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An ER retention signal explains differences in surface expression of NMDA and AMPA receptor subunits
Abstract
The molecular mechanisms that control the surface expression of NMDA receptors (NMDARs) and AMPA receptors (AMPARs) are unknown. To determine the role of the intracellular C-terminal tails of glutamate receptor subunits in the synaptic targeting of AMPARs and NMDARs, we fused the tails of the AMPAR subunits, GluR1 and GluR2, and the NMDAR subunit, NR1, to the human T lymphocyte membrane protein CD8 and expressed these constructs in HEK293 cells and cultured hippocampal neurons. The GluR1 and GluR2 fusion proteins exhibited robust surface expression in the plasma membrane of neurons at synapses as did CD8 alone. In contrast, the NR1 fusion protein was retained intracellularly in both HEK293 cells and neurons because of the presence of an ER retention signal in the C1 cassette. This ER retention signal was overridden either by the addition of a PDZ domain-binding motif or by mimicking phosphorylation at a site adjacent to the retention signal. These results provide further evidence that the intracellular trafficking of AMPAR and NMDAR subunits are regulated independently at least in part because of differences in the protein-protein interactions of their intracellular C-terminal tails.
... where X is any amino acid, has been shown to be responsible for the intracellular retention of many proteins. For example, surface expression of NMDA (N-methyl D-aspartate ) receptors was severly hampered due to the intracellular retention induced by this RXR motif [187][188][189][190] . Moreover, mutation of the RXR motifs within the ER-retained mutant cystic fibrosis transmembrane conductance regulator (∆508 CFTR) evoked its biochemical maturation and delivery to the plasma membrane 191 . ...
... It has been previously shown that ER retention mediated by the RXR motif can be overridden by phosphorylation of a neighbouring residue. For example, in the glutamate receptor, activity of the ER retention motif was overridden by the phosphorylation of an adjacent serine residue 190 . Moreover, activation of protein kinase C enhances the cell surface expression of the N-methyl-D-aspartate receptors (NMDA) 205 . ...
... Evidence also shows that in the glutamate receptor, activity of the retention motif was overridden by the phosphorylation of an adjacent serine residue 190 Whether this S447 may indeed be phosphorylated under certain conditions (like cold exposure) is not known. What we now know and report herein, however, is that 2C-AR is indeed a phosphoprotein and that cooling could increase its phosphorylation level. ...
... Different splice varients of NRl appeared to be preferentially trafficked to the cell surface, which appeared to be determined by the presence or absence of exon 21 in the C terminus. The splice variants containing exon 21 were held within the ER due to the presence of an ER retention signal, whilst the absence of exon 21 were trafficked to the surface membrane (Standley et a l, 2000;Xia et al., 2001). This retention signal could be overcome by the addition of a PDZ domain -binding motif contained in exon 22 (Standley et al., 2000;Xia et al., 2001). ...
... The splice variants containing exon 21 were held within the ER due to the presence of an ER retention signal, whilst the absence of exon 21 were trafficked to the surface membrane (Standley et a l, 2000;Xia et al., 2001). This retention signal could be overcome by the addition of a PDZ domain -binding motif contained in exon 22 (Standley et al., 2000;Xia et al., 2001). Despite this all N Rl splice variants can form functional surface receptors in Xenopus oocytes and ER retention is not required for assembly (Okabe et al., 1999). ...
The aim of this thesis is to examine the role of the N-methyl-D-aspartate (NMDA) receptor complex in the development of spinal nociception within the spinal cord of the rat. In particular the dorsal horn is crucial in the transmission and modulation of nociceptive inputs and undergoes extensive postnatal maturation. In vivo electrophysiological responses to NMDA antagonists or nitric oxide synthase (NOS) inhibitors together with in vitro immunohistochemical assessment of postnatal receptor distribution were used to assess alterations in the NMDA receptor. Immunocytochemical staining of the whole spinal cord revealed widespread NR1 subunit expression from birth, whilst NR2 subunits underwent most postnatal alteration. NR2B was the predominant subunit at birth and underwent restriction to the adult distribution in lamina II, whilst the NR2A subunit expression was low at birth and became the predominant subunit in the adult. The DRG revealed widespread expression of all the NMDA subunits from P14. In the dissociated cell culture the NR1 positive neurones in the dorsal horn increased from 25-100% with an increase in staining intensity in contrast with the DRG where all the neurones remained NR1 positive albeit with a reduction in staining intensity. These alterations in expression of the NMDA receptor subunits could account for the difference in efficacy of spinally applied 2-amino-5 phophonopentoic acid (AP5) an NMDA receptor antagonist on the responses of convergent dorsal horn neurones. AP5 was significantly more potent in pups compared with adults whilst ketamine, which binds equally to all the NR2 subunits displayed no age related alteration in efficacy. The application of 7NI (a specific nNOS inhibitor) and L-NAME (non-specific NOS inhibitor) revealed no age differences in post-synaptic evoked responses, but displayed a significantly greater inhibition of the primary evoked response at postnatal day (P) 14 compared with P21, 28 or adult. The NMDA receptor has been shown to alter its subunit composition and receptor expression on primary afferents and post-synaptic dorsal horn neurones during development. This alteration may explain the developmental differences seen in convergent dorsal horn neurones to NMDA receptor anatagonist. However in contrast the role nNOS / NO pathway in nociception does not seem to be altered during development.
... This ER retention motif was found to be regulated by phosphorylation of serine residues found adjacent to the RRR motif. Phosphomimetic mutations of S896E and S897E overcame the ER retention caused by the NRl C l cassette (Scott et a l, 2001;Xia et al, 2001). Although N R l-la ,b and NRl-3a,b subunits both contain the NRl Cl cassette, NRl-3a,b subunits were expressed on the cell surface whilst N R l-la,b subunits were not. ...
... The ER retention caused by the N R l Cl cassette was found to be overcome if the N R l C2' cassette was present in the same C-terminal domain (Scott et a l, 2001;Xia et a l, 2001). The last five amino acids present on the C2' cassette, VSTVV, were found to be vital to promote the release o f the ER retention (Scott et a l, 2001;Xia et al., 2001). It was hypothesised that the association of NRl iso forms containing the N R l C2' cassette with a post-synaptic density-95 (PSD-95), drosophila discs-large (dig) and zonal occludence 1 (ZO-1) (PDZ) domain-containing protein masked the ER retention signal found on the NRl Cl cassette, (Standley et a l, 2000). ...
N-Methyl-D-aspartate (NMDA) receptors are a subclass of excitatory ionotropic glutamate neurotransmitter receptors. There are four major subclasses of NMDA receptors formed by the co-assembly of different NMDA receptor subunits, i.e. NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D. These subclasses have distinct physiological properties and distinct temporal and spatial patterns of distribution in neurones. NMDA receptors are trafficked to dendritic spines in neurons where they are thought to be anchored in the post- synaptic membrane by their association with the postsynaptic density-95 (PSD-95) membrane associated guanylate kinase (MAGUK) family of scaffolding proteins. PSD-95 is the prototypic member of this family which also includes channel associated protein of synapse-110 (Chapsyn-110), synapse associated protein97 (SAP97) and SAP 102. The association between PSD-95 and the NR1/NR2A and NR1/NR2B receptor subtypes has been well characterized and shown to be important for the regulation of receptor cell surface expression. However there is a paucity of knowledge with regard to the association between NR1/NR2C and NR1/NR2D subtypes and PSD-95 and also all four major NMDA receptor subclasses and the other members of the PSD-95 MAGUK family. The aim of this thesis therefore was to investigate the association between the PSD-95 MAGUK proteins and the four major NMDA receptor subclasses and the effects of this association on NMDA receptor expression and cell surface trafficking. Each major NMDA receptor subclass was co-expressed with PSD-95 MAGUK family members in human embryonic kidney (HEK) 293 cells and the expressed proteins analysed by immunoprecipitation assays, quantitative immunoblotting and cell surface ELISA assays. In addition, the association between NR1/NR2A NMDA receptors and PSD-95 was studied in more detail which included the mapping of a new PSD-95 binding motif in the NR2A C-terminal domain and a study of the role of protein kinase A phosphorylation in receptor/scaffold association and cell surface trafficking. Finally, a yeast two-hybrid screen was conducted to identify novel NR1/NR2D NMDA receptor associated proteins. Differential association with the PSD-95 MAGUK family and NMDA receptors was discovered. This may be important for the regulation of cell surface receptor number, the stabilisation, clustering, turnover and compartmentalisation of NMDA receptor subtypes in neurons during development and in the mature brain.
... This construct contains the extracellular domain of CD8 fused to fibrocystin just prior to its membrane spanning domain and is predicted to have the same membrane topology as native fibrocystin. CD8 is a well characterized membrane protein often used in chimerics to identify targeting domains (Xia et al., 2001). ...
... The construct (pJAF270) used for SNAP trafficking assays was constructed by fusing the extracellular domain of mouse CD8a (Xia et al., 2001) and alanine scanning mutants used in (Follit et al., 2010), CD8-PKHD1 (pJAF268), or CD8-Megalin (pJAF281) and 48 hours later, FLAG immunoprecipitation was carried out as described in . ...
Protruding from the apical surface of nearly every cell in our body lies a specialized sensory organelle—the primary cilium. Eukaryotic cells use these ubiquitous structures to monitor the extracellular environment, defects in which result in an ever-growing list of human maladies termed ciliopathies including obesity, retinal degeneration and polycystic kidney disease. The sensory functions of primary cilia rely on the unique complement of receptors concentrated within the ciliary membrane. Vital to the proper functioning of the cilium is the cell's ability to target specific proteins to the ciliary membrane yet little is known how a cell achieves this highly polarized distribution. IFT20, a subunit of the intraflagellar transport particle is localized to the Golgi complex that is hypothesized to sort proteins to the ciliary membrane. We show that IFT20 is anchored to the Golgi complex by the golgin protein GMAP-210 and mice lacking GMAP210 die at birth with a pleiotropic phenotype that includes growth restriction and heart defects. Cilia on GMAP210 mutant cells have reduced amounts of the membrane protein polycystin-2 localized to them suggesting IFT20 and GMAP-210 function together in the sorting or transport of proteins to the ciliary membrane. To better understand the mechanism of ciliary protein trafficking, we identify a ciliary targeting sequence (CTS) contained within fibrocystin, the gene mutated in autosomal recessive polycystic kidney disease, and investigate a series of proteins required for the delivery of this sequence to the primary cilium. We demonstrate the small G protein Rab8 interacts with the CTS of fibrocystin and controls the ciliary levels of the CTS. Arf4 is another small G protein deemed a key regulator of ciliary protein trafficking. We show Arf4 binds the CTS of fibrocystin but is not absolutely required for trafficking of the fibrocystin CTS to cilia. Arf4 mutant mice are embryonic lethal and die at mid-gestation likely due to defects in the non-ciliated visceral endoderm, where the lack of Arf4 caused defects in cell structure and apical protein localization. This suggests Arf4 is not only important for the efficient transport of fibrocystin to cilia, but also plays critical roles in non-ciliary processes. Together this work aims to elucidate the mechanisms of protein targeting to the ciliary membrane.
... GluN1-3 has an ER retention signal, a PDZ-binding domain [33,37,40], and a di-valine ER-exit motif nested in the PDZ-binding domain [23]. While there is an agreement that the extreme C-terminus blocks the ER retention motif, the mechanism has been unclear. ...
... The use of chimeric reporter constructs of the NMDA receptor C-terminus has already contributed substantially to our understanding of NMDA receptor trafficking [33,37,40]. By using chimeras of VE and the NMDA receptor C-terminus, SAP97, a protein known to interact with the GluN1-3 PDZ-binding domain, was identified as a PDZ protein that blocks ER retention. ...
Keyword: SAP97 NMDA Receptor Trafficking PDZ domain SH3 domain RXR motif SAP97 is directly involved in exporting NMDA receptors with a specific subunit composition from the endoplasmic reticulum (ER). Characterization of the interactions between SAP97 and an NMDA receptor splice variant, GluN1-3, and of the effects on forward trafficking revealed that an ER-level interaction blocked the RXR ER-retention motif in the GluN1-3 cytoplasmic C-terminus in the context of both reporter molecules and full-length receptors. Binding of SAP97 to the PDZ-binding domain of GluN1-3 was required, but the blockade of ER-retention was mediated by the SH3–GuK domains coupled with the action of the N-terminus of SAP97. While other domains of SAP97 were involved in forward trafficking of GluN1-3 out of the ER, the SH3 domain was necessary and sufficient to block the ER retention. This is the first direct evidence for the masking of ER-retention signals by PDZ domain-containing proteins, and provides detailed underlying mechanistic requirements. Such a mechanism could be central to modulating the ER exit of receptors into local, non-conventional or conventional, secretory pathways in neurons.
... Previous studies identified other regions in GluN subunits besides LBDs, including ATD (Qiu et al., 2009), M3 domains (Horak et al., 2008), and CTDs (Standley et al., 2000;Scott et al., 2001;Xia et al., 2001;Horak and Wenthold, 2009), containing ER retention and export signals. Therefore, we anticipate that future studies will investigate at what level other ER retention and export signals contribute to regulating the surface expression of the GluN1/GluN2 receptors with mutated LBDs. ...
The essential role of N-methyl-D-aspartate receptors (NMDARs) in excitatory neurotransmission is underscored by numerous pathogenic variants in the GluN subunits, including those identified in their ligand-binding domains (LBDs). The prevailing hypothesis postulates that the endoplasmic reticulum (ER) quality control machinery verifies the agonist occupancy of NMDARs; however, whether it controls the structure of LBDs or the functionality of NMDARs is unknown. Using alanine substitutions combined with microscopy and electrophysiology, we found that surface expression of GluN1/GluN2A receptors, the primary NMDAR subtype in the adult forebrain, strongly correlates with EC50 values for glycine and Lglutamate. Interestingly, co-expression of both GluN1 and GluN2A subunits with alanine substitutions led
to an additive reduction in the surface number of GluN1/GluN2A receptors, as did co-expression of both GluN1 and GluN2A subunits containing closed cleft conformation of LBDs. The synchronized ER release confirmed the altered regulation of early trafficking of GluN1/GluN2A receptors bearing alanine substitutions in the LBDs. Furthermore, the human versions of GluN1/GluN2A receptors containing
pathogenic GluN1-S688Y, GluN1-S688P, GluN1-D732E, GluN2A-S511L, and GluN2A-T690M variants exhibited distinct surface expression compared to the corresponding alanine substitutions. Mutant cycles of GluN1-S688, GluN1-D732, GluN2A-S511, and GluN2A-T690 residues revealed, in most cases, a weak correlation between surface expression of the mutant GluN1/GluN2A receptors and their EC50 values for
glycine or L-glutamate. Consistent with our experimental data, molecular modeling and dynamics showed that the ER quality control machinery likely perceives structural changes of the LBDs but not the functionality of GluN1/GluN2A receptors.
