A Three Amino Acid Tail Following the TM4 Region of the N-Methyl-D-aspartate Receptor (NR) 2 Subunits Is Sufficient to Overcome Endoplasmic Reticulum Retention of NR1-1a Subunit

Yang, Wei; Zheng, Chan-Ying; Song, Qilin; Yang, Xiujuan; Qiu, Shuang; Liu, Chunqing; Zhong, Chen; Duan, Shumin; Luo, Jianhong

doi:10.1074/jbc.m700050200

Cited by 37 publications

(23 citation statements)

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“…Whole-cell Patch Clamp Recording-The electrophysiological methods have been described previously (24). The extracellular recording solution contained 147 mM NaCl, 3 mM KCl, 10 mM HEPES, 8 mM glucose, 2 mM MgCl 2 (305 mosM, pH adjusted to 7.30 with NaOH).…”

Section: Methodsmentioning

confidence: 99%

“…Immunocytochemistry-Surface-staining of HEK293 cells and cortical neurons was performed as described previously (24). Cortical neurons were plated on coverslips and transfected with the indicated plasmid on DIV 7.…”

Section: Methodsmentioning

confidence: 99%

“…Reagents-Wild-type (WT) GluN1 and GluN2B tagged with extracellular amino-terminal GFP (GFP-GluN2B WT ) in the pRK5 vector were described previously (24). The primary antibodies used were: Tyr(P)-1472 GluN2B (1:500; Millipore, Lake Placid, NY), mouse GluN2B (1:2000; Cell Signaling, Danvers, MA), Ser(P)-845-GluA1 (1:1000; Millipore), GluA1 (1:1000; Millipore), rabbit monoclonal Fyn (1:2000; Epitomics, Burlingame, CA), mouse monoclonal PSD-95 (1:1000; Abcam, Cambridge, MA), ␤-actin (1:20000; Sigma), and rabbit monoclonal GFP (1:2000; Epitomics).…”

Section: Methodsmentioning

confidence: 99%

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Phosphorylation of Tyrosine 1070 at the GluN2B Subunit Is Regulated by Synaptic Activity and Critical for Surface Expression of N-Methyl-d-aspartate (NMDA) Receptors

Fang

et al. 2015

Journal of Biological Chemistry

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Phosphorylation of Tyrosine 1070 at the GluN2B Subunit Is Regulated by Synaptic Activity and Critical for Surface Expression of N-Methyl-d-aspartate (NMDA) Receptors

Fang

et al. 2015

Journal of Biological Chemistry

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“…Surface Immunostaining and Quantitative Analysis-These methods have been described previously (20,21). Cells were examined through a 60 ϫ 1.4 (numerical aperture) oil immersion objective on a FV1000 confocal laser scanning microscope equipped with imaging analysis software (Olympus, Tokyo, Japan).…”

Section: Methodsmentioning

confidence: 99%

Transmembrane Region of N-Methyl-d-aspartate Receptor (NMDAR) Subunit Is Required for Receptor Subunit Assembly

Cao¹,

Qiu²,

Zhang

et al. 2011

Journal of Biological Chemistry

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N-Methyl-D-aspartate receptors (NMDARs), one of three main classes of ionotropic glutamate receptors, play major roles in synaptic plasticity, synaptogenesis, and excitotoxicity. Unlike non-NMDA receptors, NMDARs are thought to comprise obligatory heterotetrameric complexes mainly composed of GluN1 and GluN2 subunits. When expressed alone in heterogenous cells, such as HEK293 cells, most of the NMDAR subunits can neither leave the endoplasmic reticulum (ER) nor be expressed in the cell membrane because of the ER retention signals. Only when NMDARs are heteromerically assembled can the ER retention signals be masked and NMDARs be expressed in the surface membrane. However, the mechanisms underlying NMDAR assembly remain poorly understood. To identify regions in subunits that mediate this assembly, we made a series of truncated or chimeric cDNA constructs. Using FRET measurement in living cells combined with immunostaining and coimmunoprecipitation analysis, we examined the assemblydetermining domains of NMDAR subunits. Our results indicate that the transmembrane region of subunits is necessary for the assembly of NMDAR subunits, both for the homodimer and the heteromer.

