efficiency of solubilisation was as follows: cloned NMDA receptors expressed in mammalian cells > forebrain receptors > cerebellar receptors. Triton X-100/1 M NaCl-solubilised forebrain NMDA receptors had a molecular size of 710,000 daltons, but significant NR1 immunoreactivity (41%) migrated as a monomer of 125,000 daltons. Immunoaffinity purification of NMDA receptors from forebrain by anti-NR1 911–920 antibody affinity chromatography from 1% Triton X-100/1 M NaCl solubilised extracts yielded purification of the NR1 M_r 120,000 immunoreactive species, but no detectable NR2A or NR2B immunoreactivity. Immunoprecipitation of NMDA receptors from Triton X-100/1 M NaCl extracts with anti-NR1 911–920 antibodies also resulted in precipitation of NR1 subunits, but with no detectable NR2A or NR2B subunits. In contrast, by immunoprecipitation with anti-NR1 17–35 antibodies, which recognise all forms of NR1, NR1, NR2A, and NR2B immunoreactivities were detected in the immune pellets. Similarly, a coassociation of NR1, NR2A, and NR2B subunits was demonstrated following extraction of forebrain membranes with 1% sodium deoxycholate (pH 9) and purification by anti-NR1 911–920 antibody affinity chromatography. These results are consistent with the identification of a pool of unassembled C2 exon-containing NR1 subunits, i.e., NR1-1a, NR1-1b, NR1-2a, and NR1-2b, selectively solubilised by 1% Triton X-100/1 M NaCl.     

Characterization of NMDA Receptor Subunit-Specific Antibodies: Distribution of NR2A and NR2B Receptor Subunits in Rat Brain and Ontogenic Profile in the Cerebellum

Abstract: Selective antisera for NMDA receptor subunits NR2A and NR2B have been developed. Each antiserum identifies a single band on an immunoblot at ∼175 kDa that appears to be the appropriate subunit of the NMDA receptor. Using these antisera the relative densities of the subunits in eight areas of adult rat brain have been determined. The NR2A subunit was found to be at its highest level in hippocampus and cerebral cortex, to be at intermediate levels in striatum, olfactory tubercle, midbrain, olfactory bulb, and cerebellum, and to be at lowest levels in the pons-medulla. The NR2B subunit was found to be expressed at its highest levels in the olfactory tubercle, hippocampus, olfactory bulb, and cerebral cortex. Intermediate levels were expressed in striatum and midbrain, and low levels were detected in the pons-medulla. No signal for NR2B was found in the cerebellum. These regional distributions were compared with that for [³H]MK-801 binding sites. It was found that although the distribution of the NR2A subunit corresponds well with radioligand binding, the distribution of the NR2B subunit does not. The ontogenic profiles of NR2A and NR2B subunits in the rat cerebellum were also determined. Just following birth [postnatal day (P) 2] NR2A subunits are undetectable, whereas NR2B subunits are expressed at amounts easily measurable. Beginning at about P12 the levels of NR2A rise rapidly to reach adult levels by P22. At the same time (P12), levels of NR2B protein begin to decline rapidly to reach undetectable levels by 22 days after birth. The results suggest that NMDA receptors are likely to be composed of different subunits in different parts of the brain and that even in the same tissue the receptors are likely to show different properties at various times during development due to alterations in the subunit composition of the receptor.     