... In the hippocampus, from the prenatal period onward, functional heterotetrameric (GluN1/GluN2) NMDARs must contain GluN2B subunits [31][32][33][34]. Moreover, a lack of GluN2B subunits affects synaptogenesis and neonatal survival [35,36]. ...
Synaptic transmission is a dynamic process that requires precise regulation. Early in life, we must be able to forge appropriate connections (add and remove) to control our behavior. Neurons must recognize appropriate targets, and external soluble factors that activate specific signaling cascades provide the regulation needed to achieve this goal. Wnt signaling has been implicated in several forms of synaptic plasticity, including functional and structural changes associated with brain development. The analysis of synapses from an electrophysiological perspective allows us to characterize the functional role of cellular signaling pathways involved in brain development. The application of quantal theory to principles of developmental plasticity offers the possibility of dissecting the function of structural changes associated with the birth of new synapses as well as the maturation of immature silent synapses. Here, we focus on electrophysiological and molecular evidence that the Wnt signaling pathway regulates glutamatergic synaptic transmission, specifically N-methyl-d-aspartate receptors (NMDARs), to control the birth of new synapses. We also focus on the role of Wnts in the conversion of silent synapses into functional synapses.
... These signals must be masked for receptors to be trafficked further in the secretory pathway. This can occur through oligomerization that masks the signal, phosphorylation of key residues, or interactions with trafficking factors, such as PDZ domain-containing proteins (Xia et al., 2001). ...
N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated cation channels that mediate excitatory neurotransmission and are critical for synaptic development and plasticity in the mammalian central nervous system (CNS). Functional NMDARs typically form via the heterotetrameric assembly of GluN1 and GluN2 subunits. Variants within GRIN genes are implicated in various neurodevelopmental and neuropsychiatric disorders. Due to the significance of NMDAR subunit composition for regional and developmental signaling at synapses, properly folded receptors must reach the plasma membrane for their function. This review focuses on the protein quality control of NMDARs. Specifically, we review the quality control mechanisms that ensure receptors are correctly folded and assembled within the endoplasmic reticulum (ER) and trafficked to the plasma membrane. Further, we discuss disease-associated variants that have shown disrupted NMDAR surface expression and function. Finally, we discuss potential targeted pharmacological and therapeutic approaches to ameliorate disease phenotypes by enhancing the expression and surface trafficking of subunits harboring disease-associated variants, thereby increasing their incorporation into functional receptors.
... The assembly status is thus coupled to forward transport in a quality control process resembling "hide and run" (Michelsen et al., 2005). This process referred originally to Arg-based ER motifs in polytopic membrane proteins that are part of functional heteromultimeric complexes, including, for instance, the ATPsensitive K + channel (Zerangue et al., 1999), γ-aminobutyric acid B receptor (Margeta-Mitrovic et al., 2000), and glutamate receptors (Scott et al., 2003;Xia et al., 2001). Given the broad range of substrates and associated pathways controlled by γ-secretase, tuning and regulating its assembly after ER exit but before entrance into the Golgi provides a spatiotemporal mechanism to regulate overall intramembrane proteolysis by γ-secretase. ...
γ-Secretase affects many physiological processes through targeting >100 substrates; malfunctioning links γ-secretase to cancer and Alzheimer's disease. The spatiotemporal regulation of its stoichiometric assembly remains unresolved. Fractionation, biochemical assays, and imaging support prior formation of stable dimers in the ER, which, after ER exit, assemble into full complexes. In vitro ER budding shows that none of the subunits is required for the exit of others. However, knockout of any subunit leads to the accumulation of incomplete subcomplexes in COPII vesicles. Mutating a DPE motif in presenilin 1 (PSEN1) abrogates ER exit of PSEN1 and PEN-2 but not nicastrin. We explain this by the preferential sorting of PSEN1 and nicastrin through Sec24A and Sec24C/D, respectively, arguing against full assembly before ER exit. Thus, dimeric subcomplexes aided by Sec24 paralog selectivity support a stepwise assembly of γ-secretase, controlling final levels in post-Golgi compartments.
... The LARGE protein is a resident of the Golgi apparatus and retained AMPA receptors in their delivery to the plasma membrane [64]. AMPAR and NMDAR subunits interact with scaffold proteins such as SAP102 through the known PSD95/Dlg1/ZO-1 (PDZ) domain in the ER, and this is required for the subunit to leave the ER [58,[65][66][67]. This is consistent with the distribution of SAP102, which, in addition to being present at the synaptic zone similar to PSD-95, is also distributed throughout the cytoplasm in which it may interact with various organelles, including vesicles destined to the postsynaptic membrane [68,69]. ...
Cells comprise several intracellular membrane compartments that allow them to function properly. One of these functions is cargo movement, typically proteins and membranes within cells. These cargoes ride microtubules through vesicles from Golgi and recycling endosomes to the plasma membrane in order to be delivered and exocytosed. In neurons, synaptic functions employ this cargo trafficking to maintain inter-neuronal communication optimally. One of the complexes that oversee vesicle trafficking and tethering is the exocyst. The exocyst is a protein complex containing eight subunits first identified in yeast and then characterized in multicellular organisms. This complex is related to several cellular processes, including cellular growth, division, migration, and morphogenesis, among others. It has been associated with glutamatergic receptor trafficking and tethering into the synapse, providing the molecular machinery to deliver receptor-containing vesicles into the plasma membrane in a constitutive manner. In this review, we discuss the evidence so far published regarding receptor trafficking and the exocyst complex in both basal and stimulated levels, comparing constitutive trafficking and long-term potentiation-related trafficking.
... Immunofluorescent assays demonstrated low expression levels of NR1 in the cytoplasm and even lower levels on the membranes of untreated HK-2 cells. The cytoplasmic localization of NR1 is associated with its production in the endoplasmic reticulum and maturation, while membrane anchoring is associated with the maturation and function of NMDARs [41]. TGF-β increases the expression and localization of NR1 both in the cytoplasm and on the membrane. ...
Background:
This study aimed to understand the mechanistic role of N-methyl-D-aspartate receptor (NMDAR) in acute fibrogenesis using models of in vivo ureter obstruction and in vitro TGF-β administration.
Methods:
Acute renal fibrosis (RF) was induced in mice by unilateral ureteral obstruction (UUO). Histological changes were observed using Masson's trichrome staining. The expression levels of NR1, which is the functional subunit of NMDAR, and fibrotic and epithelial-to-mesenchymal transition markers were measured by immunohistochemical and Western blot analysis. HK-2 cells were incubated with TGF-β, and NMDAR antagonist MK-801 and Ca2+/calmodulin-dependent protein kinase II (CaMKII) antagonist KN-93 were administered for pathway determination. Chronic RF was introduced by sublethal ischemia-reperfusion injury in mice, and NMDAR inhibitor dextromethorphan hydrobromide (DXM) was administered orally.
Results:
The expression of NR1 was upregulated in obstructed kidneys, while NR1 knockdown significantly reduced both interstitial volume expansion and the changes in the expression of α-smooth muscle actin, S100A4, fibronectin, COL1A1, Snail, and E-cadherin in acute RF. TGF-β1 treatment increased the elongation phenotype of HK-2 cells and the expression of membrane-located NR1 and phosphorylated CaMKII and extracellular signal-regulated kinase (ERK). MK801 and KN93 reduced CaMKII and ERK phosphorylation levels, while MK801, but not KN93, reduced the membrane NR1 signal. The levels of phosphorylated CaMKII and ERK also increased in kidneys with obstruction but were decreased by NR1 knockdown. The 4-week administration of DXM preserved renal cortex volume in kidneys with moderate ischemic-reperfusion injury.
Conclusions:
NMDAR participates in both acute and chronic renal fibrogenesis potentially via CaMKII-induced ERK activation.
... Immuno uorescent assays demonstrated low expression levels of NR1 in the cytoplasm and even lower levels on the membranes of untreated HK-2 cells. Cytoplasmic localization of NR1 is associated with its production in the endoplasmic reticulum and maturation, while membrane anchoring is associated with maturation and function of NMDARs [41]. TGF-β increases NR1 expression and localization in both the cytoplasm and membrane. ...
Background: Renal fibrosis (RF) results in renal function impairment and eventually kidney failure. We found that N-methyl-D-aspartate receptor (NMDAR) played an important role during RF. However, its mechanism of action is yet to be deciphered.
Methods: Acute RF was induced in mice by unilateral ureteral obstruction (UUO). NR1, which is the functional subunit of NMDAR, was downregulated using lentiviral vector-mediated shRNA interference. Histological changes were observed by Masson’s trichrome staining. Expression of NR1, fibrotic and EMT markers were measured by immunohistochemistry and western blot analysis. HK-2 cells were incubated with TGF-β, and NMDAR antagonist MK-801 and Ca2+/calmodulin-dependent protein kinase II (CaMKII) antagonist KN-93 administration were further included in this study for pathway determination. Expression of NR1, total and phosphorylated CaMKII, total and phosphorylated ERK were measured using western blot and immunofluorescent assays. Chronic renal fibrosis was introduced by sublethal ischemia-reperfusion injury in mice, and oral NMDAR inhibitor dextromethorphan (DXM) administration was performed.
Results: NR1 expressions were upregulated in both obstructed kidneys and TGF-β treated HK-2 cells. NR1 knockdown, MK801 and KN93 reduced the fibrotic morphology in vivo and in vitro respectively, and companied with the downregulated ERK activation, while KN93 administration had no effect on NR1 and CaMKII levels. Mice in the DXM group had better preservation of kidney structures and corticomedullary volumes.
Conclusions: NMDAR participates in both acute and chronic renal fibrogenesis via CaMKII/ERK activation, and is a potential therapeutic target for renal fibrosis.
... Immuno uorescent assays demonstrated low expression levels of NR1 in the cytoplasm and even lower levels on the membranes of untreated HK-2 cells. Cytoplasmic localization of NR1 is associated with its production in the endoplasmic reticulum and maturation, while membrane anchoring is associated with maturation and function of NMDARs [41]. TGF-β increases NR1 expression and localization in both the cytoplasm and membrane. ...
Background: Renal fibrosis (RF) results in renal function impairment and eventually kidney failure. We found that N-methyl-D-aspartate receptor (NMDAR) played an important role during RF. However, its mechanism of action is yet to be deciphered.
Methods: RF was induced in vivo by unilateral ureteral obstruction (UUO) using 8-week-old C57BL/6 mice. The expression levels of the NMDAR’s functional subunit, NR1, was downregulated using lentiviral vector-mediated shRNA interference. Histological changes were observed using Masson’s trichrome staining. Expression of NR1, fibrotic markers (α-smooth muscle actin (α-SMA), type I collagen (COL1A4), S100A4 and fibronectin), and EMT markers (snail and E-cadherin) were measured using immunohistochemistry and western blot analysis. RF was induced after TGF-β-treatment in HK-2 cells in vitro. NMDAR antagonist MK-801 and Ca2+/calmodulin-dependent protein kinase II (CaMKII) antagonist KN-93 were included in this study for pathway determination. Expression of NR1, total and phosphorylation of CaMKII (p-CaMKII), total and p-ERK were measured using western blot and immunofluorescent assays. Results from in vitro studies were confirmed using in vivo studies for NR1, CaMKII and ERK expression levels. In addition, ischemia-reperfusion injury (IRI) mouse model was used to determine whether oral NMDAR inhibitor dextromethorphan (DXM) could inhibit chronic fibrosis.
Results: Increased NR1 expression was observed in both UUO-injured kidneys and TGF-β-treated tubular cells. NR1 knockdown and MK801 administration downregulated CaMKII/ERK activation. In vitro administered CaMKII antagonist KN93 reduced ERK phosphorylation and was not affected by NR1 expression levels. DXM protected IRI-injured kidneys from atrophy and fibrosis.
Conclusions: NMDAR participates in renal fibrogenesis by activating the CaMKII/ERK pathway. NMDAR could be a potential therapeutic target for renal fibrosis.
... Immunofluorescent assays demonstrated low expression levels of NR1 in the cytoplasm and even lower levels on the membranes of untreated HK-2 cells. Cytoplasmic localization of NR1 is associated with its production in the endoplasmic reticulum and maturation, while membrane anchoring is associated with maturation and function of NMDARs [41]. TGF-β increases NR1 expression and localization in both the cytoplasm and membrane. ...
Background: Renal fibrosis (RF) results in renal function impairment and eventually kidney failure. We found that N-methyl-D-aspartate receptor (NMDAR) played an important role during RF. However, its mechanism of action is yet to be deciphered. Methods: Acute RF was induced in mice by unilateral ureteral obstruction (UUO). NR1, which is the functional subunit of NMDAR, was downregulated using lentiviral vector-mediated shRNA interference. Histological changes were observed by Masson’s trichrome staining. Expression of NR1, fibrotic and EMT markers were measured by immunohistochemistry and western blot analysis. HK-2 cells were incubated with TGF-β, and NMDAR antagonist MK-801 and Ca2+/calmodulin-dependent protein kinase II (CaMKII) antagonist KN-93 administration were further included in this study for pathway determination. Expression of NR1, total and phosphorylated CaMKII, total and phosphorylated ERK were measured using western blot and immunofluorescent assays. Chronic renal fibrosis was introduced by sublethal ischemia-reperfusion injury in mice, and oral NMDAR inhibitor dextromethorphan (DXM) administration was performed. Results: NR1 expressions were upregulated in both obstructed kidneys and TGF-β treated HK-2 cells. NR1 knockdown, DXM, MK801, and KN93 reduced the fibrotic morphology in vivo and in vitro respectively, and companied with the downregulated ERK activation, while KN93 administration had no effect on NR1 and CaMKII levels. Conclusions: NMDAR participates in both acute and chronic renal fibrogenesis via CaMKII/ERK activation, and is a potential therapeutic target for renal fibrosis.
