Cited by 418Neuroscience and Neuropharmacology ResearchCellular transport and secretionReceptor Mechanisms and Signaling

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An NMDA Receptor ER Retention Signal Regulated by Phosphorylation and Alternative Splicing

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Scott, Derek B., Blanpied, Thomas A., Swanson, Geoffrey T., Zhang, Chi, Ehlers, Michael D.·2001·The Journal of Neuroscience

DOI 10.1523/jneurosci.21-09-03063.2001PMC PMC6762585

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Abstract

Formation of mature excitatory synapses requires the assembly and delivery of NMDA receptors to the neuronal plasma membrane. A key step in the trafficking of NMDA receptors to synapses is the exit of newly assembled receptors from the endoplasmic reticulum (ER). Here we report the identification of an RXR-type ER retention/retrieval motif in the C-terminal tail of the NMDA receptor subunit NR1 that regulates receptor surface expression in heterologous cells and in neurons. In addition, we show that PKC phosphorylation and an alternatively spliced consensus type I PDZ-binding domain suppress ER retention. These results demonstrate a novel quality control function for alternatively spliced C-terminal domains of NR1 and implicate both phosphorylation and potential PDZ-mediated interactions in the trafficking of NMDA receptors through early stages of the secretory pathway.

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Malenka, R. C., & Nicoll, A. R. A. (1999) · Science · doi:10.1126/science.285.5435.1870 · Cited by 2,729
Heteromeric NMDA receptors: molecular and functional distinction of subtypes
Monyer, H., Sprengel, R., Schoepfer, R., Herb, A., Higuchi, M., Lomeli, H., Burnashev, N., Sakmann, B., & Seeburg, P. H. (1992) · Science · doi:10.1126/science.256.5060.1217 · Cited by 2,541
Domain Interaction Between NMDA Receptor Subunits and the Postsynaptic Density Protein PSD-95
Kornau, H., Schenker, L. T., Kennedy, M. B., & Seeburg, P. H. (1995) · Science · doi:10.1126/science.7569905 · Cited by 1,916
Recognition of Unique Carboxyl-Terminal Motifs by Distinct PDZ Domains
Songyang, Z., Fanning, A. S., Fu, C., Xu, J., Marfatia, S. M., Chishti, A. H., Crompton, A., Chan, A. C., Anderson, J. M., & Cantley, L. C. (1997) · Science · doi:10.1126/science.275.5296.73 · Cited by 1,385
Setting the Standards: Quality Control in the Secretory Pathway
Ellgaard, L., Molinari, M., & Helenius, A. (1999) · Science · doi:10.1126/science.286.5446.1882 · Cited by 1,192
Glutamate receptors in the mammalian central nervous system
Ozawa, S., Kamiya, H., & Tsuzuki, K. (1998) · Progress in Neurobiology · doi:10.1016/s0301-0082(97)00085-3 · Cited by 1,133
Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase F
Tarentino, A. L., Gomez, C. M., & Plummer, T. H. (1985) · Biochemistry · doi:10.1021/bi00338a028 · Cited by 1,130
A New ER Trafficking Signal Regulates the Subunit Stoichiometry of Plasma Membrane KATP Channels
Zerangue, N., Schwappach, B., Jan, Y. N., & Jan, L. Y. (1999) · Neuron · doi:10.1016/s0896-6273(00)80708-4 · Cited by 1,025
Protein kinase C reduces Mg2+ block of NMDA-receptor channels as a mechanism of modulation
Chen, L., & Huang, L.-Y. M. (1992) · Nature · doi:10.1038/356521a0 · Cited by 974
Kinesin Superfamily Motor Protein KIF17 and mLin-10 in NMDA Receptor-Containing Vesicle Transport
Setou, M., Nakagawa, T., Seog, D., & Hirokawa, N. (2000) · Science · doi:10.1126/science.288.5472.1796 · Cited by 727
A Trafficking Checkpoint Controls GABAB Receptor Heterodimerization
Margeta, M., Jan, Y. N., & Jan, L. Y. (2000) · Neuron · doi:10.1016/s0896-6273(00)00012-x · Cited by 680
Synaptic plasticity and dynamic modulation of the postsynaptic membrane
Lüscher, C., Nicoll, R. A., Malenka, R. C., & Müller, D. (2000) · Nature Neuroscience · doi:10.1038/75714 · Cited by 662
