Appropriate NR1-NR1 Disulfide-linked Homodimer Formation Is Requisite for Efficient Expression of Functional, Cell Surface N-Methyl-D-aspartate NR1/NR2 Receptors

Papadakis, Michalis; Hawkins, Lynda M.; Stephenson, F. Anne

doi:10.1074/jbc.m313446200

Cited by 62 publications

(69 citation statements)

References 38 publications

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“…In an additional control, HEK293 cells were transfected with either NR1͞NR2A or NR1͞ NR2B subunit cDNAs, and cell surface ELISAs were carried out, as described in ref. 21 by using either anti-NR2A (44-58) or anti-NR2B (42-60) antibodies ( Fig. 1 e and f ).…”

Section: Resultsmentioning

confidence: 99%

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NMDA receptor surface mobility depends on NR2A-2B subunits

Groc

Heine

Cousins

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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The NR2 subunit composition of NMDA receptors (NMDARs) varies during development, and this change is important in NMDARdependent signaling. In particular, synaptic NMDAR switch from containing mostly NR2B subunit to a mixture of NR2B and NR2A subunits. The pathways by which neurons differentially traffic NR2A-and NR2B-containing NMDARs are poorly understood. Using single-particle and -molecule approaches and specific antibodies directed against NR2A and NR2B extracellular epitopes, we investigated the surface mobility of native NR2A and NR2B subunits at the surface of cultured neurons. The surface mobility of NMDARs depends on the NR2 subunit subtype, with NR2A-containing NMDARs being more stable than NR2B-containing ones, and NR2A subunit overexpression stabilizes surface NR2B-containing NMDARs. The developmental change in the synaptic surface content of NR2A and NR2B subunits was correlated with a developmental change in the time spent by the subunits within synapses. This suggests that the switch in synaptic NMDAR subtypes depends on the regulation of the receptor surface trafficking.development ͉ glutamate receptor ͉ lateral mobility N MDA receptors (NMDARs) are heterotetrameric cation channels composed of NR1 and NR2͞3 subunits (1). NMDARs are assembled early in the endoplasmic reticulum, and both NR1 and NR2 subunits are necessary for their association and their successful cell surface targeting (2). In addition to glutamate and glycine, NMDARs require membrane depolarization to open with high probability (3), making this receptor a pre-and postsynaptic activity coincident detector involved in the induction of Hebbian synaptic plasticity. The functional properties of NMDARs depend also on the subunit composition, and such subunit heterogeneity of synaptic NMDARs is thought to play an important role during synaptic development, maturation, and plasticity processes (4). During synaptic development, the subunit composition of synaptic NMDARs changes from heterodimers containing predominantly NR2B subunits at early stages to heterodimers containing NR1͞NR2B, NR1͞NR2A, and NR1͞NR2A͞NR2B subunits at mature stage (1,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). This change often is associated with the refinement of neuronal connections within cortical areas, although this model has been challenged and, thus, is likely incomplete (4). The pathways by which neurons differentially traffic NR2A-and NR2B-containing NMDARs remain, however, an open question of crucial importance to understand the shaping of synaptic maturation and plasticity.Changes in NR2 subunit composition of NMDARs within synapses can be triggered by mechanisms that include differences in insertion (15), internalization (16, 17), and͞or lateral diffusion. Interestingly, NMDARs diffuse laterally at the neuronal surface (18,19). In immature neurons, synaptic NMDARs are replaced rapidly by extrasynaptic ones through lateral diffusion (18), suggesting that surface mobility of NMDARs may be involved in shaping mature NMDAR synaptic components. In this study, we i...