Protein kinase C activation induces tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDA receptor

The N-methyl-D-aspartate receptor (NMDAR) is an ionotropic glutamate receptor, which plays crucial roles in synaptic plasticity and development. We have recently shown that potentiation of NMDA receptor function by protein kinase C (PKC) appears to be mediated via activation of non-receptor tyrosine kinases. The aim of this study was to test whether this effect could be mediated by direct tyrosine phosphorylation of the NR2A or NR2B subunits of the receptor. Following treatment of rat hippocampal CA1 mini-slices with 500 nM phorbol 12-myristate 13-acetate (PMA) for 15 min, samples were homogenized, immunoprecipitated with anti-NR2A or NR2B antibodies and the resulting pellets subjected to Western blotting with antiphosphotyrosine antibody. An increase in tyrosine phosphorylation of both NR2A (76 +/- 11% above control) and NR2B (41 +/- 11%) was observed. This increase was blocked by pretreatment with the selective PKC inhibitor chelerythrine, with the tyrosine kinase inhibitor Lavendustin A or with the Src family tyrosine kinase inhibitor PP2. PMA treatment also produced an increase in the phosphorylation of serine 890 on the NR1 subunit, a known PKC site, at 5 min with phosphorylation returning to near basal levels by 10 min while tyrosine phosphorylation of NR2A and NR2B was sustained for up to 15 min. These results suggest that the modulation of NMDA receptor function seen with PKC activation may be the result of tyrosine phosphorylation of NR2A and/or NR2B.     

Biochemical evidence for the co-association of three N-methyl-D-aspartate (NMDA) R2 subunits in recombinant NMDA receptors.

Functional characterization of wild-type and mutant cloned N-methyl-D-aspartate (NMDA) receptors has been used to deduce their subunit stoichiometry and quaternary structure. However, the results reported from different groups have been at variance and are thus inconclusive. This study has employed a biochemical approach to determine the number of NMDA R2 (NR2) subunits/receptor together with the NMDA R1 (NR1)/NR2 subunit ratio of both cloned and native NMDA receptors. Thus, human embryonic kidney 293 cells were transfected with the NR1-1a and NR2A NMDA receptor subunits in combination with both FLAG- and c-Myc epitope-tagged NR2B subunits. The expressed receptors were detergent-extracted and subjected to double immunoaffinity purification using anti-NR2A and anti-FLAG antibody immunoaffinity columns in series. Immunoblotting of the double immunopurified NR2A/NR2B(FLAG)-containing material demonstrated the presence of anti-NR1, anti-NR2A, anti-FLAG, and, more important, anti-c-Myc antibody immunoreactivities. The presence of anti-c-Myc antibody immunoreactivity in the double immunoaffinity-purified material showed the co-assembly of three NR2 subunits, i.e. NR2A/NR2B(FLAG)/NR2B(c-Myc), within the same NMDA receptor complex. Control experiments excluded the possibility that the co-immunopurification of the three NR2 subunits was an artifact of the solubilization procedure. These results, taken together with those previously described that showed two NR1 subunits/oligomer, suggest that the NMDA receptor is at least pentameric.     

Specific proteolysis of the NR2 subunit at multiple sites by calpain

The NMDA subtype of glutamate receptor plays an important role in the molecular mechanisms of learning, memory and excitotoxicity. NMDA receptors are highly permeable to calcium, which can lead to the activation of the calcium-dependent protease, calpain. In the present study, the ability of calpain to modulate NMDA receptor function through direct proteolytic digestion of the individual NMDA receptor subunits was examined. HEK293t cells were cotransfected with the NR1a/2A, NR1a/2B or NR1a/2C receptor combinations. Cellular homogenates of these receptor combinations were prepared and digested by purified calpain I in vitro. All three NR2 subunits could be proteolyzed by calpain I while no actin or NR1a cleavage was observed. Based on immunoblot analysis, calpain cleavage of NR2A, NR2B and NR2C subunits was limited to their C-terminal region. In vitro calpain digestion of fusion protein constructs containing the C-terminal region of NR2A yielded two cleavage sites at amino acids 1279 and 1330. Although it has been suggested that calpain cleavage of the NMDA receptor may act as a negative feedback mechanism, the current findings demonstrated that calpain cleavage did not alter [(125)I]MK801 binding and that receptors truncated to the identified cleavage sites had peak intracellular calcium levels, (45)Ca uptake rates and basal electrophysiological properties similar to wild type.     