Structure and function of the NMDA receptor channel
Mori, H., & Mishina, M. (1995) · Neuropharmacology · doi:10.1016/0028-3908(95)00109-j · Cited by 658
Kinetic Analysis of Secretory Protein Traffic and Characterization of Golgi to Plasma Membrane Transport Intermediates in Living Cells
Hirschberg, K., Miller, C. M., Ellenberg, J., Presley, J. F., Siggia, E. D., Phair, R. D., & Lippincott‐Schwartz, J. (1998) · The Journal of Cell Biology · doi:10.1083/jcb.143.6.1485 · Cited by 610
Rapid, experience-dependent expression of synaptic NMDA receptors in visual cortex in vivo
Quinlan, E. M., Philpot, B. D., Huganir, R. L., & Bear, M. F. (1999) · Nature Neuroscience · doi:10.1038/7263 · Cited by 579
PDZ domains in synapse assembly and signalling
Garner, C. C., Nash, J. E., & Huganir, R. L. (2000) · Trends in Cell Biology · doi:10.1016/s0962-8924(00)01783-9 · Cited by 520
Characterization of Protein Kinase A and Protein Kinase C Phosphorylation of the N-Methyl-D-aspartate Receptor NR1 Subunit Using Phosphorylation Site-specific Antibodies
Tingley, W. G., Ehlers, M., Kameyama, K., Doherty, C., Ptak, J., Riley, C. T., & Huganir, R. L. (1997) · Journal of Biological Chemistry · doi:10.1074/jbc.272.8.5157 · Cited by 507
SIGNAL-MEDIATED SORTING OF MEMBRANE PROTEINS BETWEEN THE ENDOPLASMIC RETICULUM AND THE GOLGI APPARATUS
Teasdale, R. D., & Jackson, M. R. (1996) · Annual Review of Cell and Developmental Biology · doi:10.1146/annurev.cellbio.12.1.27 · Cited by 500
Assembly of New Individual Excitatory Synapses
Friedman, H., Bresler, T., Garner, C. C., & Ziv, N. E. (2000) · Neuron · doi:10.1016/s0896-6273(00)00009-x · Cited by 499
Activity Regulates the Synaptic Localization of the NMDA Receptor in Hippocampal Neurons
Rao, A., & Craig, A. M. (1997) · Neuron · doi:10.1016/s0896-6273(00)80962-9 · Cited by 492
PDZ domains: fundamental building blocks in the organization of protein complexes at the plasma membrane
Fanning, A. S., & Anderson, J. M. (1999) · Journal of Clinical Investigation · doi:10.1172/jci6509 · Cited by 471
Importance of the Intracellular Domain of NR2 Subunits for NMDA Receptor Function In Vivo
Sprengel, R., Suchanek, B., Amico, C., Brusa, R., Burnashev, N., Rozov, A. V., Hvalby, Ø., Jensen, V., Paulsen, O., Andersen, P. N., et al. (1998) · Cell · doi:10.1016/s0092-8674(00)80921-6 · Cited by 460
Regulation of NMDA receptor phosphorylation by alternative splicing of the C-terminal domain
Tingley, W. G., Roche, K. W., Thompson, A. K., & Huganir, R. L. (1993) · Nature · doi:10.1038/364070a0 · Cited by 408
Regulation of morphological postsynaptic silent synapses in developing hippocampal neurons
Liao, D., Zhang, X., O’Brien, R., Ehlers, M., & Huganir, R. L. (1999) · Nature Neuroscience · doi:10.1038/4540 · Cited by 396
Role of ER Export Signals in Controlling Surface Potassium Channel Numbers
Ma, D., Zerangue, N., Lin, Y., Collins, A., Yu, M. Y., Jan, Y. N., & Jan, L. Y. (2001) · Science · doi:10.1126/science.291.5502.316 · Cited by 396
LTP mechanisms: from silence to four-lane traffic
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The I-II Loop of the Ca 2+ Channel α 1 Subunit Contains an Endoplasmic Reticulum Retention Signal Antagonized by the β Subunit
Bichet, D., Cornet, V., Geib, S., Carlier, E., Volsen, S. G., Hoshi, T., Mori, Y., & WAARD, M. M. D. (2000) · Neuron · doi:10.1016/s0896-6273(00)80881-8 · Cited by 377
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Standley, S., Roche, K. W., McCallum, J. D., Sans, N., & Wenthold, R. J. (2000) · Neuron · doi:10.1016/s0896-6273(00)00161-6 · Cited by 352
Activity Coregulates Quantal AMPA and NMDA Currents at Neocortical Synapses
Watt, A. J., van Rossum, M. C. W., MacLeod, K. M., Nelson, S. B., & Turrigiano, G. G. (2000) · Neuron · doi:10.1016/s0896-6273(00)81202-7 · Cited by 350
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Brodsky, J. L., & McCracken, A. A. (1999) · Seminars in Cell and Developmental Biology · doi:10.1006/scdb.1999.0321 · Cited by 343