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Section: Resultsmentioning

confidence: 99%

“…To selectively track surface NR2A-and NR2B-containing NMDARs, polyclonal antibodies directed against extracellular epitopes of NR2A subunit was developed, and a previously described antibody directed against NR2B subunit were used (20,21). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

NMDA receptor surface mobility depends on NR2A-2B subunits

Groc

Heine

Cousins

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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317

290

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“…1, closed circles; Fig. 5, yellow ball-and-stick), as shown for Cys 140 in mGluR1 and as recently proposed for Cys 79 in NMDA receptor subunit NR1 (38). Indeed, mutating either or both of these cysteines to alanine in GluR3 (C85A/C111A) resulted in an almost complete block of activity.…”

Section: Figmentioning

confidence: 99%

“…Finally, it has been also shown that the mGluR1-NTD homodimer is stabilized by a disulfide bond formed by cysteine 140 from each monomer. In NMDA receptors, it was recently proposed that a similar bond may be formed by Cys 79 between two NR1 subunits (38). In AMPA and kainate receptors there are four conserved cysteines, which in GluR3 correspond to Cys 85 , Cys 111 , Cys 215 , and Cys 334 .…”

Section: Computer Modeling Of Glur3-ntd and Glur6-ntd Suggests An Expmentioning

confidence: 99%

Two Regions in the N-terminal Domain of Ionotropic Glutamate Receptor 3 Form the Subunit Oligomerization Interfaces That Control Subtype-specific Receptor Assembly

Ayalon¹,

Segev²,

Elgavish

et al. 2005

Journal of Biological Chemistry

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The N-terminal domain (NTD) of ␣-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) and kainate glutamate receptors plays an important role in controlling subtype specific receptor assembly. To identify NTD subdomains involved in this process we generated AMPA glutamate receptor 3 (GluR3) mutants having intra-NTD substitutions with the corresponding regions of the kainate receptor GluR6 and tested their ability to form functional heteromers with wild-type subunits. The chimeric design was based on the homology of the NTD to the NTD of the metabotropic GluR1, shown to form two globular lobes and to assemble in dimers. Accordingly, the NTD was divided into four regions, termed here N1-N4, of which N1 and N3 correspond to the regions forming lobe-1 and N2 and N4 to those forming lobe-2. Substituting N1 or N3 impaired functional heteromerization but allowed protein-protein interactions. Conversely, exchanging N2 or N4 preserved functional heteromerization, although it significantly decreased homomeric activity, indicating a role in subunit folding. Moreover, a deletion in GluR3 corresponding to the hotfoot mouse mutation of the glutamate receptor ␦2, covering part of N2, N3, and N4, impaired both homomeric and heteromeric oligomerization, thus explaining the null-like mouse phenotype. Finally, computer modeling suggested that the dimer interface, largely formed by N1, is highly hydrophobic in GluR3, whereas in GluR6 it contains electrostatic interactions, hence offering an explanation for the subtype assembly specificity conferred by this region. N3, however, is positioned perpendicular to the dimer interface and therefore may be involved in secondary interactions between dimers in the assembled tetrameric receptor.Glutamate, the major excitatory neurotransmitter, activates two receptor families: metabotropic glutamate receptors (mGluRs), 1 which are coupled to G-proteins, and ionotropic glutamate receptors (iGluRs), which form cation selective ion channels. The iGluRs are further divided into three subtypes: AMPA, kainate, and NMDA receptors, originally named after their most selective agonist (1). Each subtype, in turn, consists of several subunits that assemble as tetramers in various combinations to form channels with distinct properties (2). Of the cloned subunits, GluR1-4 form the AMPA receptors, GluR5-7 and KA1-2 form the kainate receptors, and NR1, NR2A-D, and NR3A-B form the NMDA receptors. The ␦1-2 subunits constitute an orphan group of iGluRs, and their relation to the three principal subtypes is unclear.Overall, all iGluR subunits have a common domain organization (3). Each subunit contains four membrane regions (M1-M4), of which M1, M3, and M4 traverse the plasma membrane and M2 is a reentrant loop aligning the channel pore. The N terminus is extracellular and comprises an ϳ400-residue Nterminal domain (NTD; also termed "X domain"), which is homologous to bacterial periplasmic amino acid-binding proteins and to the N-terminal domain of mGluRs (4). Following the NTD is S1, an ϳ150-residue segment, which ...