Immunolocalization and Biochemical Characterization of N-methyl-D-aspartate Receptor Subunit NR1 from Rat Brain

The N-methyl-D-aspartate (NMDA) receptor subunit NR1 gene can produce eight isoforms in rat brain. A novel methodology for purifying NMDA receptor NR1 subunit from rat brain is reported here using chicken polyclonal antibodies (IgYs) against synthetic peptides corresponding to N1, C1 and C2′ cassettes. The isolated protein was recognized by produced IgYs and commercial anti-NR1 IgGs, shown by MALDI-TOF MS a MW = 131,192 Da (glycosylated form); the enzymatically deglycosylated protein revealed a MW = 102,754 Da. The NMDA receptor NR1 subunit was characterized as being a heavily N-glycosylated protein. The isoelectric point was determined (6.3) as being different from that predicted for any of the isoforms (7.9–9.02). Attempts to separate the isoforms from the purified NR1 were unsuccessful, indicating the presence of just one isoform (NR1₁₁₁). Immunohistochemistry on hippocampus regions CA1, CA3 and Dentate gyrus with anti-N1, anti-N2 and anti-C2′ IgYs showed different staining intensity, depending upon the antibody assayed.     

N-Methyl-d-Aspartate Receptor Proteins NR2A and NR2B Are Differentially Distributed in the Developing Rat Central Nervous System as Revealed by Subunit-Specific Antibodies

Abstract: We have identified the regional distributions and developmental expression of NMDA-receptor proteins NR2A and NR2B in rat CNS, using two subunit-specific affinity-purified polyclonal antibodies that recognize NR2A and NR2B. In western blots of cells transfected with NR2A or NR2B cDNAs, and of brain homogenates, each antibody detects a single predominant 172-kDa protein corresponding to its homologous subunit. Both subunits are glycoproteins that are enriched in synaptic membranes. In adult rat CNS, NR2A and NR2B are enriched in cortex and hippocampus but are present in other forebrain regions. In hindbrain, NR2A is present at low levels but NR2B is barely detectable. These subunits are differentially expressed in postnatal CNS development. In cortex and striatum, NR2A is absent at birth but expression increases thereafter, whereas NR2B is expressed at nearly adult levels during forebrain development. In hindbrain, low levels of NR2A are present throughout development, whereas NR2B is expressed only transiently in the first postnatal weeks. These results suggest that native NMDA receptors are modulated by NR2A and NR2B in adult forebrain but not appreciably in hindbrain. In contrast, during early postnatal development, NR2B may have a more dominant role than NR2A in modulating NMDA receptors throughout the CNS. Thus, transient changes in NMDA-receptor function may occur during maturation of certain neuronal and/or glial populations via differential expression of NR2A and NR2B subunits.     

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.     

Phosphorylation of an alpha 1-like subunit of an omega-conotoxin-sensitive brain calcium channel by cAMP-dependent protein kinase and protein kinase.