... The in vivo expressions of ion channels in neurons were subtly tuned and the dysregulations were closely related to diseases, as those of NaVs in neuropathic pain and ASIC1a in ischemia (Lai et al., 2003;Chai et al., 2010). It was shown that some motifs in ion channels were important in determining their subcellular locations (Standley et al., 2000;Xia et al., 2001;Garrido et al., 2003). In this study, by conducting a screening analysis, we confirmed that the NaV1.8 ...
Among the nine voltage-gated sodium channel (NaV) subtypes, NaV1.8 is an attractive therapeutic target for pain. The heterologous expression of recombinant NaV1.8 currents is of particular importance for its electrophysiological and pharmacological studies. However, NaV1.8 expresses no or low-level functional currents when transiently transfected into non-neuronal cell lines. The present study aims to explore the molecular determinants limiting its functional expression and accordingly establish a functional NaV1.8 expression system. We conducted screening analysis of the NaV1.8 intracellular loops by constructing NaV chimeric channels and confirmed that the NaV1.8 C-terminus was the only limiting factor. Replacing this sequence with that of NaV1.4, NaV1.5, or NaV1.7 constructed functional channels (NaV1.8/1.4L5, NaV1.8/1.5L5, and NaV1.8/1.7L5, respectively), which expressed high-level NaV1.8-like currents in HEK293T cells. The chimeric channel NaV1.8/1.7L5 displayed much faster inactivation of its macroscopic currents than NaV1.8/1.4L5 and NaV1.8/1.5L5, and it was the most similar to wild-type NaV1.8 expressed in ND7/23 cells. Its currents were very stable during repetitive depolarizations, while its repriming kinetic was different from wild-type NaV1.8. Most importantly, NaV1.8/1.7L5 pharmacologically resembled wild-type NaV1.8 as revealed by testing their susceptibility to two NaV1.8 selective antagonists, APETx-2 and MrVIB. NaV chimeras study showed that at least the domain 2 and domain 4 of NaV1.8 were involved in binding with APETx-2. Our study provided new insights into the function of NaV1.8 intracellular loops, as well as a reliable and convenient expression system which could be useful in NaV1.8 studies.
... • Burying RXR motifs as CFTR folds prevents ER retention and ERAD (Hegedus et al. 2006) • GABA B (Margeta-Mitrovic et al. 2000) and NMDA (Xia et al. 2001) receptors fold properly to hide RXR motifs for ER exit and plasma membrane expression Posttranslational modifications ...
The eukaryotic cell is organized into membrane-delineated compartments that are characterized by specific cadres of proteins sustaining biochemically distinct cellular processes. The appropriate subcellular localization of proteins is key to proper organelle function and provides a physiological context for cellular processes. Disruption of normal trafficking pathways for proteins is seen in several genetic diseases, where a protein’s absence for a specific subcellular compartment leads to organelle disruption, and in the context of an individual, a disruption of normal physiology. Importantly, several drug therapies can also alter protein trafficking, causing unwanted side effects. Thus, a deeper understanding of trafficking pathways needs to be appreciated as novel therapeutic modalities are proposed. Despite the promising efficacy of novel therapeutic agents, the intracellular bioavailability of these compounds has proved to be a potential barrier, leading to failures in treatments for various diseases and disorders. While endocytosis of drug moieties provides an efficient means of getting material into cells, the subsequent release and endosomal escape of materials into the cytosol where they need to act has been a barrier. An understanding of cellular protein/lipid trafficking pathways has opened up strategies for increasing drug bioavailability. Approaches to enhance endosomal exit have greatly increased the cytosolic bioavailability of drugs and will provide a means of investigating previous drugs that may have been shelved due to their low cytosolic concentration.
... NMDAR is a tetramer containing 2 GluN1 and 2 GluN2 subunits. GluN1 is retained in the endoplasmic reticulum when it is expressed alone; however, when it is expressed with GluN2, together they assemble to form a functional receptor that localizes to the plasma membrane (McIlhinney et al., 1998;Scott et al., 2001;Standley et al., 2000;Wenthold et al., 2003;Xia et al., 2001). Thus, if the expression level of GluN1 or GluN2 decreases, the number of functional NMDARs is potentially reduced, even when those of other subunits increase. ...
Early-life stress can induce several neuropsychological disorders in adulthood. However, the underlying mechanisms inducing such disorders are still not fully understood. Furthermore, the effects of early-life stress on the changes in cognitive function with age are still not clarified. In this study, we used maternal deprivation (MD) to examine the cognitive function in middle-aged mice using a touchscreen-equipped operant chamber. In the visual-discrimination task, the aged (∼1.4 years old) control mice could accurately learn to discriminate between different visual stimuli. In contrast, the correct response rate of aged MD mice increased to ∼60% by day 10; it was still significantly lower than that of the control mice (85%). In the hippocampus of aged MD mice, the expression level of the N-methyl-d-aspartate receptor subunit GluN1 decreased significantly as compared to that in control mice. On the other hand, no significant difference in GluN1 expression level was detected in young (2.5 months old) mice. These findings indicate that early-life stress accelerates cognitive impairment in middle-aged mice.
... Furthermore, tight control of protein maturation and stability is required for plasticity to develop. Several lines of evidence have indicated that local ER compartments regulate the availability of synaptic proteins, including glutamate receptors, in an activity-driven manner (Hanus et al. 2014, Cui-Wang et al. 2012, Pick et al. 2017, Xia et al. 2001, Mu et al. 2003. However, the precise mechanisms of subsequent Golgi sorting and additional post-translational modification have still been a matter of debate. ...
One of the most intriguing features of the brain is its ability to be malleable, allowing it to adapt continually to changes in the environment. Specific neuronal activity patterns drive long‐lasting increases or decreases in the strength of synaptic connections, referred to as long‐term potentiation and long‐term depression, respectively. Such phenomena have been described in a variety of model organisms, which are used to study molecular, structural, and functional aspects of synaptic plasticity. This review originated from the first International Society for Neurochemistry ( ISN ) and Journal of Neurochemistry ( JNC ) Flagship School held in Alpbach, Austria (Sep 2016), and will use its curriculum and discussions as a framework to review some of the current knowledge in the field of synaptic plasticity. First, we describe the role of plasticity during development and the persistent changes of neural circuitry occurring when sensory input is altered during critical developmental stages. We then outline the signaling cascades resulting in the synthesis of new plasticity‐related proteins, which ultimately enable sustained changes in synaptic strength. Going beyond the traditional understanding of synaptic plasticity conceptualized by long‐term potentiation and long‐term depression, we discuss system‐wide modifications and recently unveiled homeostatic mechanisms, such as synaptic scaling. Finally, we describe the neural circuits and synaptic plasticity mechanisms driving associative memory and motor learning. Evidence summarized in this review provides a current view of synaptic plasticity in its various forms, offers new insights into the underlying mechanisms and behavioral relevance, and provides directions for future research in the field of synaptic plasticity.
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... However, it was demonstrated that the activation of NMDA receptors during neuropathic pain leads to excitation of protein kinase C (PKC), which in turn phosphorylates serine residue 896 of the NMDA receptor NR1 subunit (Gao et al., 2005(Gao et al., , 2007Roh et al., 2008;Tingley et al., 1997;Ultenius et al., 2006). This allows for transfer of NMDA receptors from the cell interior to the cell membrane (Scott et al., 2003;Xia et al., 2001), induces membrane insertion (Scott et al., 2003), and leads to central sensitization. It was reported that oral administration of magnesium sulfate (MgSO 4 ) prevents spinal cord NMDA receptor NR1 subunit phosphorylation, as well as alleviates hyperalgesia and allodynia in a streptozotocin-induced diabetic neuropathy in rats (Rondón et al., 2010). ...
Opioids are increasingly used in alleviating pain, including cancer-related pain and postoperative pain. Unfortunately, the development of tolerance, the resistance of neuropathic pain on opioid analgesia or other undesirable effects may limit their utility. In order to reduce opioid doses, and thereby to avoid the risk of side effects and sudden deaths due to overdosing, attempts have been made to introduce co-analgesics. Due to an increasing amount of data concerning a potential enhance of opioid analgesia by the physiological antagonist of N-methyl-d-aspartate receptors, magnesium ions (Mg2 +), a concomitant use of such a combination seems to be interesting from a clinical point of view. Therefore, the aim of this review is to provide an analysis of existing preclinical and clinical studies in the context of the benefits of using this combination in clinical practice. A potential mechanism of magnesium - opioid interaction is also suggested. The potential influence of Mg on opioid adverse/side effects as well as conclusions on the safety of combined administration of magnesium and opioid drugs were also summarized. Data from animal studies indicate that magnesium increases opioid analgesia in chronic (e.g., neuropathic, inflammatory) as well as acute pain. In clinical trials, most authors confirmed that magnesium reduces opioid consumption and alleviates postoperative pain scores while not increasing the risk of side effects after opioids. However, more clinical studies are needed concerning an influence of Mg on opioid activity in other difficult to treat types of pain, especially neuropathic and inflammatory.
... So, to assess changes in PKC phosphorylation levels, we used an antibody against phosphorylation at the carboxy-terminal residue serine 660 (this occurs in the hydrophobic motif of cPKC and nPKC, but not in aPKC) and demonstrated sharper up-regulation of cPKC and nPKC after HG incubation of podocytes than the total elevation of PKC expression after the first 6 h of HG incubation. After NR1 is expressed, it is normally retained in the ER, but not in the Golgi apparatus, in early, recycling, and late endosomes [28], and its maturation and trafficking to the cell surface are activated by PKC [29]. In studies of neurons, phosphorylation of both serines 890 and 896 of NR1 is necessary for its fine-tuning and to allow ER secretion and membrane trafficking; the PKC α, β (I and II), and γ isoforms play specific and different roles in the phosphorylation of Ser890 and/or Ser896 residues during the process [30]. ...
Podocytes play important roles in the progression of diabetic kidney disease (DKD) and these roles are closely associated with cytoskeletal actin dynamics. N-methyl-D-aspartate receptors (NMDARs), which consist of two functional NR1 subunits and two regulatory NR2 subunits, are widely expressed in the brain but are also found in podocytes. Here, we found increased NR1 expression in two diabetic mouse models and in podocytes incubated in high glucose (HG). In diabetic mice, knockdown of NR1 using lentivirus carrying NR1-shRNA ameliorated the pathological features associated with DKD, and reversed the decreased expression of synaptopodin and Wilms' tumor-1. In podocytes incubated with HG, NR1 was secreted from the endoplasmic reticulum. and this was blocked by bisindolylmaleimide I. NR1 knockdown decreased the cell shape remodelling, cell collapse, bovine serum albumin permeability, and migration induced by HG. After HG incubation, levels of cell division control protein 42 (Cdc42) and its active form increased, and a significantly higher Cdc42-GTP level, increased Cdc42 translocation onto the leading edges, and lower migration ability were found in podocytes with NR1-knockdown. Increases in the number and length of filopodia were found in podocytes with NR1-knockdown but these were abolished by Cdc42-GTP blockade with ML141. In conclusion, the activation of NMDARs plays an important role in DKD by reducing Cdc42-GTP activation.
... It has been proposed that several transmembrane proteins contain a short amino acid sequence on their cytoplasmic domains that act as an ER retention signal and the presence or absence of this domain is governed by alternate splicing (Sprengel, et al. 1998). Cell surface expression also depends on the splice variant of the C-terminus and this mechanism appears to govern NR1 release from the ER (Scott, et al 2001;Xia, et al. 2001). Longer splice variants of NR1 are retained in the ER due to the presence of the ER retention motif in the alternatively spliced C-terminus. ...
... These data suggest that levels of NR1 are generally decreased in the absence of PSD95, while its localisation at synaptic sites may also be affected by loss of both SAP102. The C1 cassette at the C terminus of NR1 splice variant contains an endoplasmic reticulum (ER) retention signal, and exit of NR1 from ER requires suppression of C1 by the PDZ interacting domain of C2' cassette (Standley et al., 2000;Xia et al., 2001). Standley et al., (2000) show that all PSD95 family MAGUKs interact with specific amino acid sequences in C2', and that SAP102 and NR1 coimmunoprecipitate in microsomal fractions suggesting that they associate early in the secretory pathway of NR1. ...
The accurate development of the wiring between the billions of neurons in our
brain is fundamental to brain function. Development of this connectivity
relies on activity-dependent modification of synapses similar to those that
underlie learning and memory. Glutamate is the principal excitatory
neurotransmitter in the mammalian brain and several brain disorders result
from altered glutamatergic receptor signalling (Catania et al., 2007; Lau and
Zukin, 2007). Genes encoding glutamate receptor associated proteins have a
high incidence of mutation in cognitive disorders, especially X-linked mental
retardation (MR)(Laumonnier et al., 2007). MR has long been associated
with altered cortical connectivity, particularly dendritic spine dysgenesis.
There is also an emerging view that aberrant local protein synthesis within
dendrites and protein trafficking to dendrites underlies some forms of MR
(Kelleher and Bear, 2008; Pfeiffer and Huber, 2006; Zalfa and Bagni, 2005).
Most studies examining the role of glutamatergic receptors in MR have
focused on adults. Little is known about how these MR genes regulate brain
development despite their neurodevelopmental aetiology.
Fragile X mental retardation (FXS) is the most common form of inherited MR
and results from the loss of fragile X mental retardation protein (FMRP).
FMRP is a RNA binding protein and is hypothesised to have a role in protein
trafficking from nucleus to sites of synapses, and regulating local protein
synthesis at sites of synapses (Bagni and Greenough, 2005). A prevalent
theory of FXS causation is ‘metabotropic glutamate receptor (mGluR) theory
of fragile X’, which postulates that all functional consequences of mGluR
(predominantly mGluR5)-dependent protein synthesis maybe exaggerated in
FXS (Bear et al., 2004).