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“…Previous studies have shown that homodimer formation may be the first step in the assembly process of the NMDA receptor complex, because dimers of NR1 can be detected as intermediates in this process (32)(33). However, the importance of homodimer formation in the processing of the NR2 subunit remains unclear.…”

Section: Discussionmentioning

confidence: 99%

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

Zheng

Song

et al. 2007

Journal of Biological Chemistry

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The cytoplasmic C-terminal domains of NR2 subunits have been proposed to modulate the assembly and trafficking of NMDA receptors. However, questions remain concerning which domains in the C terminus of NR2 subunits control the assembly of receptor complexes and how the assembled complexes are selectively trafficked through the various cellular compartments such as endoplasmic reticulum (ER) to the cell surface. In the present study, we found that the three amino acid tail after the TM4 region of NR2 subunits is necessary for surface expression of functional NMDA receptors, while truncations with only two amino acids following the TM4 region (NR2⌬2) completely eliminated surface expression of the NMDA receptor on co-expression with NR1-1a in HEK293 cells. FRET (fluorescence resonance energy transfer) analysis showed that these NR2⌬2 truncations are able to form homomers and heteromers on co-expression with NR1-1a. Furthermore, when NR2⌬2 subunits were cotransfected with either the NR1-4a or NR1-1a AAA mutant, lacking the ER retention motif (RRR), functional NMDA receptors were detected in the transfected HEK293 cells. Unexpectedly, we found that the replacement of five residues after TM4 with alanines gave results indistinguishable from those of NR2B⌬5 (EHLFY), demonstrating the short tail following the TM4 of NR2 subunits is not sequence-specific-dependent. Taken together, our results show that the C terminus of the NR2 subunits is not necessary for the assembly of NMDA receptor complexes, whereas a three amino acid long cytoplasmic tail following the TM4 of NR2 subunits is sufficient to overcome the ER retention existing in the C terminus of NR1, allowing the assembled NMDA receptors to reach the cell surface. N-methyl-D-aspartate (NMDA)3 receptors are heteromeric complexes primarily assembled from two subunit classes: NR1 and NR2. Co-assembly of NR1 and NR2 subunits is essential for formation of a functional channel, presumed to be a tetramer containing two NR1 and two NR2 subunits (1-2). NR1 is a single subunit with eight splicing variants, which have distinct trafficking and functional properties (3). NR2 subunits are coded by four separate genes, NR2A-D, each of which can endow the receptor channel with different properties (4). Thus, the subunit composition of NMDA receptors is a major determinant of NMDA receptor-mediated activity in the central nervous system. Although much is known about the physiological roles that NMDARs play in long term potentiation (LTP), learning and memory (5-7), much remains to be learned about the mechanisms by which these receptors are assembled, sorted, targeted, and anchored to the appropriate location.The C-terminal domains of the receptor subunits contain critical determinants of subcellular receptor localization. Regulation of receptor trafficking by these determinants ensures that only fully assembled multimeric receptors are expressed on the plasma membrane. When expressed alone in heterogeneous cells, the major NR1 isoform (NR1-1) is retained in the ER because of an RR...

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Appropriate NR1-NR1 Disulfide-linked Homodimer Formation Is Requisite for Efficient Expression of Functional, Cell Surface N-Methyl-D-aspartate NR1/NR2 Receptors

Cited by 62 publications

References 38 publications

NMDA receptor surface mobility depends on NR2A-2B subunits

NMDA receptor surface mobility depends on NR2A-2B subunits

Two Regions in the N-terminal Domain of Ionotropic Glutamate Receptor 3 Form the Subunit Oligomerization Interfaces That Control Subtype-specific Receptor Assembly

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

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