Antibodies that recognize the alpha 2 delta and alpha 1 subunits of skeletal muscle L-type calcium channels have been used to investigate the subunit components and phosphorylation of omega-conotoxin (omega-CgTx)-sensitive N-type calcium channels from rabbit brain. Photolabeling of the N-type channel with a photoreactive derivative of 125I-omega-CgTx results in the identification of a single polypeptide of 240 kDa. MANC-1, a monoclonal antibody recognizing alpha 2 delta subunits of L-type calcium channels from skeletal muscle, immunoprecipitates the omega-CgTx-labeled 240-kDa polypeptide and approximately 6% of the digitonin-solubilized 125I-omega-CgTx-labeled N-type channels. MANC-1 also immunoprecipitates a phosphoprotein of 240 kDa that comigrates with 125I-omega-CgTx-labeled N-type calcium channels, but not with L-type calcium channels, in sucrose gradients. Both cAMP-dependent protein kinase and protein kinase C are effective in the phosphorylation of this polypeptide. Similar to the alpha 1 subunits of skeletal muscle L-type calcium channels, the immunoprecipitation of the 240-kDa phosphoprotein by MANC-1 is prevented by the detergent Triton X-100. Anti-CP-(1382-1400), an antipeptide antibody against a highly conserved segment of the alpha 1 subunits of calcium channels, immunoprecipitates the 240-kDa phosphopeptide in Triton X-100. The 240-kDa protein is phosphorylated to a stoichiometry of approximately 1 mol of phosphate/mol of omega-CgTx-binding N-type calcium channels by both cAMP-dependent protein kinase and protein kinase C. Our results show that the 240-kDa polypeptide is an alpha 1-like subunit of an omega-CgTx-sensitive N-type calcium channel. The N-type calcium channels containing this subunit are phosphorylated by cAMP-dependent protein kinase and protein kinase C and contain noncovalently associated alpha 1-like and alpha 2 delta-like subunits as part of their oligomeric structure.     

Formation of NR1/NR2 and NR1/NR3 heterodimers constitutes the initial step in N-methyl-D-aspartate receptor assembly

N-Methyl-D-aspartate (NMDA) receptors are tetrameric protein complexes composed of the glycine-binding NR1 subunit with a glutamate-binding NR2 and/or glycine-binding NR3 subunit. Tri-heteromeric receptors containing NR1, NR2, and NR3 subunits reconstitute channels, which differ strikingly in many properties from the respective glycine- and glutamate-gated NR1/NR2 complexes and the NR1/NR3 receptors gated by glycine alone. Therefore, an accurate oligomerization process of the different subunits has to assure proper NMDA receptor assembly, which has been assumed to occur via the oligomerization of homodimers. Indeed, using fluorescence resonance energy transfer analysis of differentially fluorescence-tagged subunits and blue native polyacrylamide gel electrophoresis after metabolic labeling and affinity purification revealed that the NR1 subunit is capable of forming homo-oligomeric aggregates. In contrast, both the NR2 and the NR3 subunits formed homo- and hetero-oligomers only in the presence of the NR1 subunit indicating differential roles of the subunits in NMDA receptor assembly. However, co-expression of the NR3A subunit with an N-terminal domain-deleted NR1 subunit (NR1(DeltaNTD)) abrogating NR1 homo-oligomerization did not affect NR1/NR3A receptor stoichiometry or function. Hence, homo-oligomerization of the NR1 subunit is not essential for proper NR1/NR3 receptor assembly. Because identical results were obtained for NR1(DeltaNTD)/NR2 NMDA receptors (Madry, C., Mesic, I., Betz, H., and Laube, B. (2007) Mol. Pharmacol., 72, 1535-1544) and NR1-containing hetero-oligomers are readily formed, we assume that heterodimerization of the NR1 with an NR3 or NR2 subunit, which is followed by the subsequent association of two heterodimers, is the key step in determining proper NMDA receptor subunit assembly and stoichiometry.     