Primary somatosensory cortex (S1) of rodents provides an excellent model
system to study the role of MR genes in development because of its
stereotypic, glutamate receptor-dependent, anatomical development (Barnett
et al., 2006b; Erzurumlu and Kind, 2001). Hannan et al., (2001) reported
that genetic deletion of mGluR5 results in loss of ‘barrels’, the anatomical
correlates of rodent whiskers in S1. Chapter 3 extends these findings to show
that there is expression of mGluR5 as early as P4 in S1 prior to segregation of
layer 4 cells into barrels suggesting a tropic role for glutamate in barrel
formation. The expression of mGluR5 is postsynaptic during barrel formation
and does not regulate tangential or radial cortical development. Its effects on
barrel segregation are dose dependent and are not due to a developmental
delay. During late S1 development, loss of mGluR5 results in decreased spine
density suggesting a role in synaptogenesis. Supporting this hypothesis in
mGluR5 mutant mice there is a general decrease in expression of synaptic
markers in early S1 development. Chapter 4 explores the role of FMRP in
cortical development. FMRP is expressed early in S1 development with peak
expression prior to synaptogenesis at P14. It is expressed postsynaptically at
P7 and pre and postsynaptically at P14. FMRP does not regulate cortical
arealisation during barrel formation but results in decreased barrel
segregation. In the absence of FMRP, biochemical studies show altered
expression of glutamatergic receptors in the neocortex P7 and P14
suggesting altered glutamatergic receptor composition at synaptic sites.
During late S1 development, loss of FMRP results in increased spine density
in layer 4 spiny cells. Together these data indicate a role for FMRP during
early and late S1 development. Chapter 5 directly tests the mGluR theory of
FXS by examining whether genetic reduction of mGluR5 levels rescues
anatomical phenotypes characterised in Fmr1-/y mice. The defect in barrel
formation in Fmr1-/y mice is partially rescued by reducing mGluR5 levels.
However, layer 4 spine density in Fmr1-/y mice does not appear to be rescued.
Chapter 6 explores the expression patterns of three key synaptic MAGUKs
(Membrane associated guanylate kinases) PSD95, SAP102 and PSD93, one of
which (PSD95) is regulated by FMRP (Zalfa et al., 2007) and the others
which have putative binding sites for FMRP. MAGUKs tether glutamatergic
receptors to their associated signalling complexes at the postsynaptic
membrane and also regulate glutamatergic receptor trafficking (Collins and
Grant, 2007; Kim and Sheng, 2004). The immunohistochemical expression
profiles of PSD95, SAP102 and PSD93 show dynamic regulation during S1
development that is unaffected by loss of FMRP (at P7), and biochemical data
indicates that basal levels of these MAGUKs in neocortex are unaltered at P7
and P14 in Fmr1-/y mice. In Sap102-/y and Psd95-/- mice, there is altered
expression of several synaptic proteins biochemically providing evidence for
differential roles of SAP102 and PSD95 in regulating expression of
glutamatergic receptors at synaptic sites during early S1 development.
This thesis demonstrates that synaptic proteins associated with MR are
expressed early in development and display regulatory roles in cellular
processes governing S1 formation. An understanding of their role in early
brain development would be critical in fully appreciating when and where
they exert their regulatory effects, and this in turn would be beneficial in
designing therapeutic interventions.
... For NR1-3 variants, however, lower export efficiency might be compensated by the presence of a PDZ binding motif in the C2' cassette and the subsequent interaction with PDZ proteins. As neither NR1-2 nor NR1-4 contain the C1 cassette, none features the retention signal, and NR1-4 in addition has the C2' cassette PDZ interacting motif (Standley et al., 2000;Scott et al., 2001;Xia et al., 2001). Splicing between exons 2 and 4 generates the truncated 181-amino acid subunit NR1-5 (Sugihara et al., 1992;Hollmann et al., 1993), for which no function has yet been demonstrated. ...
... The construct (pJAF270) used for SNAP trafficking assays was constructed by fusing the extracellular domain of mouse CD8a [44] to the last 17 extracellular residues of mouse fibrocystin through the first 27 intracellular residues, the SNAP tag was cloned onto the c-terminal end of the CTS creating CD8-CTS-SNAP. Mouse kidney cells stably expressing CD8-CTS-SNAP were incubated with 0.04 mM cell permeable non-fluorescent BG-Block (New England Biolabs) for 30 minutes to block all SNAP epitopes. ...
The primary cilium is a sensory organelle, defects in which cause a wide range of human diseases including retinal degeneration, polycystic kidney disease and birth defects. The sensory functions of cilia require specific receptors to be targeted to the ciliary subdomain of the plasma membrane. Arf4 has been proposed to sort cargo destined for the cilium at the Golgi complex and deemed a key regulator of ciliary protein trafficking. In this work, we show that Arf4 binds to the ciliary targeting sequence (CTS) of fibrocystin. Knockdown of Arf4 indicates that it is not absolutely required for trafficking of the fibrocystin CTS to cilia as steady-state CTS levels are unaffected. However, we did observe a delay in delivery of newly synthesized CTS from the Golgi complex to the cilium when Arf4 was reduced. Arf4 mutant mice are embryonic lethal and die at mid-gestation shortly after node formation. Nodal cilia appeared normal and functioned properly to break left-right symmetry in Arf4 mutant embryos. At this stage of development Arf4 expression is highest in the visceral endoderm but we did not detect cilia on these cells. In the visceral endoderm, the lack of Arf4 caused defects in cell structure and apical protein localization. This work suggests that while Arf4 is not required for ciliary assembly, it is important for the efficient transport of fibrocystin to cilia, and also plays critical roles in non-ciliary processes.
... Curiosity about the remarkable ability of a cell to enforce octomeric assembly of ATP-sensitive potassium (K ATP ) channels containing four Kir6.1/2 subunits of the Kir family and four SUR1/2 subunits of an equally ancient transporter family led to the finding that COPI recognition of arginine-based motifs on these a and b subunits in partially assembled K ATP channel complexes causes their retrieval from the Golgi back to the ER (Heusser et al., 2006;Yuan et al., 2003;Zerangue et al., 1999). Similar arginine-based ER retrieval motifs have been found in TASK channels (O'Kelly et al., 2002), sodium channels (Zhang et al., 2008), glutamate receptors (Horak et al., 2008;Nasu-Nishimura et al., 2006;Ren et al., 2003;Scott et al., 2001;Vivithanaporn et al., 2006;Xia et al., 2001), acetylcholine receptors (Keller et al., 2001;Srinivasan et al., 2011), and the ER resident calcium channel localization factor-1 (CALF-1) that promotes surface expression of calcium channels (Saheki and Bargmann, 2009). ...
Heartbeats, muscle twitches, and lightning-fast thoughts are all manifestations of bioelectricity and rely on the activity of a class of membrane proteins known as ion channels. The basic function of an ion channel can be distilled into, "The hole opens. Ions go through. The hole closes." Studies of the fundamental mechanisms by which this process happens and the consequences of such activity in the setting of excitable cells remains the central focus of much of the field. One might wonder after so many years of detailed poking at such a seemingly simple process, is there anything left to learn?
... Mutating all five arginines to alanines released this effect and led to cell surface expression (33). Similar argininebased motifs function as ER retention and retrieval signals in other receptors (5,29,36,45,46). In combination with the ER export motif in ␣ 2C -ARs, the arginine-rich region may mediate regulated delivery to the cell surface under certain physiological stimuli. ...
The second messenger cyclic AMP (cAMP) plays a vital role in vascular physiology, including vasodilation of large blood vessels. We recently demonstrated cAMP activation of Epac-Rap1A and RhoA-ROCK-F-actin signaling in arteriolar derived smooth muscle cells to increase expression and cell surface translocation of functional α2C-adrenoceptors (α2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expression of α2C-ARs and also increased translocation of perinuclear α2C-ARs to intracellular F-actin and to the plasma membrane. This study examined the mechanism of translocation to better understand the role of these newly discovered mediators of blood flow control, potentially activated in peripheral vascular disorders. We utilized a yeast two-hybrid screen with human microVSM cDNA library and the α2C-AR C-terminus to identify a novel interaction with the actin cross-linker, filamin-2. Yeast α-galactosidase assays, site-directed mutagenesis, and co-immunoprecipitation experiments in heterologous HEK 293 cells and in human microVSM demonstrated that α2C-ARs, but not α2A-AR subtype, interacted with filamin. In Rap1-stimulated human microVSMs, α2C-ARs co-localized with filamin on intracellular filaments and at the plasma membrane. siRNA mediated knockdown of filamin-2 inhibited Rap1-induced redistribution of α2C-ARs to the cell surface and inhibited receptor function. The studies suggest that cAMP-Rap1-Rho-ROCK signaling facilitates receptor translocation and function via phosphorylation of filamin-2 Ser-2113. Together, these studies extend our previous findings to show that functional rescue of α2C-ARs is mediated through Rap1-filamin signaling. Perturbation of this signaling pathway may lead to alterations in α2C-AR trafficking and physiological function.
... For NR1-3 variants, however, lower export efficiency might be compensated by the presence of a PDZ binding motif in the C2 cassette and the subsequent interaction with PDZ proteins (Cavara et al., 2009). As neither NR1-2 nor NR1-4 contains the C1 cassette or features the retention signal, and in addition, NR1-4 has the C2 cassette PDZ interacting motif (Xia et al., 2001). Bradley (2006) have demonstrated that the removal of C0 and C1 domains from the NR1 subunit is required for downstream signaling to the CRE-dependent gene expression. ...
The brain is one of the major targets of alcohol actions. Most of the excitatory synaptic transmission in the central nervous system is mediated by N-methyl-D-aspartate (NMDA) receptors. However, one of the most devastating effects of alcohol leads to brain shrinkage, loss of nerve cells at specific regions through a mechanism involving excitotoxicity, oxidative stress. Earlier studies have indicated that chronic exposure to ethanol both in vivo and in vitro, increases NR1 and NR2B gene expression and their polypeptide levels. The effect of alcohol and molecular changes on the regulatory process, which modulates NMDAR functions including factors altering transcription, translation, post-translational modifications, and protein expression, as well as those influencing their interactions with different regulatory proteins (downstream effectors) are incessantly increasing at the cellular level. Further, I discuss the various genetically altered mice approaches that have been used to study NMDA receptor subunits and their functional implication. In a recent countable review, epigenetic dimension (i.e., histone modification-induced chromatin remodeling and DNA methylation, in the process of alcohol related neuroadaptation) is one of the key molecular mechanisms in alcohol mediated NMDAR alteration. Here, I provide a recount on what has already been achieved, current trends and how the future research/studies of the NMDA receptor might lead to even greater engagement with many possible new insights into the neurobiology and treatment of alcoholism.
... This has been observed for Kir6 channels, where the channel-forming subunit is retained inside the cell unless it is co-assembled with SUR (Sakura et al., 1995; Zerangue et al., 1999 ). It is also seen with the GABA B receptor , the nicotinic acetylcholine receptor, kainate receptors, and the N-methyl-D-aspartate (NMDA) receptor (Okabe et al., 1999; Standley et al., 2000; Xia et al., 2001 ). In these cases of obligatory heteromerization, the only condition required to use the PCS approach is to endow a subunit with photosensitivity that is able to regulate the entire protein complex. ...
Optogenetic tools were originally designed to target specific neurons for remote control of their activity by light and have largely been built around opsin-based channels and pumps. These naturally photosensitive opsins are microbial in origin and are unable to mimic the properties of native neuronal receptors and channels. Over the last 8 years, photoswitchable tethered ligands (PTLs) have enabled fast and reversible control of mammalian ion channels, allowing optical control of neuronal activity. One such PTL, maleimide-azobenzene-quaternary ammonium (MAQ), contains a maleimide (M) to tether the molecule to a genetically engineered cysteine, a photoisomerizable azobenzene (A) linker and a pore-blocking quaternary ammonium group (Q). MAQ was originally used to photocontrol SPARK, an engineered light-gated potassium channel derived from Shaker. Potassium channel photoblock by MAQ has recently been extended to a diverse set of mammalian potassium channels including channels in the voltage-gated and K2P families. Photoswitchable potassium channels, which maintain native properties, pave the way for the optical control of specific aspects of neuronal function and for high precision probing of a specific channel's physiological functions. To extend optical control to natively expressed channels, without overexpression, one possibility is to develop a knock-in mouse in which the wild-type channel gene is replaced by its light-gated version. Alternatively, the recently developed photoswitchable conditional subunit technique provides photocontrol of the channel of interest by molecular replacement of wild-type complexes. Finally, photochromic ligands also allow photocontrol of potassium channels without genetic manipulation using soluble compounds. In this review we discuss different techniques for optical control of native potassium channels and their associated advantages and disadvantages.
... This protein is a type 1 membrane protein that contains a nearly 4000 amino acid N-terminal extracellular domain, a single transmembrane span and a short cytoplasmiclocalized C-terminal tail (Ward et al., 2002). To study the movement of this protein through the endomembrane system, we developed constructs where the large extracellular domain is replaced by a similar, but much smaller, extracellular domain of CD8 (Xia et al., 2001). The CD8 fragment serves to ensure the correct membrane topology of the fusion protein and is also an epitope tag that can be detected by a monoclonal antibody. ...
The sensory functions of the primary cilium rely on receptors and other membrane proteins that are specifically sorted to the ciliary compartment, which is a subdomain of the plasma membrane. Defects in this process underlie a large number of human diseases, yet it is poorly understood. Thus, it is of great interest to understand the mechanisms by which the cell sorts and traffics proteins to the ciliary membrane. Here, we provide an overview of our method to study the sorting and trafficking of ciliary membrane proteins using SNAP technology. This technology enables pulse-chase analysis of the movement of proteins through the endomembrane system and onto the cilium.
... However, we do not show this directly by live-imaging, nor do we know if the additional SAP102 proteins become a permanent part of the postsynaptic architecture. Several studies have indicated that PDZ proteins interact at the level of the ER [202122, [44,45]], and play an important role in the early trafficking of membrane proteins. PDZ protein interactions appear to be required for ER exit in the case of pro-TGFa [44] and of the NMDA receptor subunits, NR1-3 [20– 22]. ...
NMDA receptor NR2A/B subunits have PDZ-binding domains on their extreme C-termini that are known to interact with the PSD-95 family and other PDZ proteins. We explore the interactions between PSD-95 family proteins and the NR2A/B cytoplasmic tails, and the consequences of these interactions, from the endoplasmic reticulum (ER) through delivery to the synapse in primary rat hippocampal and cortical cultured neurons. We find that the NR2A/B cytoplasmic tails cluster very early in the secretory pathway and interact serially with SAP102 beginning at the intermediate compartment, and then PSD-95. We further establish that colocalization of the distal C-terminus of NR2B and PSD-95 begins at the trans-Golgi Network (TGN). Formation of NR2B/PSD-95/SAP102 complexes is dependent on the PDZ binding domain of NR2B subunits, but association with SAP102 and PSD-95 plays no distinguishable role in cluster pre-formation or initial targeting to the vicinity of the synapse. Instead the PDZ binding domain plays a role in restricting cell-surface clusters to postsynaptic targets.