C-Terminal Interaction Is Essential for Surface Trafficking But Not for Heteromeric Assembly of GABA B Receptors
Pagano, A., Rovelli, G., Mosbacher, J., Lohmann, T., Duthey, B., Stauffer, D., Ristig, D., Schuler, V., Meigel, I., Lampert, C., et al. (2001) · Journal of Neuroscience · doi:10.1523/jneurosci.21-04-01189.2001 · Cited by 310
Regulated Subcellular Distribution of the NR1 Subunit of the NMDA Receptor
Ehlers, M., Tingley, W. G., & Huganir, R. L. (1995) · Science · doi:10.1126/science.7569904 · Cited by 290
Molecular mechanisms of glutamate receptor clustering at excitatory synapses
O’Brien, R., Lau, L., & Huganir, R. L. (1998) · Current Opinion in Neurobiology · doi:10.1016/s0959-4388(98)80062-7 · Cited by 273
Optimizing hydrolysis of N-linked high-mannose oligosaccharides by endo-β-N-acetylglucosaminidase H
Trimble, R. B., & Maley, F. (1984) · Analytical Biochemistry · doi:10.1016/0003-2697(84)90080-0 · Cited by 270
A monoclonal antibody against a 135‐K Golgi membrane protein.
Burke, B. E., Griffiths, G. W., Reggio, H., Louvard, D., & Warren, W. G. (1982) · The EMBO Journal · doi:10.1002/j.1460-2075.1982.tb01364.x · Cited by 260
Turnover Analysis of Glutamate Receptors Identifies a Rapidly Degraded Pool of the N-Methyl-d-aspartate Receptor Subunit, NR1, in Cultured Cerebellar Granule Cells
Huh, K.-H., & Wenthold, R. J. (1999) · Journal of Biological Chemistry · doi:10.1074/jbc.274.1.151 · Cited by 248
An Endocytosed TGN38 Chimeric Protein Is Delivered to the TGN after Trafficking through the Endocytic Recycling Compartment in CHO Cells
Ghosh, R. N., Mallet, W. G., Soe, T. T., McGraw, T. E., & Maxfield, F. R. (1998) · The Journal of Cell Biology · doi:10.1083/jcb.142.4.923 · Cited by 243
Characterization of the human receptor for T-cell growth factor.
Leonard, W. J., Depper, J. M., Robb, R. J., Waldmann, T. A., & Greene, W. C. (1983) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.80.22.6957 · Cited by 234
C-Terminal Truncation of NR2A Subunits Impairs Synaptic But Not Extrasynaptic Localization of NMDA Receptors
Steigerwald, F., Schulz, T. W., Schenker, L. T., Kennedy, M., Seeburg, P., & Köhr, G. (2000) · Journal of Neuroscience · doi:10.1523/jneurosci.20-12-04573.2000 · Cited by 204
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Okabe, S., Miwa, A., & Okado, H. (1999) · Journal of Neuroscience · doi:10.1523/jneurosci.19-18-07781.1999 · Cited by 187
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Calver, A. R., Robbins, M. J., Cosio, C., Rice, S. Q. J., Babbs, A. J., Hirst, W. D., Boyfield, I., Wood, M. D., Russell, R. B., Price, G. W., et al. (2001) · Journal of Neuroscience · doi:10.1523/jneurosci.21-04-01203.2001 · Cited by 164
Assembly intracellular targeting and cell surface expression of the human N-methyl-d-aspartate receptor subunits NR1a and NR2A in transfected cells
McIlhinney, R. A. J., Bourdellès, B. L., Molnár, E., Tricaud, N., Streit, P., & Whiting, P. J. (1998) · Neuropharmacology · doi:10.1016/s0028-3908(98)00121-x · Cited by 160
Molecular Basis for K ATP Assembly
Schwappach, B., Zerangue, N., Jan, Y. N., & Jan, L. Y. (2000) · Neuron · doi:10.1016/s0896-6273(00)81146-0 · Cited by 157
A Role for a PDZ Protein in the Early Secretory Pathway for the Targeting of proTGF-α to the Cell Surface
Fernández-Larrea, J., Merlos‐Suárez, A., Ureña, J. M., Baselga, J. M., & Arribas, J. (1999) · Molecular Cell · doi:10.1016/s1097-2765(00)80470-0 · Cited by 151
Acetylcholine receptor assembly: Subunit folding and oligomerization occur sequentially
Green, W. N., & Claudio, T. (1993) · Cell · doi:10.1016/0092-8674(93)90294-z · Cited by 149
Differential Surface Expression and Phosphorylation of the N-Methyl-D-aspartate Receptor Subunits NR1 and NR2 in Cultured Hippocampal Neurons