Regulation of NR1/NR2C N-methyl-D-aspartate (NMDA) receptors by phosphorylation

NR2C-containing N-methyl-D-aspartate (NMDA) receptors are highly expressed in cerebellar granule cells where they mediate the majority of current in the adult. NMDA receptors composed of NR1/NR2C exhibit a low conductance and reduced sensitivity to Mg(2+), compared with the more commonly studied NR2A- and NR2B-containing receptors. Despite these interesting features, very little is known about the regulation of NR2C function. Here we investigate the role of phosphorylation of NR2C in regulating NMDA receptor trafficking and ion channel properties. We identify a phosphorylation site, serine 1244 (Ser(1244)), near the extreme COOH terminus of NR2C, which is phosphorylated by both cAMP-dependent protein kinase and protein kinase C. This residue is located adjacent to the consensus PDZ ligand, a region that regulates protein-protein interactions and receptor trafficking in NR2A and NR2B. We show that Ser(1244) on NR2C is phosphorylated in vitro, in heterologous cells, and in neurons. Moreover, we demonstrate for the first time that NR2C interacts with the PSD-95 family of PDZ domain-containing proteins but that phosphorylation of Ser(1244) does not influence this PDZ interaction. Furthermore, Ser(1244) phosphorylation does not regulate surface expression of NR1/NR2C receptors. However, we find that this site does regulate the kinetics of the ion channel: a phosphomimetic mutation at Ser(1244) accelerates both the rise and decay of NMDA-evoked currents in excised patches from HEK-293 cells. Therefore, phosphorylation of Ser(1244) does not regulate trafficking but unexpectedly affects ion channel function, suggesting that phosphorylation of Ser(1244) on NR2C may be important in defining the functional properties of NMDA receptor-mediated currents in the cerebellum.     

NMDA Receptor Heterogeneity During Postnatal Development of the Rat Brain: Differential Expression of the NR2A, NR2B, and NR2C Subunit Proteins

Abstract: Changes in the expression of the NMDA receptor subunits (NRs) NR2A, 2B, and 2C were investigated in histo blots of the developing rat brain with subunit-specific antisera. At birth, the NR2B subunit was detected almost ubiquitously, the NR2A subunit staining was faint and restricted to the hippocampus, cerebral cortex, and striatum, and no NR2C subunit immunoreactivity was detected. During the first 3 postnatal weeks, the NR2B subunit became confined to forebrain structures, whereas the NR2A immunoreactivity became abundantly expressed throughout the brain. The NR2C immunoreactivity emerged 5 days after birth in the olfactory bulb, thalamus, and vestibular nuclei and became very intense after 10 days in cerebellar granule cells, its primary site of expression in adulthood. After 3 weeks, NR2A and NR2B immunoreactivity decreased to adult levels, whereas NR2C immunoreactivity remained unchanged. The patterns of distribution of the subunit proteins were in agreement with those of their corresponding mRNAs, as monitored by in situ hybridization histochemistry, although the mRNA translation appeared to be delayed by several days in certain areas. Our results reveal a progressive increase in the heterogeneity of NMDA receptors due to the comparably late onset of NR2A and NR2C subunit expression and by the area-specific rearrangement of NR2B subunit expression following birth.     

Subunit Composition of the Zinc Proteins α- and β-Lipovitellin from Chicken

Chicken α- and β-lipovitellin are derived from parent vitellogenin proteins and contain four subunits (125, 80, 40, and 30 kDa) and two subunits (125 and 30 kDa), respectively. Metal analyses demonstrate both are zinc proteins containing 2.1 ± 0.2 mol of zinc/275 kDa per α-lipovitellin and 1.4 ± 0.2 mol of zinc/155 kDa per β-lipovitellin, respectively. The subunits of β-lipovitellin, Lv 1 (MW 125 kDa) and Lv 2 (MW 30 kDa), are separated by gel exclusion chromatography in the presence of zwittergent 3–16. Zinc elutes with Lv 1, suggesting that this subunit binds zinc in the absence of Lv 2. The subunits of α- and β-lipovitellin were separated by SDS-PAGE, digested with trypsin, and mapped by reverse-phase HPLC. The peptide maps of the 125-kDa subunits from α- and β-lipovitellin are essentially identical. Similar results are obtained for the 30-kDa subunits of both lipovitellins. The sequences of five and four peptides of the 125-kDa subunit of α- and β-Lv, respectively, and two peptides of the 30-kDa subunit of α- and β-lipovitellin were determined and match those predicted from the gene for vitellogenin II, Vtg II. Comparison of the amino acid composition of the 125- and 30-kDa subunits of α- and β-lipovitellin support the conclusion that they originate from the same gene. The sequences of peptides from the 80- and 40-kDa subunits of α-lipovitellin have not been found in the NCBI nonredundant data bank. The 27-amino acid N-terminal sequence of the 40-kDa protein is 56% similar to the last third of the Lv 1-coding region of the Vtg II gene, suggesting it may come from an analogous region of the Vtg I gene. We propose a scheme for the precursor—product relationship of Vtg I.     

