Trafficking and surface expression of the glutamate receptor subunit, KA2

Hayes, Dayna M; Braud, Stephanie; Hurtado, David; McCallum, Jennifer; Standley, Steve; Isaac, John T.R.; Roche, Katherine W.

doi:10.1016/j.bbrc.2003.08.115

Cited by 27 publications

(36 citation statements)

References 34 publications

(45 reference statements)

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“…Electrophysiology Methods-HEK-293 cells were transiently transfected with NR1-1a and FLAG-NR2C-IRES-EGFP as described previously (17). Between 2 and 3 days after transfection, cells were used for electrophysiology with transfected cells identified by visualizing GFP fluorescence.…”

Section: Methodsmentioning

confidence: 99%

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Regulation of NR1/NR2C N-Methyl-D-aspartate (NMDA) Receptors by Phosphorylation*

Chen

Braud

Badger

et al. 2006

Journal of Biological Chemistry

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NR2C-containing N-methyl-D-aspartate NMDA2 receptors are glutamate-gated ion channels that are critical for synapse formation, synaptic plasticity, and learning and memory (1, 2). They are members of the larger family of ionotropic glutamate receptors that also include AMPA and kainate receptors. Endogenous NMDA receptors are heteromeric channels containing two NR1 subunits combined with additional NR2 (NR2A-D) and NR3 (NR3A-B) subunits (2, 3). In contrast to the widespread distribution for NR1, NR2 subunits display unique spatiotemporal expression patterns throughout the central nervous system (4) and confer distinct pharmacological and functional properties on NMDA receptors (2). NMDA receptors in the cerebellum are composed of NR1 and NR2A-C subunits; the NR2C subunit is notably enriched in cerebellar granule cells and is up-regulated developmentally such that in the adult, NR1, NR2A, and NR2C are the predominant NMDA receptor subunits assembling to form functional receptors (2, 5). Moreover, NR2C-containing receptors have unique properties of low conductance and reduced sensitivity to Mg 2ϩ compared with NR2A-or NR2B-containing receptors (2,4,6). Despite this, the specific regulation of NR2C-containing receptors has not been studied extensively.Phosphorylation is an important mechanism regulating glutamate receptors and synaptic plasticity and occurs primarily in the cytosolic COOH-terminal domain of the receptor subunits. Phosphorylation of AMPA receptors by PKA and CaMKII directly affects channel function (7-9), receptor trafficking to synapses (10), and is critical for the expression of long term potentiation and long term depression (11, 12). Phosphorylation is an important regulator of NMDA receptor function and trafficking (13-15); however, the direct phosphorylation of NR2C has not been reported. Interestingly, NR2C contains a prototypic PKA phosphorylation site in the distal COOH-terminal region that is not conserved in NR2A and NR2B, suggesting that phosphorylation at this location might play a specific role in regulating NR2C-containing NMDA receptor complexes.In this study, we have investigated the regulation of NR2C by phosphorylation. We demonstrate that Ser 1244 is phosphorylated by both PKA and PKC in vitro and also on native NR2C in neurons. This serine is just upstream of the PDZ binding domain, which is conserved in NR2 subunits and is critical for NMDA receptor binding to PSD-95. We demonstrate that NR2C interacts directly with PSD-95; however, we find no effect of Ser 1244 phosphorylation on PSD-95 binding to NR2C. Unlike the PKA regulation of AMPA receptors (10), we find no effect of Ser 1244 phosphorylation on surface expression of NR1/NR2C NMDA receptors. However, we find that this site alters NMDA receptor channel function, because a phosphomimetic mutation (S1244E) accelerates channel kinetics. Thus, we find that this phosphorylation site in the distal COOH terminus regulates NR2C channel function.