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“…Second, using truncated and chimeric NMDA receptor subunits, we showed recently that M3 of both NR1 and NR2B possess ER retention signals and that these domains as well as M4 of NR1 are responsible for masking the ER retention signals in the M3 domains.10 Third, NR1/NR2 complexes can reach the cell surface membrane even when either one or both C-terminal regions are deleted five or six amino acid residues after M4.10 -12 However, full-length NR2B constructs lacking the HLFY motif, located one amino acid residue after the M4 domain, are not delivered to the surface membrane.6 Fourth, a later study showed that replacement of the HLFY motif with alanines in the NR2B subunit, truncated immediately after the mutated HLFY motif, does not affect the formation of functional channels. 13 Since this study also showed that at least three amino acid residues after NR2B M4 are necessary for surface trafficking of the receptors, this indicates that the NR2B M4 domain and surrounding segments are critical for correct assembly and formation of functional receptors. Finally, co-expression of the NR2 subunit truncated before M4 (NR2 preM4) with the full-length NR1 subunit fails to produce functional receptors.…”

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confidence: 99%

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

Hořák¹,

Al‐Hallaq²,

Chang³

et al. 2008

Channels

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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. KeywordsER retention; glutamate receptor; ion channel; masking; patch-clamp; recombinant; subunit assembly N-methyl-D-aspartate (NMDA) receptors are glutamate-gated ionotropic receptors mediating excitatory neurotransmission in the central nervous system.1 It is believed that these receptors are heterotetramers composed of two NR1 and two NR2 and/or NR3 subunits.2 All NMDA receptor subtypes share the same structural topology with three membrane domains (M1, M3 and M4), a re-entrant pore-forming domain (M2), and extracellular (N-terminus and loop between M3 and M4) and intracellular (C-terminus) regions. The most abundant NR1 splice variant (NR1-1) and all NR2 subunits are retained in the ER when expressed by themselves in various heterologous cells and neurons; however, co-expression of NR1-1 and NR2 leads to surface expression of assembled complexes.3 -5 Thus, these data suggest the presence of a mechanism(s) regulating ER retention of unassembled subunits, correct assembly of the subunits into functional receptors, as well as release of the functional receptors from the ER.There have been a number of reports studying the role of ER retention in the assembly of NMDA receptors. First, the localization of ER retention signals was studied using chimeric proteins composed of tac (an interleukin-2 receptor subunit) and NMDA receptor C-termini. 6-9 Since both NR1-1 and NR2B C-termini cause the chimeric protein to be retained in the ER, an extensive mutagenesis study was performed. Hence, an RXR ER retention motif in the NR1-1 C-terminus was identified. Efforts to localize a particular motif responsible for the ER retention of tac-NR2B chimera have been unsuccessful. Second, using truncated and chimeric NMDA receptor subunits, we showe...

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A Three Amino Acid Tail Following the TM4 Region of the N-Methyl-D-aspartate Receptor (NR) 2 Subunits Is Sufficient to Overcome Endoplasmic Reticulum Retention of NR1-1a Subunit

Cited by 37 publications

References 36 publications

Phosphorylation of Tyrosine 1070 at the GluN2B Subunit Is Regulated by Synaptic Activity and Critical for Surface Expression of N-Methyl-d-aspartate (NMDA) Receptors

Phosphorylation of Tyrosine 1070 at the GluN2B Subunit Is Regulated by Synaptic Activity and Critical for Surface Expression of N-Methyl-d-aspartate (NMDA) Receptors

Transmembrane Region of N-Methyl-d-aspartate Receptor (NMDAR) Subunit Is Required for Receptor Subunit Assembly

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

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