... It is also what is seen with the GABA B receptor, a GPCR that is composed of GB1 and GB2 subunits, in which RXR endoplasmic reticulum retention motifs, which prevent trafficking to the cell surface, are masked in a complementary manner to allow for surface trafficking in the GB1/GB2 heteromer (Margeta-Mitrovic et al., 2000). A functionally analogous scheme operates in NMDA receptors, which are composed of two NR1 and two NR2 subunits, with neither subtype arriving on the cell surface on its own (Okabe et al., 1999;Standley et al., 2000;Xia et al., 2001). ...
Selective ligands are lacking for many neuronal signaling proteins. Photoswitched tethered ligands (PTLs) have enabled fast and reversible control of specific proteins containing a PTL anchoring site and have been used to remote control overexpressed proteins. We report here a scheme for optical remote control of native proteins using a "photoswitchable conditional subunit" (PCS), which contains the PTL anchoring site as well as a mutation that prevents it from reaching the plasma membrane. In cells lacking native subunits for the protein, the PCS remains nonfunctional internally. However, in cells expressing native subunits, the native subunit and PCS coassemble, traffic to the plasma membrane, and place the native protein under optical control provided by the coassembled PCS. We apply this approach to the TREK1 potassium channel, which lacks selective, reversible blockers. We find that TREK1, typically considered to be a leak channel, contributes to the hippocampal GABA(B) response.
... Although a primary role of PDZ proteins may be to anchor and organize receptors at the synapse, considerable evidence implicates a chaperone role for PDZ proteins in the early events of assembly, processing, and delivery of receptor proteins. For example, the presence of a putative PDZ binding motif in the C2Ј cassette of NR1-3 splice variants overcomes (masks) an Arginine-Arginine-Arginine motif in the C1 cassette that serves as an endoplasmic reticulum (ER) retention signal and promotes forward trafficking of the receptors out of the ER (Standley et al., 2000;Scott et al., 2001;Xia et al., 2001). Thus, the two signals differentially regulate the surface expression of NR1 splice variants. ...
NMDA receptors (NMDARs) colocalize with postsynaptic density protein-95 (PSD-95), a multivalent synaptic scaffolding protein and core component of the postsynaptic density, at excitatory synapses. Although much is known about the identity and properties of scaffolding proteins, little is known about their actions on NMDAR function. Here we show that association of PSD-95 with NMDARs modulates channel gating and surface expression. PSD-95 increases the number of functional channels at the cell surface and channel opening rate of NMDARs, with little or no change in conductance, reversal potential, or mean open time. We show further that PSD-95 increases NMDAR surface expression by increasing the rate of channel insertion and decreasing the rate of channel internalization. The PDZ (PSD-95, discs large, zona occludens-1) binding motif at the distal end of the NR2 C-terminal tail is critical to the actions of PSD-95 on NMDAR function and surface expression. Given that activity bi-directionally modifies synaptic levels of PSD-95, our findings suggest a novel mechanism for activity-dependent regulation of NMDARs at central synapses.
... Receptors lacking the N1 cassette are partially inhibited (~50%) at physiological pH, potentiated by polyamines and inhibited by zinc ions, while those containing N1 are insensitive to these modulators (Traynelis et al., 1998; Traynelis et al., 1995). The C1 cassette contains phosphorylation sites for protein kinase A and protein kinase C (Tingley et al., 1997; Tingley et al., 1993), an endoplasmic reticulum retention signal (Scott et al., 2001; Xia et al., 2001) and binding sites for various intracellular proteins [calmodulin (Ehlers et al., 1996); neurofilaments (Ehlers et al., 1998); yotiao (Lin et al., 1998)]. The C2' cassette contains a PDZ binding domain which interacts with post synaptic density (PSD)-95. ...
The supraoptic nucleus (SON) of the hypothalamus contains magnocellular neurosecretory neurons (MNC) which synthesize and release the peptide hormones vasopressin and oxytocin. Glutamate is a prominent excitatory neurotransmitter in the SON and regulates MNC excitability. NMDA receptors (NMDAR), a type of ionotropic glutamate receptor, mediate synaptic plasticity of MNCs and are necessary for characteristic burst firing patterns which serve to maximize hormone release. NMDARs are di- or tri-heteromeric complexes of NR1 and NR2 subunits. Receptor properties depend on NR2 subunit composition and variable splicing of NR1. We investigated the expression profile of NR1 and NR2 subunits in the SON at the mRNA and protein levels plus protein expression of NR1 splice variants in control and salt-loaded adult rats. There was robust mRNA expression of all subunits, with NR2D levels being the highest. At the protein level, NR1, NR2B, and NR2D were robustly expressed, while NR2A was weakly expressed. NR2C protein was not detected with either of the two antibodies tested. All four NR1 splice variant cassettes (N1, C1, C2, C2') were detected in the SON, although NR1 N1 expression was too low for accurate analysis. Three days of salt-loading did not alter mRNA, protein, or splice variant expression of NMDAR subunits in the SON. Robust NR2D protein expression has not been previously shown in MNCs and is uncommon in the adult brain. Although the functional significance of this unusual expression profile is unknown, it may contribute to important physiological characteristics of SON neurons, such as burst firing and resistance to excitotoxicity.
... AMPA receptor antagonists also have been valuable tools to understand receptor gating mechanisms. Pharmacological studies have identified non-competitive antagonists including the 2,3-benzodiazepines, such as GYKI-53655 (59), and quinazolinones, such as CP-465,022 (60). Radioligand binding indicates that the 2,3-benzodiazepines and the quinazolinones interact with overlapping sites on the receptor; however, their binding site remains uncertain. ...
AMPA receptors mediate fast excitatory transmission in the brain. Neuronal AMPA receptors comprise GluA pore-forming principal subunits and can associate with multiple modulatory components, including transmembrane AMPA receptor regulatory proteins (TARPs) and CNIHs (cornichons). AMPA receptor potentiators and non-competitive antagonists represent potential targets for a variety of neuropsychiatric disorders. Previous studies showed that the AMPA receptor antagonist GYKI-53655 displaces binding of a potentiator from brain receptors but not from recombinant GluA subunits. Here, we asked whether AMPA receptor modulatory subunits might resolve this discrepancy. We find that the cerebellar TARP, stargazin (γ-2), enhances the binding affinity of the AMPA receptor potentiator [(3)H]-LY450295 and confers sensitivity to displacement by non-competitive antagonists. In cerebellar membranes from stargazer mice, [(3)H]-LY450295 binding is reduced and relatively resistant to displacement by non-competitive antagonists. Coexpression of AMPA receptors with CNIH-2, which is expressed in the hippocampus and at low levels in the cerebellar Purkinje neurons, confers partial sensitivity of [(3)H]-LY450295 potentiator binding to displacement by non-competitive antagonists. Autoradiography of [(3)H]-LY450295 binding to stargazer and γ-8-deficient mouse brain sections, demonstrates that TARPs regulate the pharmacology of allosteric AMPA potentiators and antagonists in the cerebellum and hippocampus, respectively. These studies demonstrate that accessory proteins define AMPA receptor pharmacology by functionally linking allosteric AMPA receptor potentiator and antagonist sites.
... Despite this limitation, there is ample evidence from a variety of studies that phospho-site mutants produce effects that are consistent with those produced by a kinase. For example, introducing aspartate residues at S896 and S897 of the NR1 subunit enhances surface expression of this protein when it is expressed alone in HEK293 cells or neurons (Scott et al., 2003; Xia et al., 2001). This is presumably due to masking of an ER retention domain that serves as a quality control check for mis-folded or mismatched subunits. ...
N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels activated by the neurotransmitter glutamate. These channels are highly expressed by brain neurons and are critically involved in excitatory synaptic transmission. Results from previous studies show that both native and recombinant NMDA receptors are inhibited by ethanol at concentrations associated with signs of behavioral impairment and intoxication. Given the important role that NMDA receptors play in synaptic transmission and brain function, it is important to understand the factors that regulate the ethanol inhibition of these receptors. One dynamic mechanism for regulating ethanol action may be via phosphorylation of NMDA subunits by serine-threonine and tyrosine kinases. Both NR1 and NR2 subunits contain multiple sites of phosphorylation; and in the NR1 subunit, most of these are contained within the C1 domain, a carboxy-terminal cassette that is subject to alternative splicing. Although results from our previous studies suggest that single phosphorylation sites do not greatly affect ethanol sensitivity of NMDA receptors, it is likely that in vivo, these subunits are phosphorylated at multiple sites by different kinases. In the present study, we constructed a series of NMDA receptor mutants at serine (S) or threonine (T) residues proposed to be sites of phosphorylation by protein kinase A and various isoforms of protein kinase C. Ethanol (100mM) inhibited currents from wild-type NR1/2A and NR1/2B receptors expressed in human embryonic kidney 293 cells by approximately 25 and 30%, respectively. This inhibition was not different in single-site mutants expressing alanine (A) or aspartate/glutamate (D/E) at positions T879, S896, or T900. The mutant NR1(S890D) showed greater ethanol inhibition than NR1(890A) containing receptors, although this was only observed when it was combined with the NR2A subunit. Ethanol inhibition was not altered by aspartate substitution at four serines (positions 889, 890, 896, and 897) or when T879D was added to the four serine-substituted mutant. Ethanol inhibition was increased when T900E was added to the five serine-/threonine-substituted mutants, but again this was selective for NR2A containing receptors. Together with previously published data, these findings suggest that modification of putative phosphorylation sites could contribute to the overall acute ethanol sensitivity of recombinant NMDA receptors. Supported by R37AA009986.
... Presumably, the activated NMDA receptors lead to protein kinase C activation, which in turn phosphorylates serine residue 896 of the NR1 subunit (Tingley et al. 1997). NR1 phosphorylation at Ser 896 may promote the trafficking of NMDA receptors from intracellular stores to the cell membrane (Xia et al. 2001;Scott et al. 2003) and, in coordination with PKA phosphorylation of Ser 897, may also induce membrane insertion (Scott et al. 2003), enhancing synaptic activity and inducing central sensitization in chronic neuropathic pain. Several studies have reported that Mg deficiency induces a mechanical hyperalgesia that involves NMDA receptors (Begon et al. 2002;Alloui et al. 2003). ...
Neuropathic pain is a common diabetic complication affecting 8-16% of diabetic patients. It is characterized by aberrant symptoms of spontaneous and stimulus-evoked pain including hyperalgesia and allodynia. Magnesium (Mg) deficiency has been proposed as a factor in the pathogenesis of diabetes-related complications, including neuropathy. In the central nervous system, Mg is also a voltage-dependent blocker of the N-methyl-d-aspartate receptor channels involved in abnormal processing of sensory information. We hypothesized that Mg deficiency might contribute to the development of neuropathic pain and the worsening of clinical and biological signs of diabetes and consequently, that Mg administration could prevent or improve its complications. We examined the effects of oral Mg supplementation (296 mg l(-1) in drinking water for 3 weeks) on the development of neuropathic pain and on biological and clinical parameters of diabetes in streptozocin (STZ)-induced diabetic rats. STZ administration induced typical symptoms of type 1 diabetes. The diabetic rats also displayed mechanical hypersensitivity and tactile and thermal allodynia. The level of phosphorylated NMDA receptor NR1 subunit (pNR1) was higher in the spinal dorsal horn of diabetic hyperalgesic/allodynic rats. Magnesium supplementation failed to reduce hyperglycaemia, polyphagia and hypermagnesiuria, or to restore intracellular Mg levels and body growth, but increased insulinaemia and reduced polydipsia. Moreover, it abolished thermal and tactile allodynia, delayed the development of mechanical hypersensitivity, and prevented the increase in spinal cord dorsal horn pNR1. Thus, neuropathic pain symptoms can be attenuated by targeting the Mg-mediated blockade of NMDA receptors, offering new therapeutic opportunities for the management of chronic neuropathic pain.
The efficient targeting of ionotropic receptors to postsynaptic sites is essential for the function of chemical excitatory and inhibitory synapses, constituting the majority of synapses in the brain. A growing body of evidence indicates that cell adhesion molecules (CAMs), which accumulate at synapses at the earliest stages of synaptogenesis, are critical for this process. A diverse variety of CAMs assemble into complexes with glutamate and GABA receptors and regulate the targeting of these receptors to the cell surface and synapses. Presynaptically localized CAMs provide an additional level of regulation, sending a trans-synaptic signal that can regulate synaptic strength at the level of receptor trafficking. Apart from controlling the numbers of receptors present at postsynaptic sites, CAMs can also influence synaptic strength by modulating the conductivity of single receptor channels. CAMs thus act to maintain basal synaptic transmission and are essential for many forms of activity dependent synaptic plasticity. These activities of CAMs may underlie the association between CAM gene mutations and synaptic pathology and represent fundamental mechanisms by which synaptic strength is dynamically tuned at both excitatory and inhibitory synapses.
Individual synapses vary enormously in their biochemical properties and functionality. The neurotransmitters released, the receptors activated, and the ensuing physiological responses are all variable, and susceptible to modification through a variety of mechanisms. The regulation of neurotransmitter receptors is a central mechanism by which the signaling properties of a synapse and a cell may be controlled. Excitatory neurotransmitter receptors are primarily localized to the postsynaptic dendritic compartment, anchored within the postsynaptic density (PSD). In their lifetime, receptors are subject to alternative splicing, differential sorting and compartmentalization. Moreover, these processes are influenced by activity driven protein trafficking, post-translational modifications and interaction with an overwhelmingly increasing number of proteins enriched at excitatory synapses. The diverse repertoire of regulatory mechanisms contributes to the amazing heterogeneity of synapse morphology and function in the central nervous system. Changes in receptor trafficking has been also implicated in shaping synaptic activity and plasticity, and defects in these processes may underlie several pathological states.
