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

Nasu‐Nishimura, Yukiko; Hurtado, David; Braud, Stephanie; Tang, Tina Tze-Tsang; Isaac, John T.R.; Roche, Katherine W.

doi:10.1523/jneurosci.0573-06.2006

Cited by 58 publications

(72 citation statements)

References 23 publications

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“…A recent paper has shown that the replacement of the RRR ER retention motif with three alanines is sufficient for a slight release of the full-length NR1-1 subunits to the cell surface of HEK-293 cells (17), which is not in agreement with our results. A similar mechanism of ER retention employing multiple ER retention motifs has been proposed for the kainate receptor subunit KA2 (18). The phosphorylation of serine residues adjacent to the RRR ER retention motif can suppress ER retention of single transmembrane chimeric proteins containing the C terminus of the NR1-1 subunit (6,7,9).…”

Section: Discussionmentioning

confidence: 63%

Different Roles of C-terminal Cassettes in the Trafficking of Full-length NR1 Subunits to the Cell Surface

Hořák

Wenthold

2009

Journal of Biological Chemistry

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N-Methyl-D-aspartate (NMDA) receptors are glutamategated ion channels composed of NR1 and NR2 subunits. When expressed alone, the most prevalent NR1 splice variant and all NR2 subunits are retained in the endoplasmic reticulum (ER), whereas other NR1 splice variants reach the cell surface to varying degrees. Because similar trafficking patterns have been seen for single transmembrane domain chimeric proteins with appended C termini of NMDA receptor subunits, these chimeric proteins have been used as a model for studying the mechanisms underlying the ER retention and surface trafficking of NMDA receptors. Using this approach, an RRR motif in the C1 cassette has been identified as a major ER retention signal present in NR1 subunits, and the surface localization of other NR1 splice variants has been explained by the absence of the C1 cassette or by the presence of a PDZ/coatomer protein complex II-binding domain in the C2 cassette. However, when we tested these conclusions using full-length NR1 constructs, a more complex role of the C-terminal cassettes in the trafficking of NR1 subunits emerged. Our experiments showed that two independent ER retention motifs in the C1 cassette, KKK and RRR, are the signals mediating ER retention of the full-length NR1 subunits and that the C2 cassette has an additional inhibitory effect on the forward trafficking of NR1 subunits. On the other hand, C0 and C2 cassettes had an enhancing effect on the trafficking of NR1 subunits to the cell surface. Our observations identify the unique roles of C-terminal cassettes in the trafficking of fulllength NR1 subunits. N-Methyl-D-aspartate (NMDA)2 receptors are glutamategated ion channels that play a central role in excitatory synaptic transmission as well as excitotoxicity (1, 2). The functional NMDA receptors are composed of NR1, NR2, and/or NR3 subunits. The NR1 subunit exists in eight different splice variants derived from a single gene, whereas NR2 subunits are products of four different genes, NR2A-D, and NR3 subunits are products of two different genes, NR3A and NR3B. All subunits have the same transmembrane topology with four membrane regions (M1-4), two extracellular regions, and an intracellular C-terminal region. Recent evidence suggests that abnormal surface expression and synaptic targeting of NMDA receptors play fundamental roles in the development of many disorders, including Alzheimer disease, Parkinson disease, and schizophrenia (1, 2). Although much is known about the biophysical properties of NMDA receptors, the mechanisms underlying their trafficking to the cell surface remain poorly understood.Previous studies have shown that full-length NR1-1 and NR2 subunits are retained intracellularly when expressed by themselves in heterologous cells, whereas other NR1 splice variants reach the cell surface (3-5). Similar results have been obtained using single transmembrane domain chimeras with the appended C termini of NMDA receptor subunits, and thus, these chimeras have been used as a model for studying the mechanisms governing ...

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Section: Discussionmentioning

confidence: 63%

Different Roles of C-terminal Cassettes in the Trafficking of Full-length NR1 Subunits to the Cell Surface

Hořák

Wenthold

2009

Journal of Biological Chemistry

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“…Previous studies showed that after cotransfection of GluR6 and KA2 in heterologous systems, the amount of KA2 subunit expressed at the cell membrane surface critically depends on the GluR6:KA2 cDNA ratio (Nasu-Nishimura et al, 2006). Glutamate pulses (1 mM, 100 ms) delivered to cells transfected with GluR6 elicited current responses of large amplitude (1650 Ϯ 65 pA, at a holding potential of Ϫ40 mV; n ϭ 25) (Fig.…”

Section: Resultsmentioning

confidence: 88%

“…GluR6 and GluR6/KA2 receptors expressed in HEK293 cells mediate currents with distinct I-V relationships Cotransfection of GluR6 and KA2 cDNAs is expected to yield a mixture of homomeric GluR6 and heteromeric GluR6/KA2 [because KA2 subunits do not form functional homomers (Herb et al, 1992;Ren et al, 2003;Nasu-Nishimura et al, 2006)]. Previous studies showed that after cotransfection of GluR6 and KA2 in heterologous systems, the amount of KA2 subunit expressed at the cell membrane surface critically depends on the GluR6:KA2 cDNA ratio (Nasu-Nishimura et al, 2006).…”