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Section: Methodsmentioning

confidence: 99%

“…Fluorescence-activated Cell Sorting (FACS) Analysis-HEK-293 cells were transiently transfected with NR1-1a and FLAG-NR2C-IRES-EGFP as described previously (17). Transfected HEK-293 cells were rinsed twice with PBS containing 5 mM EDTA (PBS/EDTA) and then incubated with PBS/EDTA for 10 min at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Regulation of NR1/NR2C N-Methyl-D-aspartate (NMDA) Receptors by Phosphorylation*

Chen

Braud

Badger

et al. 2006

Journal of Biological Chemistry

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“…Unlike GluR6, KA2 does not exist as functional homomers, and it is likely that neurons use precise quality control mechanisms to regulate the oligomerization and trafficking of KA2. Recently, it was demonstrated that KA2 is endoplasmic reticulum (ER) retained when expressed in heterologous cells (Gallyas et al, 2003;Hayes et al, 2003;Ren et al, 2003). The molecular mechanisms regulating KA2 ER-retention likely play a critical role in regulating the complement of synaptic kainate receptors, because they ensure that KA2 only reaches the plasma membrane as a heteromeric channel.…”

Section: Introductionmentioning

confidence: 99%

Identification of an Endoplasmic Reticulum-Retention Motif in an Intracellular Loop of the Kainate Receptor Subunit KA2

et al. 2006

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Neuronal kainate receptors are typically heteromeric complexes composed of GluR5-7 and KA1-2 subunits. Although GluR5-7 can exist as functional homomeric channels, the KA subunits cannot. KA2 is widely expressed in the CNS, and KA2/GluR6 heteromers are the most prevalent subunit composition in brain. Previous work has identified endoplasmic reticulum (ER)-retention motifs in the C terminus of KA2, which prevent surface expression of KA2 homomers. However, we find that, when these motifs are mutated, only a small fraction of KA2 is surface expressed. We now identify an additional ER retention motif in the intracellular loop region of KA2, which, when mutated together with the C-terminal motifs, significantly increases the level of KA2 surface expression. However, electrophysiological analysis of surface-expressed KA2 homomers indicates that they do not form functional ion channels. In heterologous cells, a large fraction of KA2 remains intracellular even when the trafficking motifs are mutated or when GluR6 is coexpressed. Therefore, we analyzed the trafficking of endogenous KA2 in vivo. We find that native KA2 surface expression is dramatically reduced in GluR6 knock-out mice compared with wild-type mice. In contrast, KA2 trafficking was unaffected in the GluR5 knock-out. Thus, our study demonstrates that trafficking motifs in both the intracellular loop and C terminus regulate KA2 surface expression; however, in neurons, GluR6 oligomerization is required for egress of KA2 from the ER and transport to the cell surface. The combination of these mechanisms likely prevents surface expression of nonfunctional KA2 homomers and ensures a high level of GluR6/KA2 heteromeric kainate receptors.

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“…76 GluK5 subunits can form homomeric receptors, but these are not surface expressed because they are retained in the ER. [77][78][79] These subunits contain an RXR motif in the intracellular region between the second and third membrane domains 80 and an intracellular C-terminal arginine-rich (RRRRR) ER retention/retrieval motif. This motif is a binding site for the coatomer protein I (COPI) 81 a component of the retrograde trafficking protein complex that mediates the transport of defective proteins back from the Golgi to the ER.…”

Section: Molecular Determinants Of Kar Traffic Er Retention/retrievalmentioning

confidence: 99%

Kainate receptor trafficking

González‐González

Konopacki

Rocca

et al. 2011

WIREs Membr Transp Signal

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Kainate receptors (KARs) are ligand-gated ion channels that can regulate neuronal network activity and are involved in processes ranging from neuronal development and differentiation to neurodegeneration and neuronal cell death. They are tetrameric assemblies which can comprise different subunit combinations and are differentially targeted to specific cell surface compartments including preand postsynaptic membranes where they perform distinct functional roles. The processes regulating the constitutive and activity-dependent trafficking of KARs are subtle and complex. Intricate combinations of protein-protein interactions and post-translational modifications orchestrate their surface membrane delivery, residence time, internalization, and recycling or degradation. Furthermore, in addition to regulating surface expression, interacting proteins connect receptors to anchoring and signaling pathways. Although our knowledge of the processes that regulate KAR trafficking is far from complete, there has been significant progress toward defining the roles of many of these protein partners and post-translational modifications. 

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Trafficking and surface expression of the glutamate receptor subunit, KA2

Cited by 27 publications

References 34 publications

Regulation of NR1/NR2C N-Methyl-D-aspartate (NMDA) Receptors by Phosphorylation*

Regulation of NR1/NR2C N-Methyl-D-aspartate (NMDA) Receptors by Phosphorylation*

Identification of an Endoplasmic Reticulum-Retention Motif in an Intracellular Loop of the Kainate Receptor Subunit KA2

Kainate receptor trafficking

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