The N-methyl-D-aspartate receptor (NMDA-R) is a member of the glutamate-activated ion channel family. The receptor forms a cation-selective channel with high calcium permeability that is tightly regulated by oxidizing agents, protons, zinc, polyamines, protein kinases, calmodulin, and most notably, magnesium. As NMDA-R is critically involved in synaptic transmission, its trafficking is a tightly regulated process. The NMDA-R subunit composition controls aspects of the biophysical properties of the ligand-activated ionotropic receptor as well as its trafficking. NMDA-R is a tetrameric complex and an obligate heteromultimer composed of NR1 subunits and one or more NR2 subunits. The carboxy termini domain of NMDA-R subunits, which are intracellular domains capable of interacting with different scaffolding and signaling proteins, controls trafficking and stabilization of NMDA-Rs at synapses. In particular, the carboxy termini of NR2 subunits influence trafficking, localization, and internalization of the receptor. The retention of NR1 in the endoplasmic reticulum is an important checkpoint, ensuring that only assembled heterocomplexes are processed. Despite the knowledge that NMDA-Rs interact with proteins that may aid in their trafficking, the precise role of these proteins in receptor delivery is unclear. The expression of NMDA-R interacting proteins is regionally and developmentally regulated, suggesting that elucidating the dynamics of these interactions may aid in the understanding of NMDA-R trafficking.
Leiomyoma is the most common uterine disease in women in age of procreation. Previous result have shown that in ELT3 cells, which is a rat leiomyoma-derived cell line, lysophosphatidic acid (LPA) was able to activate ERK1/2 MAP kinases through the activation of the LPA1 receptor via the classical MAP kinase pathway involving Raf, Ras and MEK. We have observed that LPA was dephosphorylated in ELT3 cells by Lipid-phosphate phosphatase 1 (LPP1) which is the only LPP isoform expressed in these cells. In a second part, we investigated the effect of diacylglycerol pyrophosphate (DGPP). DGPP, in its octanoyl form (DGPP8:0), is described as an LPA1, 3-antognist. Here, we show that DGPP had no antagonistic effect on LPA-ERK activation, but was able to induce ERK activation by itself. This activation occurred through the activation of PKC, Raf and MEK. On the other hand, we show that DGPP-ERK activation required its dephosphorylation by LPP. Next we observed that the DGPP-ERK activation was inhibited by VPC32183, which we showed to inhibit LPP activity, and by siRNA treatment targeting LPP1. This results show that DGPP needs to be dephosphorylated into PA to induce ERK phosphorylation, and this dephosphorylation is catalyzed by LPP1. Finally, in the third part of this study, we were interested in the differential LPP expression between ELT3 cells and myometrium cells. Indeed, we have previously observed that ELT3 cell express only LPP1 while myometrial cells express LPP1 and LPP3. We observed that when LPP3 is expressed in ELT3 cells, it can interact with LPP1 and may restrain its plasma membrane localization reducing ectoLPP activity in favor of intracellular LPP activity. This may result in a negative regulation of intracellular phospholipidic second messengers. All these results show the significance of LPP in the regulation of bioactive phospholipid effects and suggest a relationship between the tumoral phenotype of leiomyoma cells and the absence of LPP3.
A modified invertebrate glutamate-gated Cl⁻ channel (GluCl αβ) was previously employed to allow pharmacologically induced silencing of electrical activity in CNS neurons upon exposure to the anthelmintic drug ivermectin (IVM). Usefulness of the previous receptor was limited by 1) the high concentration of IVM necessary to elicit a consistent silencing phenotype, raising concern about potential side effects, and 2) the variable extent of neuronal spike suppression, due to variations in the co-expression levels of the fluorescent protein-tagged α and β subunits. To address these issues, mutant receptors generated via rational protein engineering strategies were examined for improvement. Introduction of a gain-of-function mutation (L9′F) in the second transmembrane domain of the α subunit appears to facilitate β subunit incorporation and substantially increase heteromeric GluCl αβ sensitivity to IVM. Removal of an arginine-based endoplasmic reticulum retention motif (RSR mutated to AAA) from the intracellular loop of the β subunit further promotes heteromeric expression at the plasma membrane possibly by preventing endoplasmic reticulum-associated degradation of the β subunit rather than simply reducing endoplasmic reticulum retention. A monomeric XFP (mXFP) mutation that prevents fluorescent protein dimerization complements the mutant channel effects. Expression of the newly engineered GluCl opt α-mXFP L9′F + opt β-mXFP Y182F RSR_AAA receptor in dissociated neuronal cultures markedly increases conductance and reduces variability in spike suppression at 1 nm IVM. This receptor, named “GluClv2.0,” is an improved tool for IVM-induced silencing.
Background: Ivermectin (IVM) silences activity in neurons expressing glutamate-gated chloride channels (GluCl).
Results: Rational point mutations in GluCl robustly increase IVM-induced conductance and reduce the variability of spike suppression in cultured neurons.
Conclusion: The newly engineered receptor improves heteromeric receptor expression and sensitivity to IVM.
Significance: GluClv2.0 is an improved tool for IVM-induced silencing.
In order to carry out their physiological functions, ion transport proteins must be targeted to the appropriate domains of cell membranes. Regulation of ion transport activity frequently involves the tightly controlled delivery of intracellular populations of transport proteins to the plasma membrane or the endocytic retrieval of transport proteins from the cell surface. Transport proteins carry signals embedded within their structures that specify their subcellular distributions and endow them with the capacity to participate in regulated membrane trafficking processes. Recently, a great deal has been learned about the biochemical nature of these signals, as well as about the cellular machinery that interprets them and acts upon their messages.
NMDA receptors (NMDARs) play a distinct role at excitatory glutamatergic synapses, where they are usually localized with other
ionotropic glutamate receptors, including tors. Two features are essential to their specialized roles in synaptic plasticity
and the excitodependent magnesium block, the removal of which requires depolarization of the membrane potential. Second, upon
activation, the NMDAR channel passes sodium and, importantly, calcium into the neuron. Calcium is the universal second messenger
in numerous intracellular signaling cascades and is critical in synaptic plasticity and mechanisms of neurotoxicity (288).
One of the major classes of ionotropic glutamate receptors is made up of the N-methyl-D-aspartate (NMDA) receptors, which require two agonists, glycine and glutamate, for activation and can pass calcium ions that
may mediate synaptic and neuronal plasticity. They are formed from complexes made by various combinations of the subunits
NR1 (with eight isoforms), NR2A-D, and NR3A-B and are found in most neurons of the brain and in various other cells. During
development, generally NMDA receptors with NR2B, NR2D, and NR3A are abundant and decrease during maturation, whereas those
with NR2A and NR2C increase. The function of NMDA receptors has been explored with a wide range of in vitro and in vivo studies,
employing both recombinant gene constructs and native receptors. NMDA receptor subunits contain various motifs that control
retention in the endoplasmic reticulum and trafficking through Golgi and other organelles to reach the cell membrane. Association
of NMDA receptors with PDZ domain–containing proteins such as PSD-95 and SAP102 may be particularly important to trafficking
and/or stabilization and function on the cell membrane. NMDA receptors on the cell membrane are sequestered mainly to the
postsynaptic membrane of synapses, but some populations remain in extrasynaptic domains, especially those receptors that contain
NR2B or NR2D.
To understand the molecular methods by which synapse formation occurs, it is critical to know the mechanisms by which all
the synaptic components can possibly arrive at a newly forming synapse. Methods of trafficking can range from diffusion through
the cytoplasm or in the plasma membrane to exo/endocytic cycling between bouts of transport along microtubules. In the case
of the postsynaptic density (PSD) of glutamatergic synapses, it appears that many of the necessary proteins arrive separately
and by different means. Here we examine the modes of transport and recruitment of a number of postsynaptic components to newly
forming, glutamatergic synapses.
Recent structural and functional studies of ionotropic glutamate receptors (iGluRs) have begun to offer rare insight into
the structure–function relationship for an integral membrane protein. In particular, advances in our understanding of iGluR
structure are providing an opportunity to interpret functional work in terms of potential conformational changes. Moreover,
working hypotheses derived from structural insight offer an opportunity to enrich and guide functional studies. This chapter
summarizes knowledge of glutamate receptor structure, with an emphasis on how it has shaped our functional understanding of
these receptors.
Synaptic adhesion-like molecules (SALMs) are a family of cell adhesion molecules involved in neurite outgrowth and synapse formation. Of the five family members, only SALM1, -2, and -3 contain a cytoplasmic C-terminal PDZ-binding motif. We have found that SALM1 is unique among the SALMs because deletion of its PDZ-binding motif (SALM1ΔPDZ) blocks its surface expression in heterologous cells. When expressed in hippocampal neurons, SALM1ΔPDZ had decreased surface expression in dendrites and the cell soma but not in axons, suggesting that the PDZ-binding domain may influence cellular trafficking of SALMs to specific neuronal locations. Endoglycosidase H digestion assays indicated that SALM1ΔPDZ is retained in the endoplasmic reticulum (ER) in heterologous cells. However, when the entire C-terminal tail of SALM1 was deleted, SALM1 was detected on the cell surface. Using serial deletions, we identified a region of SALM1 that contains a putative dileucine ER retention motif, which is not present in the other SALMs. Mutation of this DXXXLL motif allowed SALM1 to leave the ER and enhanced its surface expression in heterologous cells and neurons. An increase in the number of protrusions at the dendrites and cell body was observed when this SALM1 mutant was expressed in hippocampal neurons. With electron microscopy, these protrusions appeared to be irregular, enlarged spines and filopodia. Thus, enrichment of SALM1 on the cell surface affects dendritic arborization, and intracellular motifs regulate its dendritic versus axonal localization.
At glutamatergic synapses, NMDA receptors (NMDARs) are localized with other ionotropic glutamate receptors [AMPA receptors (AMPARs) and kainate receptors] and with metabotropic glutamate receptors. Targeting the necessary number of NMDARs to the proper sites at synapses is critical for normal glutamatergic neurotransmission and synaptic plasticity. Additional diversity of NMDAR responses arises from the complexity of subunit composition and variations in localization. Thus, the mechanisms of NMDAR trafficking and targeting must address the complex needs of neurons. For example, NMDARs must be transported to different subcellular sites because some receptors are localized to synaptic sites (pre- and postsynaptic) while others are localized extrasynaptically [1–4]. NMDAR subunit expression differs as a function of brain region and developmental age [5–7]. Subunit composition may determine subcellular localization, i.e., in adults, NR2A-containing receptors are enriched at synapses while extrasynaptic receptors are predominantly NR2B-containing complexes [1,2,4]. Furthermore, evidence indicates the incorporation of more than one type of NR2 subunit in each complex (tri-heteromeric NR1/NR2X/NR2Y) such as NR1/NR2A/NR2B receptors in hippocampal neuron synapses, NR1/NR2A/NR2C in cerebellar granule cell synapses, and NR1/NR2B/NR2D in substantia nigra dopaminergic neurons [8]. Finally, different NMDARs may be expressed at different synapses within the same neuron [4]. Thus, the mechanisms of NMDAR trafficking must be varied and well regulated to meet the needs of neurons.
The AMPA receptor, which is involved in most fast glutamatergic transmission in the mammalian brain and is expressed in most neurons, is made up of four subunits, GluR1-4. In situ hybridzation, immunocytochemistry studies, and single-cell PCR analyses show that the number and type of AMPA receptor subunits expressed vary among neuronal populations and that two to four subunits usually are expressed in each neuron. Neurons that express two or more subunits theoretically could produce multiple pentameric receptor complexes that differ in their subunit compositions, and these complexes could be targeted to different synaptic populations. To determine whether a single neuronal population produces multiple AMPA receptor complexes, we used a preparation of CA1/CA2 hippocampal pyramidal neurons and immunoprecipitation with subunit-specific antibodies to characterize the receptor complexes. The CA1/CA2 pyramidal neurons express high levels of GluR1-3 and receive multiple excitatory inputs, offering the possibility that distinct receptor complexes may be assembled and expressed selectively at different synaptic populations. Our results suggest the presence of two major populations of AMPA receptor complexes: those made up of GluR1 and GluR2 and those made up of GluR2 and GluR3. Very few complexes contained both GluR1 and GluR3, whereas approximately 8% of the total AMPA receptor complexes was homomeric GluR1. The integrity of the receptor complex was verified by measuring [3H]AMPA binding activity in the immunoprecipitated fractions. These results show that AMPA receptor complexes with different subunit compositions are present in CA1/CA2 pyramidal neurons and suggest an additional mechanism to regulate receptor expression in neurons.
The amino acids L-glutamate and glycine are essential agonists of the excitatory NMDA receptor, a subtype of the ionotropic glutamate receptor family. The native NMDA receptor is composed of two types of homologous membrane-spanning subunits, NR1 and NR2. Here, the numbers of glycine-binding NR1 and glutamate-binding NR2 subunits in the NMDA receptor hetero-oligomer were determined by coexpressing the wild-type (wt) NR1 with the low-affinity mutant NR1(Q387K), and the wt NR2B with the low-affinity mutant NR2BE387A, subunits in Xenopus oocytes. In both cases, analysis of the resulting dose-response curves revealed three independent components of glycine and glutamate sensitivity. These correspond to the respective wild-type and mutant affinities and an additional intermediate hybrid affinity, indicating the existence of three discrete receptor populations. Binomial analysis of these data indicates the presence of two glycine and two glutamate binding subunits in the functional receptor. In addition, we analyzed the inhibitory effects of the negative dominant NR1(R505K) and NR2BR493K mutants on maximal inducible whole-cell currents of wt NR1/NR2B receptors. The inhibition profiles obtained on expression of increasing amounts of these mutant proteins again were fitted best by assuming an incorporation of two NR1 and two NR2 subunits into the receptor hetero-oligomer. Our data are consistent with NMDA receptors being tetrameric proteins that are composed of four homologous subunits.