Hall, R. A., & Soderling, T. R. (1997) · Journal of Biological Chemistry · doi:10.1074/jbc.272.7.4135 · Cited by 143
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Mori, H., Manabe, T., Watanabe, M., Satoh, Y., Suzuki, N., Toki, S., Nakamura, K., Yagi, T., Kushiya, E., Takahashi, T., et al. (1998) · Neuron · doi:10.1016/s0896-6273(00)80567-x · Cited by 136
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Tan, P. K., Waites, C. L., Liu, Y., Krantz, D. E., & Edwards, R. H. (1998) · Journal of Biological Chemistry · doi:10.1074/jbc.273.28.17351 · Cited by 116
Subtype-specific Assembly of α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid Receptor Subunits Is Mediated by Their N-terminal Domains
Leuschner, W. D., & Hoch, W. (1999) · Journal of Biological Chemistry · doi:10.1074/jbc.274.24.16907 · Cited by 112
Protein kinase C potentiation of N -methyl- d -aspartate receptor activity is not mediated by phosphorylation of N -methyl- d -aspartate receptor subunits
Zheng, X., Zhang, L., Wang, A. P., Bennett, M. V. L., & Zukin, R. S. (1999) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.96.26.15262 · Cited by 111
Assembly intermediates of the mouse muscle nicotinic acetylcholine receptor in stably transfected fibroblasts.
Blount, P., Smith, M. M., & Merlie, J. P. (1990) · The Journal of Cell Biology · doi:10.1083/jcb.111.6.2601 · Cited by 102
Polypeptide translocation across the endoplasmic reticulum membrane.
Sanders, S. L., & Schekman, R. W. (1992) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(19)49634-8 · Cited by 96
Cell surface expression of the human N-methyl-d-aspartate receptor subunit 1a requires the co-expression of the NR2A subunit in transfected cells
McIlhinney, R. A. J., Molna´r, E., Atack, J. R., & Whiting, P. J. (1996) · Neuroscience · doi:10.1016/0306-4522(95)00419-x · Cited by 95
Differential interaction of the tSXV motifs of the NR1 and NR2A NMDA receptor subunits with PSD‐95 and SAP97
Bassand, P., Bernard, A., Rafiki, A., Gayet, D., & Khrestchatisky, M. (1999) · European Journal of Neuroscience · doi:10.1046/j.1460-9568.1999.00611.x · Cited by 78
Identification of Routing Determinants in the Cytosolic Domain of a Secretory Granule-associated Integral Membrane Protein*
Milgram, S. L., Mains, R., & Eipper, B. (1996) · Journal of Biological Chemistry · doi:10.1074/jbc.271.29.17526 · Cited by 75
Growth of the NMDA receptor industrial complex
Sheng, M., & Lee, S. H. (2000) · Nature Neuroscience · doi:10.1038/76576 · Cited by 45
Protein kinase C transiently activated heteromeric N-methyl-D-aspartate receptor channels independent of the phosphorylatable C-terminal splice domain and of consensus phosphorylation sites.
Sigel, E., Baur, R., & Malherbe, P. (1994) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(17)37180-6 · Cited by 44
Synaptic Targeting of the Postsynaptic Density Protein PSD-95 Mediated by a Tyrosine-based Trafficking Signal
Craven, S. E., & Bredt, D. S. (2000) · Journal of Biological Chemistry · doi:10.1074/jbc.m910153199 · Cited by 36
Targeting of Membrane Proteins to the Regulated Secretory Pathway in Anterior Pituitary Endocrine Cells*
Meskini, R. E., Galano, G. J., Marx, R., Mains, R., & Eipper, B. (2001) · Journal of Biological Chemistry · doi:10.1074/jbc.m008062200 · Cited by 32
Intracellular trafficking of the vacuolar H+-ATPase accessory subunit Ac45
Jansen, E. J. R., Holthuis, J. C. M., McGrouther, C., Burbach, J. P. H., & Martens, G. J. M. (1998) · Journal of Cell Science · doi:10.1242/jcs.111.20.2999 · Cited by 29
Chapter 13 Modulation of NMDA receptors
MacDonald, J. F., Xiong, X.-G., Lu, W.-Y., Raouf, R., & Orser, B. A. (1998) · Progress in brain research · doi:10.1016/s0079-6123(08)60438-0 · Cited by 23
The N‐terminal region of the α‐subunit of the TRAP complex has a conserved cluster of negative charges
Hartmann, E., & Prehn, S. (1994) · FEBS Letters · doi:10.1016/0014-5793(94)00693-8 · Cited by 15