Differential modulation of NR1-NR2A and NR1-NR2B subtypes of NMDA receptor by PDZ domain-containing proteins

The PSD-95/Dlg/ZO-1 (PDZ) domain-containing proteins MALS and PSD-95 localize to post-synaptic densities and bind the COOH-termini of NR2 subunits of the NMDA receptor. The effects of MALS-2 and PSD-95 on the channel activity of NMDA receptors were compared using the Xenopus oocyte expression system. Both MALS-2 and PSD-95 increased the current response of the NR1-NR2B receptor to l-glutamate. In contrast, the current response of the NR1-NR2A receptor was increased by PSD-95 but not by MALS-2. MALS-2 had no effect either on the potentiation of NR1-NR2A or NR1-NR2B channel activity by protein kinase C, or on Src-mediated potentiation of NR1-NR2A activity, whereas PSD-95 almost completely inhibited the effects of these protein kinases. Construction of chimeras of MALS-2 and PSD-95 revealed that the first two PDZ domains and two NH(2)-terminal cysteine residues are essential for the inhibitory effects of PSD-95 on protein kinase C-mediated potentiation of NR1-NR2A and NR1-NR2B channel activity, respectively. The second of the three PDZ domains of PSD-95 was required for its inhibition of Src-mediated potentiation of NR1-NR2A activity. These results indicate that the NR1-NR2A and NR1-NR2B receptors are modulated differentially by MALS-2 and PSD-95, and that similar regulatory effects of PSD-95 on these receptors are achieved by distinct mechanisms.     

Pharmacological Characterization of Heterodimeric NMDA Receptors Composed of NR 1a and 2B Subunits: Differences with Receptors Formed from NR 1a and 2A

Abstract: Pharmacological and molecular biological evidence indicates the existence of multiple types of NMDA receptors within the CNS. We have characterized pharmacological properties of receptors assembled from the combination of NR 1a and NR 2B subunits (NR 1a/2B) expressed in transfected cells using both ¹²⁵I-MK-801 binding assays and electrophysiological measures. Binding of ¹²⁵I-MK-801 to cells transfected with NR 1a/2B is saturable with a K _D of 440 p M . The binding is potently inhibited by ketamine, dextromethorphan, phencyclidine, and MK-801 and is stimulated by low concentrations of magnesium. These properties resemble those of native receptors and receptors produced by NR 1a/2A. However, ¹²⁵I-MK-801 binding to membranes from cells transfected with NR 1a/2B is inhibited with high affinity by ifenprodil and is stimulated by spermidine, unlike receptors assembled from NR 1a/2A. NMDA-induced currents measured in cells transfected with either NR 1a/2A or NR 1a/2B have pharmacological properties that correlate well with the binding studies. Currents in cells transfected with NR 1a/2B are potentiated by spermidine and blocked with high affinity by ifenprodil, whereas currents in cells transfected with NR 1a/2A are not enhanced by spermidine and are weakly inhibited by ifenprodil. These data suggest that pharmacological heterogeneity in native NMDA receptors may be explained by combinations of different subunits.     