The AMPA receptor, which is involved in most fast glutamatergic transmission in the mammalian brain and is expressed in most neurons, is made up of four subunits, GluR1-4. In situ hybridzation, immunocytochemistry studies, and single-cell PCR analyses show that the number and type of AMPA receptor subunits expressed vary among neuronal populations and that two to four subunits usually are expressed in each neuron. Neurons that express two or more subunits theoretically could produce multiple pentameric receptor complexes that differ in their subunit compositions, and these complexes could be targeted to different synaptic populations. To determine whether a single neuronal population produces multiple AMPA receptor complexes, we used a preparation of CA1/CA2 hippocampal pyramidal neurons and immunoprecipitation with subunit-specific antibodies to characterize the receptor complexes. The CA1/CA2 pyramidal neurons express high levels of GluR1-3 and receive multiple excitatory inputs, offering the possibility that distinct receptor complexes may be assembled and expressed selectively at different synaptic populations. Our results suggest the presence of two major populations of AMPA receptor complexes: those made up of GluR1 and GluR2 and those made up of GluR2 and GluR3. Very few complexes contained both GluR1 and GluR3, whereas approximately 8% of the total AMPA receptor complexes was homomeric GluR1. The integrity of the receptor complex was verified by measuring [3H]AMPA binding activity in the immunoprecipitated fractions. These results show that AMPA receptor complexes with different subunit compositions are present in CA1/CA2 pyramidal neurons and suggest an additional mechanism to regulate receptor expression in neurons.
Cell surface expression of the NR1a subunit has been examined in mouse L cell lines permanently transfected with the complementary DNA for human NR1a or with the complementary DNAs for NR1a and NR2A. The expression of the subunits was under the control of the murine mammary tumour virus promoter and following induction of expression by dexamethazone both cell lines expressed high levels of the NR1a subunit as determined by immunofluorescence using permeabilized cells and immunoblotting of cell membranes with subunit specific antibodies. However, cell surface expression of the NR1a subunit was found only in the cells expressing both the NR1a and NR2A subunits. This was confirmed by cell surface biotinylation of the two cell lines and affinity isolation of the receptor subunits. To determine if this result was solely due to the use of a particular cell line and or the choice of expression vector, Cos-7 cells were transiently transfected with either NR1a or NR1a plus NR2A. Here too cell surface expression was only found following co-transfection of both subunits.
To investigate the mechanism of assembly of the mouse muscle acetylcholine receptor, we have expressed truncated N-terminal
fragments of the α and δ subunits in COS cells and have examined their ability to fold, to associate into heterodimers, and
to form a ligand-binding site. Truncated fragments of the α subunit that include all, part, or none of the first transmembrane
domain (M1) folded to acquire α-bungarotoxin binding activity. Neither the full-length α subunit nor any of the fragments
were expressed on the cell surface, although the shortest folded fragment lacking a transmembrane domain was secreted into
the medium. When coexpressed with the δ subunit, the α subunit fragment possessing M1 formed a heterodimer containing a ligand-binding
site, but shorter fragments, which lack transmembrane segments, did not associate with the δ subunit. N-terminal δ subunit
fragments gave similar results. An N-terminal δ subunit fragment that contains M1 associated with the α subunit to form a
heterodimer, while a fragment lacking M1 did not. These results show that a complete M1 domain is necessary for association
of truncated N-terminal α and δ subunits into a heterodimer with high affinity ligand binding activity.
Each organelle of the secretory pathway is required to selectively allow transit of newly synthesized secretory and plasma membrane proteins and also to maintain a unique set of resident proteins that define its structural and functional properties. In the case of the endoplasmic reticulum (ER), residency is achieved in two ways: (a) prevention of residents from entering newly forming transport vesicles and (b) retrieval of those residents that escape. The latter mechanism is directed by discrete retrieval motifs: Soluble proteins have a H/KDEL sequence at their carboxy-terminus; membrane proteins have a dibasic motif, either di-lysine or di-arginine, located close to the terminus of their cytoplasmic domain. Recently it was found that di-lysine motifs bind the complex of cytosolic coat proteins, COP I, and that this interaction functions in the retrieval of proteins from the Golgi to the ER. Also discussed are the potential roles this interaction may have in vesicular trafficking.
Modulation of N-methyl-D-aspartate receptors in the brain by protein phosphorylation may play a central role in the regulation of synaptic plasticity.
To examine the phosphorylation of the NR1 subunit of N-methyl-D-aspartate receptors in situ, we have generated several polyclonal antibodies that recognize the NR1 subunit only when specific serine residues are phosphorylated.
Using these antibodies, we demonstrate that protein kinase C (PKC) phosphorylates serine residues 890 and 896 and cAMP-dependent
protein kinase (PKA) phosphorylates serine residue 897 of the NR1 subunit. Activation of PKC and PKA together lead to the
simultaneous phosphorylation of neighboring serine residues 896 and 897. Phosphorylation of serine 890 by PKC results in the
dispersion of surface-associated clusters of the NR1 subunit expressed in fibroblasts, while phosphorylation of serine 896
and 897 has no effect on the subcellular distribution of NR1. The PKC-induced redistribution of the NR1 subunit in cells occurs
within minutes of serine 890 phosphorylation and reverses upon dephosphorylation. These results demonstrate that PKA and PKC
phosphorylate distinct residues within a small region of the NR1 subunit and differentially affect the subcellular distribution
of the NR1 subunit.
PDZ motifs are protein-protein interaction domains that often bind to COOH-terminal peptide sequences. The two PDZ proteins characterized in skeletal muscle, syntrophin and neuronal nitric oxide synthase, occur in the dystrophin complex, suggesting a role for PDZ proteins in muscular dystrophy. Here, we identify actinin-associated LIM protein (ALP), a novel protein in skeletal muscle that contains an NH2-terminal PDZ domain and a COOH-terminal LIM motif. ALP is expressed at high levels only in differentiated skeletal muscle, while an alternatively spliced form occurs at low levels in the heart. ALP is not a component of the dystrophin complex, but occurs in association with alpha-actinin-2 at the Z lines of myofibers. Biochemical and yeast two-hybrid analyses demonstrate that the PDZ domain of ALP binds to the spectrin-like motifs of alpha-actinin-2, defining a new mode for PDZ domain interactions. Fine genetic mapping studies demonstrate that ALP occurs on chromosome 4q35, near the heterochromatic locus that is mutated in fascioscapulohumeral muscular dystrophy.
Single-channel currents were recorded from mouse NR1-NR2B (zeta-epsilon2) receptors containing mixtures of wild-type and mutant subunits expressed in Xenopus oocytes. Mutant subunits had an asparagine-to-glutamine (N-to-Q) mutation at the N0 site of the M2 segment (NR1:598, NR2B:589). Receptors with pure N or Q NR1 and NR2 subunits generated single-channel currents with distinctive current patterns. Based on main and sublevel amplitudes, occupancy probabilities, and lifetimes, four patterns of current were identified, corresponding to receptors with the following subunit compositions (NR1/NR2): N/N, N/Q, Q/N, and Q/Q. Only one current pattern was apparent for each composition. When a mixture of N and Q NR2 subunits was coexpressed with pure mutant NR1 subunits, three single-channel current patterns were apparent. One pattern was the same as Q/Q receptors and another was the same as Q/N receptors. The third, novel pattern presumably arose from hybrid receptors having both N and Q NR2 subunits. When a mixture of N and Q NR1 subunits was coexpressed with pure mutant NR2 subunits, six single-channel current patterns were apparent. One pattern was the same as Q/Q receptors and another was the same as N/Q receptors. The four novel patterns presumably arose from hybrid receptors having both N and Q NR1 subunits. The relative frequency of NR1 hybrid receptor current patterns depended on the relative amounts of Q and N subunits that were injected into the oocytes. The number of hybrid receptor patterns suggests that there are two NR2 subunits per receptor and is consistent with either three or five NR1 subunits per receptor, depending on whether or not the order of mutant and wild-type subunits influences the current pattern. When considered in relation to other studies, the most straightforward interpretation of the results is that N-methyl-D-aspartate receptors are pentamers composed of three NR1 and two NR2 subunits.
Stargazer mice have spike-wave seizures characteristic of absence epilepsy, with accompanying defects in the cerebellum and inner ear. We describe here a novel gene, Cacng2, whose expression is disrupted in two stargazer alleles. It encodes a 36-kD protein (stargazin) with structural similarity to the gamma subunit of skeletal muscle voltage-gated calcium (Ca2+) channels. Stargazin is brain-specific and, like other neuronal Ca2+-channel subunits, is enriched in synaptic plasma membranes. In vitro, stargazin increases steady-state inactivation of alpha1 class A Ca2+ channels. The anticipated effect in stargazer mutants, inappropriate Ca2+ entry, may contribute to their more pronounced seizure phenotype compared with other mouse absence models with Ca2+-channel defects. The discovery that the stargazer gene encodes a gamma subunit completes the identification of the major subunit types for neuronal Ca2+ channels, namely alpha1, alpha2delta, beta and gamma, providing a new opportunity to understand how these channels function in the mammalian brain and how they may be targeted in the treatment of neuroexcitability disorders.
The intracellular trafficking, assembly, and cell surface targeting of the human N-methyl-D-aspartate receptor subunits NR1a and NR2A has been studied using both transiently and permanently transfected mammalian cell lines. The expression of either NR1a or NR2A alone does not result in significant cell surface expression of either subunit as determined by cell surface biotinylation and immunofluorescence staining. When NR1a is expressed alone large intracellular accumulations of the subunit are formed which do not co-localize with the golgi apparatus markers protein p58 and wheat germ agglutinin, but do co-localize with the endoplasmic reticulum marker calreticulin. Co-expression of NR1a and NR2A results in a reduction of these intracellular accumulations and the appearance of both subunits on the cell surface. Immunoprecipitation of NR1a from in vitro translated subunit proteins showed that NR2A could only be immunoprecipitated with NR1a when both subunits were co-synthesized in the presence of microsomes. When cells expressing NR1a and NR2A were incubated with [35S]methionine in the presence of Brefeldin-A, a drug which prevents protein transport from the endoplasmic reticulum, NR2A could be immunoprecipitated by an antiserum specific for NR1a. Together these results suggest that the NMDA receptor subunits are assembled in the endoplasmic reticulum and that co-synthesis of the subunits is necessary for their association and their successful cell surface targeting.
Glutamate receptors (GluR) are oligomeric protein complexes formed by the assembly of four or perhaps five subunits. The rules that govern the selectivity of this process are not well understood. Here, we expressed combinations of subunits from two related GluR subfamilies in COS7 cells, the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors. By co-immunoprecipitation experiments, we assessed the ability of AMPA receptor subunits to assemble into multimeric complexes. Subunits GluR1-4 associated with indistinguishable efficiency with each other, whereas the kainate receptor subunits GluR6 and 7 showed a much lower degree of association with GluR1. Using chimeric receptors and truncation fragments of subunits, we show that this assembly specificity is determined by N-terminal regions of these subunits and that the most N-terminal domain of GluR2 together with a membrane anchor efficiently associates with GluR1.
The extracellular part of ionotropic glutamate receptor (iGluR) subunits can be divided into a conserved two-lobed ligand-binding
domain (“S1S2”) and an N-terminal ∼400-residue segment of unknown function (“X domain”) which shows high sequence variation
among subunits. To investigate the structure and properties of the N-terminal domain, we have now produced affinity-tagged
recombinant fragments which represent the X domain of the GluRD subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic
acid (AMPA)-selective glutamate receptors either alone or covalently linked to the ligand-binding domain (“XS1S2”). These
fragments were expressed in insect cells as secreted soluble proteins and were recognized by a conformation-specific anti-GluRD
monoclonal antibody. A hydrodynamic analysis of the purified fragments revealed them to be dimers, in contrast to the S1S2
ligand-binding domain which is monomeric. The X domain did not bind radiolabeled AMPA or glutamate nor did its presence affect
the ligand binding properties of the S1S2 domain. Our findings demonstrate that the N-terminal domain of AMPA receptor can
be expressed as a soluble polypeptide and suggest that subunit interactions in iGluR may involve the extracellular domains.
A variety of quality control mechanisms operate in the endoplasmic reticulum and in downstream compartments of the secretory
pathway to ensure the fidelity and regulation of protein expression during cell life and differentiation. As a rule, only
proteins that pass a stringent selection process are transported to their target organelles and compartments. If proper maturation
fails, the aberrant products are degraded. Quality control improves folding efficiency by retaining proteins in the special
folding environment of the endoplasmic reticulum, and it prevents harmful effects that could be caused by the deployment of
incompletely folded or assembled proteins.
Postsynaptic density-95 (PSD-95/SAP-90) is a palmitoylated peripheral membrane protein that scaffolds ion channels at excitatory synapses. To elucidate mechanisms for postsynaptic ion channel clustering, we analyzed the cellular trafficking of PSD-95. We find that PSD-95 transiently associates with a perinuclear membranous compartment and traffics with vesiculotubular structures, which migrate in a microtubule-dependent manner. Trafficking of PSD-95 with these vesiculotubular structures requires dual palmitoylation, which is specified by five consecutive hydrophobic residues at the NH(2) terminus. Mutations that disrupt dual palmitoylation of PSD-95 block both ion channel clustering by PSD-95 and its synaptic targeting. Replacing the palmitoylated NH(2) terminus of PSD-95 with alternative palmitoylation motifs at either the NH(2) or COOH termini restores ion channel clustering also induces postsynaptic targeting, respectively. In brain, we find that PSD-95 occurs not only at PSDs but also in association with intracellular smooth tubular structures in dendrites and spines. These data imply that PSD-95 is an itinerant vesicular protein; initial targeting of PSD-95 to an intracellular membrane compartment may participate in postsynaptic ion channel clustering by PSD-95.
PSD-95, DLG, ZO-1 (PDZ) domain-mediated protein interactions have been shown to play important roles in the regulation of glutamate receptor function at excitatory synapses. Recent studies demonstrating the rapid regulation of AMPA receptor function during synaptic plasticity have suggested that AMPA receptor interaction with PDZ domain-containing proteins may be dynamically modulated. Here we show that PKC phosphorylation of the AMPA receptor GluR2 subunit differentially modulates its interaction with the PDZ domain-containing proteins GRIP1 and PICK1. The serine residue [serine-880 (Ser880)] in the GluR2 C-terminal sequence (IESVKI) critical for PDZ domain binding is a substrate of PKC and is phosphorylated in vivo. In vitro binding and coimmunoprecipitation studies show that phosphorylation of serine-880 within the GluR2 PDZ ligand significantly decreases GluR2 binding to GRIP1 but not to PICK1. Immunostaining of cultured hippocampal neurons demonstrates that the Ser880-phosphorylated GluR2 subunits are enriched and colocalized with PICK1 in the dendrites, with very little staining observed at excitatory synapses. Interestingly, PKC activation in neurons increases the Ser880 phosphorylation of GluR2 subunits and recruits PICK1 to excitatory synapses. Moreover, PKC stimulation in neurons results in rapid internalization of surface GluR2 subunits. These results suggest that GluR2 phosphorylation of serine-880 may be important in the regulation of the AMPA receptor internalization during synaptic plasticity.