Section: Resultsmentioning

confidence: 99%

GluR6/KA2 Kainate Receptors Mediate Slow-Deactivating Currents

Barberis

Sachidhanandam²,

Mulle³

2008

Journal of Neuroscience

126

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Kainate receptors (KARs) are ionotropic glutamate receptors contributing to EPSCs with a slow-decaying component that is likely essential for synaptic integration. The slow kinetics of KAR-EPSCs markedly contrasts with the fast kinetics reported for recombinant KARs expressed in heterologous systems, for reasons that remain unexplained. Here we have studied the properties of recombinant heteromeric GluR6/KA2 receptors, which compose synaptic KARs. We report that, in response to brief glutamate applications, currents mediated by recombinant GluR6/KA2 receptors, but not GluR6 receptors, decay with a time course similar to KAR-EPSCs. Model simulations suggest that, after brief agonist exposures, GluR6/KA2 currents undergo slow deactivation caused by the stabilization of partially bound open states. We propose, therefore, that the GluR6/KA2 gating features could contribute to the slow KAR-EPSC decay kinetics.

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“…Surface Biotinylation Assays-Biotinylation assays of cell surface proteins were performed as described (17). siRNAtransfected cells were washed 3 times with ice-cold phosphate-buffered saline (PBS) containing 1 mM CaCl 2 and 0.5 mM MgCl 2 (PBS(ϩ)) and incubated with 0.25 mg/ml EZ-Link Sulfo-NHS-LC-Biotin (Pierce) in PBS(ϩ) for 20 min at 4°C with gentle agitation.…”

Section: Identification Of Px-rics-interactingmentioning

confidence: 99%

The PX-RICS-14-3-3ζ/θ Complex Couples N-cadherin-β-Catenin with Dynein-Dynactin to Mediate Its Export from the Endoplasmic Reticulum

Nakamura

Hayashi

Mimori-Kiyosue³

et al. 2010

Journal of Biological Chemistry

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We have recently shown that ␤-catenin-facilitated export of cadherins from the endoplasmic reticulum requires PX-RICS, a ␤-catenin-interacting GTPase-activating protein for Cdc42. Here we show that PX-RICS interacts with isoforms of 14-3-3 and couples the N-cadherin-␤-catenin complex to the microtubule-based molecular motor dynein-dynactin. Similar to knockdown of PX-RICS, knockdown of either 14-3-3 or -resulted in the disappearance of N-cadherin and ␤-catenin from the cellcell boundaries. Furthermore, we found that PX-RICS and 14-3-3/ are present in a large multiprotein complex that contains dynein-dynactin components as well as N-cadherin and ␤-catenin. Both RNAi-and dynamitin-mediated inhibition of dyneindynactin function also led to the absence of N-cadherin and ␤-catenin at the cell-cell contact sites. Our results suggest that the PX-RICS-14-3-3/ complex links the N-cadherin-␤-catenin cargo with the dynein-dynactin motor and thereby mediates its endoplasmic reticulum export.In general, cargo proteins to be exported from the endoplasmic reticulum (ER) 2 have characteristic amino acid sequences called "ER export motifs" that facilitate their ER exit (1-4). Classic cadherins (simply referred to cadherins hereafter) (5-9), however, are known to have no functional ER export motifs, and their efficient ER exit requires complex formation with ␤-catenin at the ER immediately after cadherin synthesis (10 -12). We have recently shown that this ␤-catenin-facilitated ER export of cadherins requires PX-RICS, a ␤-catenininteracting GTPase-activating protein (GAP) for Cdc42 (13).PX-RICS is an alternatively spliced isoform of RICS (RhoGAP involved in the ␤-catenin-N-cadherin and N-methyl-D-aspartate receptor signaling) and contains phox homology (PX) and Src homology 3 domains in its N-terminal region (14,15). RICS is expressed predominantly in neurons of the brain and localized to the growth cone and postsynaptic density, where it regulates neurite extension and presumably N-methyl-D-aspartate signaling (14). In contrast, PX-RICS is expressed in a wide variety of tissues and cell lines and localized to the ER and cis-Golgi (13, 15). Our recent study has revealed that PX-RICS facilitates ER-to-Golgi transport of the N-cadherin-␤-catenin complex through its direct interaction with ␤-catenin, Cdc42, ␥-aminobutyric acid type A receptor-associated protein (GABARAP) and phosphatidylinositol 4-phosphate (PI4P) (13). This finding suggests that PX-RICS is a key molecule in a novel intracellular transport system that is independent of known ER export motifs and provides a molecular basis that explains why the assembly of cadherins with ␤-catenin is essential for efficient ER exit of cadherins. However, the precise molecular mechanisms by which PX-RICS triggers ER exit of the N-cadherin-␤-catenin complex remain to be elucidated. To address this issue, we attempted to identify PX-RICS-interacting scaffold proteins involved in the PX-RICS-dependent forward transport mechanism. Here we report that PX-RICS and its novel binding partne...

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

Cited by 58 publications

References 23 publications

Different Roles of C-terminal Cassettes in the Trafficking of Full-length NR1 Subunits to the Cell Surface

Different Roles of C-terminal Cassettes in the Trafficking of Full-length NR1 Subunits to the Cell Surface

GluR6/KA2 Kainate Receptors Mediate Slow-Deactivating Currents

The PX-RICS-14-3-3ζ/θ Complex Couples N-cadherin-β-Catenin with Dynein-Dynactin to Mediate Its Export from the Endoplasmic Reticulum

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