Mass spectrometrical identification of hippocampal NMDA receptor subunits NR1, NR2A-D and five novel phosphorylation sites on NR2A and NR2B

The NMDA receptor (NMDA-R) is a key element in neural transmission and mediating a vast variety of physiological and pathological processes in the nervous system. It is well-known that phosphorylation is required for functioning of the NMDA-R, and we therefore decided to study this post-translational modification in subunits NR1 and NR2A-D. Immunoprecipitation with an antibody against NR1 was carried out from rat hippocampi and SDS-PAGEs were run. Bands were punched, destained, and digested with trypsin and chymotrypsin and peptides were identified by nano-LC-ESI-MS/MS using an ion trap (HCT). Proteins were identified using specific software. Phosphorylations were verified by phosphatase treatment and reanalysis by mass spectrometry. The NMDA-R subunits NR1 and 2A-D were identified. On NR2A, a novel phosphorylation site was observed at S511, and on NR2B, four novel phosphorylation sites were revealed at S886, S917, S1303, and S1323 by mass spectrometry and verified by phosphatase treatment with mass spectrometrical reanalysis. A series of NMDA-R phosphorylations have been reported and these serve different functions as receptor activation, localization, and protein-protein interactions. Herein, findings of novel phosphorylation sites are extending knowledge on chemical characterization of the NMDA-R and warrant studying function of site-specific receptor phosphorylation in health and disease.     

Relative extent of tyrosine phosphorylation of the NR2A and NR2B subunits in the rat forebrain postsynaptic density fraction

The activity of the N-methyl-D-aspartate (NMDA) receptor, a subclass of ionotropic glutamate receptor, is modulated by a complex network of phosphorylation and dephosphorylation. I investigated the relative extent of tyrosine phosphorylation of NMDA receptor subunit 2A (NR2A) and 2B (NR2B) subunits in the rat forebrain postsynaptic density (PSD) fraction. Immunoblot analysis of immunoprecipitates with antiphosphotyrosine antibodies indicated that tyrosine phosphorylation of NR2A was only 28.6% of that of NR2B. When phosphotyrosine-containing peptides were isolated by affinity-purification or immunoprecipitation, and probed for the two subunits, NR2B was detected but not NR2A. Furthermore, depletion of NR2B removed the phosphotyrosine-containing 180 kDa peptide from the solution while the converse was not true. The small extent of tyrosine phosphorylation of NR2A in the unstimulated condition may explain the dramatic increase in tyrosine phosphorylation in various physiological and pathological conditions.     

Amyloid precursor protein 695 associates with assembled NR2A‐ and NR2B‐containing NMDA receptors to result in the enhancement of their cell surface delivery

This is a study of the interaction between the two NMDA neurotransmitter receptor subtypes, NR1/NR2A and NR1/NR2B, and amyloid precursor protein (APP) 695, the major APP variant expressed in neurones. APP695 co‐immunoprecipitated with assembled NR1‐1a/NR2A and NR1‐1a/NR2B NMDA receptors following expression in mammalian cells. Single NR1‐1a, NR1‐2a, NR1‐4b^c‐Myc, or NR2 subunit transfections revealed that co‐association of APP695 with assembled NMDA receptors was mediated via the NR1 subunit; it was independent of the NR1 C1, C2, and C2′ cassettes and, the use of an NR1‐2a^c‐Myc‐trafficking mutant suggested that interaction between the two proteins occurs in the endoplasmic reticulum. The use of antibodies directed against extracellular and intracellular NR2 subunit epitopes for immunoprecipitations suggested that APP/NMDA receptor association was mediated via N‐terminal domains. Anti‐APP antibodies immunoprecipitated NR1, NR2A, and NR2B immunoreactive bands from detergent extracts of mammalian brain; reciprocally, anti‐NR1 or anti‐NR2A antibodies co‐immunoprecipitated APP immunoreactivity. Immune pellets from brain were sensitive to endoglycosidase H suggesting that, as for heterologous expression, APP and NMDA receptor association occurs in the endoplasmic reticulum. Co‐expression of APP695 in mammalian cells resulted in enhanced cell surface expression of both NR1‐1a/NR2A and NR1‐1a/NR2B NMDA receptors with no increase in total subunit expression. These findings are further evidence for a role of APP in intracellular trafficking mechanisms. Further, they provide a link between two major brain proteins that have both been implicated in Alzheimer’s disease.