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Authors: Scott, D. B., Blanpied, T. A., Swanson, G. T., Zhang, C., & Ehlers, M. D.
Affiliations: Duke Medical Center (US)
Salk Institute for Biological Studies (US)
ORCID: Blanpied, Thomas A.
Swanson, Geoffrey T.
Zhang, Chi
Ehlers, Michael D.
Venue: The Journal of Neuroscience
Publisher: Society for Neuroscience
Published: 2001-05-01
Volume: 21
Issue: 9
Pages: 3063-3072
ISSN: 0270-6474 (ISSN-L)
1529-2401
DOI: 10.1523/jneurosci.21-09-03063.2001
PMC: PMC6762585
OpenAlex: W1527134354
Cited by: 418
References: 62
Citations by year: 2026: 4 · 2025: 5 · 2024: 5 · 2023: 5 · 2022: 10 · 2021: 10 · 2020: 10 · 2019: 8 · 2018: 11 · 2017: 10 · 2016: 3 · 2015: 12 · 2014: 13 · 2013: 10 · 2012: 10
FWCI: 12.93
Type: journal article
Language: en
Open access: hybrid
doi.org
Licence: cc-by-nc-sa
creativecommons.org/licenses/by-nc-sa/4.0/ (vor, 184 d)
Locations: Journal of Neuroscience (publishedVersion, cc-by-nc-sa)
PubMed Central (publishedVersion)
PubMed (publishedVersion)
Funders: Alfred P. Sloan Foundation
American Health Assistance Foundation
McKnight Foundation
North Carolina Biotechnology Center
Triangle Community Foundation
National Alliance for Research on Schizophrenia and Depression
National Institutes of Health
National Institute of Neurological Disorders and Stroke (R01 NS39402, R01NS039402)
Topics: Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
Keywords: NMDA receptor, NR1 subunit, endoplasmic reticulum retention signal, PKC phosphorylation, alternative splicing, PDZ-binding domain, receptor surface expression, excitatory synapse formation, secretory pathway, quality control, receptor trafficking
MeSH terms: Animals, Cells, Cultured, Endoplasmic Reticulum, Hippocampus, Humans, Neurons, Phosphorylation, Protein Kinase C, Recombinant Fusion Proteins, Transcription Factors, Transfection, Receptors, Interleukin-2, Receptors, N-Methyl-D-Aspartate, Alternative Splicing, Protein Structure, Tertiary, Receptors, Retinoic Acid, Amino Acid Motifs, Protein Subunits, Protein Sorting Signals, Retinoid X Receptors, Rats

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An NMDA Receptor ER Retention Signal Regulated by Phosphorylation and Alternative Splicing

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