Stargazer, an ataxic and epileptic mutant mouse, lacks functional AMPA (alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate) receptors on cerebellar granule cells. Stargazin, the mutated protein, interacts with both AMPA receptor subunits and synaptic PDZ proteins, such as PSD-95. The interaction of stargazin with AMPA receptor subunits is essential for delivering functional receptors to the surface membrane of granule cells, whereas its binding with PSD-95 and related PDZ proteins through a carboxy-terminal PDZ-binding domain is required for targeting the AMPA receptor to synapses. Expression of a mutant stargazin lacking the PDZ-binding domain in hippocampal pyramidal cells disrupts synaptic AMPA receptors, indicating that stargazin-like mechanisms for targeting AMPA receptors may be widespread in the central nervous system.
PDZ domains are modular protein interaction domains that bind in a sequence-specific fashion to short C-terminal peptides or internal peptides that fold in a beta-finger. The diversity of PDZ binding specificities can be explained by variable amino acids lining the peptide-binding groove of the PDZ domain. Abundantly represented in Caenorhabditis elegans, Drosophila melanogaster, and mammalian genomes, PDZ domains are frequently found in multiple copies or are associated with other protein-binding motifs in multidomain scaffold proteins. PDZ-containing proteins are typically involved in the assembly of supramolecular complexes that perform localized signaling functions at particular subcellular locations. Organization around a PDZ-based scaffold allows the stable localization of interacting proteins and enhances the rate and fidelity of signal transduction within the complex. Some PDZ-containing proteins are more dynamically regulated in distribution and may also be involved in the trafficking of interacting proteins within the cell.
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.
Activity-mediated changes in the strength of synaptic communication are
important for the establishment of proper neuronal connections during development
and for the experience-dependent modification of neural circuitry that is
believed to underlie all forms of behavioural plasticity. Owing to the wide-ranging
significance of synaptic plasticity, considerable efforts have been made to
identify the mechanisms by which synaptic changes are triggered and expressed.
New evidence indicates that one important expression mechanism of several
long-lasting forms of synaptic plasticity might involve the physical transport
of AMPA-type glutamate receptors in and out of the synaptic membrane. Here,
we focus on the rapidly accumulating evidence that AMPA receptors undergo
regulated endocytosis, which is important for long-term depression.
Activity-dependent changes in synaptic function are believed to underlie the formation of memories. Two prominent examples are long-term potentiation (LTP) and long-term depression (LTD), whose mechanisms have been the subject of considerable scrutiny over the past few decades. Here we review the growing literature that supports a critical role for AMPA receptor trafficking in LTP and LTD, focusing on the roles proposed for specific AMPA receptor subunits and their interacting proteins. While much work remains to understand the molecular basis for synaptic plasticity, recent results on AMPA receptor trafficking provide a clear conceptual framework for future studies.
Abstract : Phosphorylation of the glutamate receptor is an important mechanism of synaptic plasticity. Here, we show that the C terminus of GluR2 of the α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor is phosphorylated by protein kinase C and that serine-880 is the major phosphorylation site. This phosphorylation also occurs in human embryonic kidney (HEK) cells by addition of 12-O-tetradecanoylphorbol 13-acetate. Our immunoprecipitation experiment revealed that the phosphorylation of serine-880 in GluR2 drastically reduced the affinity for glutamate receptor-interacting protein (GRIP), a synaptic PDZ domain-containing protein, in vitro and in HEK cells. This result suggests that modulation of serine-880 phosphorylation in GluR2 controls the clustering of AMPA receptors at excitatory synapses and consequently contributes to synaptic plasticity.
Synaptic junctions are highly specialized structures designed to promote the rapid and efficient transmission of signals from the presynaptic terminal to the postsynaptic membrane within the central nervous system. Proteins containing PDZ domains play a fundamental organizational role at both the pre- and postsynaptic plasma membranes. This review focuses on recent advances in our understanding of the mechanisms underlying the assembly of synapses in the central nervous system.
Brief periods of strong neuronal activity induce long-lasting changes in synaptic function. This synaptic plasticity is thought to play important roles in learning and memory. One example — long-term potentation in the CA1 region of the hippocampus — has been studied extensively, and conflicting views regarding the underlying mechanisms have emerged. Recent findings, regarding basic properties of synaptic transmission, appear to reconcile these diverging views.
Recent findings suggest that a combination of chaperonin-assisted and unassisted mechanisms operate in protein folding in the cytosol. While nascent chain-binding chaperones, such as Hsp70, could have a general role in maintaining the folding competence of translating polypeptide chains, the contribution of the cylindrical chaperonin complexes to overall folding is limited to a subset of aggregation-sensitive polypeptides. The majority of bacterial proteins are relatively small and they are synthesized rapidly and folded independently of the chaperonin GroEL in a posttranslational manner. Eukaryotes have a proportionally larger number of multi-domain proteins than bacteria. The individual domains of these proteins can be folded cotranslationally and sequentially. The use of this mechanism explains how large proteins fold independently of a chaperonin and could have been crucial in the evolution of a wide array of modular polypeptides in eukaryotes.
Activation of the transcription factor CREB is thought to be important in the formation of long-term memory in several animal species. The phosphorylation of a serine residue at position 133 of CREB is critical for activation of CREB. This phosphorylation is rapid when driven by brief synaptic activity in hippocampal neurons. It is initiated by a highly local, rise in calcium ion concentrations near the cell membrane, but culminates in the activation of a specific calmodulin-dependent kinase known as CaMK IV, which is constitutively present in the neuronal nucleus. It is unclear how the signal is conveyed from the synapse to the nucleus. We show here that brief bursts of activity cause a swift (approximately 1 min) translocation of calmodulin from the cytoplasm to the nucleus, and that this translocation is important for the rapid phosphorylation of CREB. Certain Ca2+ entry systems (L-type Ca2+ channels and NMDA receptors) are able to cause mobilization of calmodulin, whereas others (N- and P/Q-type Ca2+ channels) are not. This translocation of calmodulin provides a form of cellular communication that combines the specificity of local Ca2+ signalling with the ability to produce action at a distance.
The structure of glutamate receptor-channel (GluR) subunits has recently been shown to differ from that of other ligand-gated channels and to contain a voltage-gated channel-like pore-forming motif. The view that the structure of GluR complexes is similar to the pentameric structure of other ligand-gated channels was questioned here. Studies of the response properties of the GluR1 subunit of the AMPA subtype of GluRs, co-expressed in Xenopus oocytes with its L646A mutant, which differs only by a greatly reduced sensitivity to quisqualate, provide new evidence suggesting that the GluR1 homomeric receptor channel has a tetrameric structure.
The T cell surface glycoproteins T4 and T8 are thought to mediate efficient cell-cell interactions in the immune system and in this way may be responsible for the appropriate targeting of subpopulations of T cells. We have used gene transfer combined with subtractive hybridization to isolate both cDNA and functional genomic clones encoding the T8 protein. The sequence of the cDNA reveals that T8 is a transmembrane protein with an N-terminal domain which shares significant homology to immunoglobulin variable region light chains. This immunoglobulin-like structure is likely to be important in the function of T8 during differentiation and in the course of the immune response.
An in situ study of mRNAs encoding NMDA receptor subunits in the developing rat CNS revealed that, at all stages, the NR1 gene is expressed in virtually all neurons, whereas the four NR2 transcripts display distinct expression patterns. NR2B and NR2D mRNAs occur prenatally, whereas NR2A and NR2C mRNAs are first detected near birth. All transcripts except NR2D peak around P20. NR2D mRNA, present mainly in midbrain structures, peaks around P7 and thereafter decreases to adult levels. Postnatally, NR2B and NR2C transcript levels change in opposite directions in the cerebellar internal granule cell layer. In the adult hippocampus, NR2A and NR2B mRNAs are prominent in CA1 and CA3 pyramidal cells, but NR2C and NR2D mRNAs occur in different subsets of interneurons. Recombinant binary NR1-NR2 channels show comparable Ca2+ permeabilities, but marked differences in voltage-dependent Mg2+ block and in offset decay time constants. Thus, the distinct expression profiles and functional properties of NR2 subunits provide a basis for NMDA channel heterogeneity in the brain.
The temperature sensitivity of nicotinic acetylcholine receptors (AChRs) from T. californica was used to identify steps in AChR subunit folding and oligomerization. Assembly intermediates were isolated by lowering to an assembly-permissive temperature. The earliest identifiable assembly intermediates, alpha beta gamma trimers, form minutes after subunit synthesis. alpha beta gamma delta tetramers are formed slowly by the addition of delta subunits to trimers, and finally a second alpha subunit is added to form alpha 2 beta gamma delta pentamers. Between these oligomerization steps, subunits fold as monitored by alpha-bungarotoxin-binding site formation, appearance of antigenic epitopes, changes in apparent molecular weight, and changes in detergent solubility. Subunit folding requires specific combinations of subunits and correlates in time with subunit additions, suggesting that these subunit folding events contribute to subunit recognition site formation during assembly.
Exciting new milestones in glutamate receptor (GluR) channel research include the following: the cloning of N-methyl-D-aspartate (NMDA) receptors; delineation of molecular determinants for ion flow through glutamate-gated channels; the discovery that Ca2+ permeability of non-NMDA receptor channels is determined by RNA editing; the construction of antibodies and their use in immunocytochemical localizations of alpha-amino-3-hydroxy-5-methyl isoxazole-4-propionic acid (AMPA) receptor subunits in the rat brain; and the return to prominence of the high-affinity kainate site with the publication of cDNA sequences for subunits (GluR-5, -6, -7; KA-1, -2) constituting subtypes of this site. Major unresolved issues comprise the transmembrane topology and subunit stoichiometries of native receptor channels.
Stimulus-linked RNA and protein synthesis is required for establishment of long-term neuroplasticity. To identify molecular mechanisms underlying long-term neuroplasticity, we have used differential cDNA techniques to clone a novel immediate-early gene (IEG) that is rapidly induced in neurons of the hippocampus and cortex by physiological synaptic activity. Analysis of the deduced amino acid sequence indicates homology to members of the pentraxin family of secreted lectins that include C-reactive protein and serum amyloid P component. Regions of homology include an 8 amino acid "pentraxin signature" sequence and a characteristic pentraxin calcium-binding domain. We have termed this gene and the encoded protein Narp (from neuronal activity-regulated pentraxin). Biochemical analyses confirm the presence of a functional signal sequence, and Narp is secreted by transfected COS-1 cells in culture. Additionally, Narp binds to agar matrix in a calcium-dependent manner consistent with the lectin properties of the pentraxin family. When cocultured with Narp-secreting COS-1 cells, neurons of cortical explants exhibit enhanced growth of neuronal dendritic processes. Neurite outgrowth-promoting activity is also observed using partially purified Narp and can be specifically immunodepleted, demonstrating that Narp is the active principle. Narp is fully active at a concentration of approximately 40 ng/ml, indicating a potency similar to known peptide growth factors. Because Narp is rapidly regulated by neuronal activity, its lectin and growth-promoting activities are likely to play role in the modification of cellular properties that underlie long-term plasticity.
AMPA glutamate receptors mediate the majority of rapid excitatory synaptic transmission in the central nervous system and play a role in the synaptic plasticity underlying learning and memory. AMPA receptors are heteromeric complexes of four homologous subunits (GluR1-4) that differentially combine to form a variety of AMPA receptor subtypes. These subunits are thought to have a large extracellular amino-terminal domain, three transmembrane domains and an intracellular carboxy-terminal domain. AMPA receptors are localized at excitatory synapses and are not found on adjacent inhibitory synapses enriched in GABA(A) receptors. The targeting of neurotransmitter receptors, such as AMPA receptors, and ion channels to synapses is essential for efficient transmission. A protein motif called a PDZ domain is important in the targeting of a variety of membrane proteins to cell-cell junctions including synapses. Here we identify a synaptic PDZ domain-containing protein GRIP (glutamate receptor interacting protein) that specifically interacts with the C termini of AMPA receptors. GRIP is a new member of the PDZ domain-containing protein family which has seven PDZ domains and no catalytic domain. GRIP appears to serve as an adapter protein that links AMPA receptors to other proteins and may be critical for the clustering of AMPA receptors at excitatory synapses in the brain.
Previous studies have shown that both the Fasciclin II (Fas II) cell adhesion molecule and the Shaker potassium channel are localized at the Drosophila neuromuscular junction, where they function in the growth and plasticity of the synapse. Here, we use the GAL4-UAS system to drive expression of the chimeric proteins CD8-Fas II and CD8-Shaker and show that the C-terminal sequences of both Fas II and Shaker are necessary and sufficient to drive the synaptic localization of a heterologous protein. Moreover, we show that the PDZ-containing protein Discs-Large (Dlg) controls the localization of these proteins, most likely through a direct interaction with their C-terminal amino acids. Finally, transient expression studies show that the pathway these proteins take to the synapse involves either an active clustering or a selective stabilization in the synaptic membrane.
Ion channels and associated signal transduction cascades are clustered at excitatory synapses by PSD-95 and related PDZ-containing proteins. Mechanisms that target PSD-95 to synaptic membranes, however, are unknown. Here, PSD-95 is shown to partition as an integral membrane protein in brain homogenates. Metabolic labeling of brain slices or cultured cells demonstrates that PSD-95 is modified by thioester-linked palmitate, a long chain fatty acid that targets proteins to cell membranes. In fact, PSD-95 is a major palmitoylated protein in intact cells, and palmitoylated PSD-95 partitions exclusively with cell membranes. Mutagenesis indicates that palmitoylation of PSD-95 occurs on conserved N-terminal cysteines 3 and 5. Palmitoylation-deficient mutants of PSD-95 do not partition as integral membrane proteins and do not participate in PDZ-ion channel interactions in vivo. This work identifies palmitoylation as a critical regulatory mechanism for receptor interactions with PSD-95.
Proper ion channel function often requires specific combinations of pore-forming alpha and regulatory beta subunits, but little is known about the mechanisms that regulate the surface expression of different channel combinations. Our studies of ATP-sensitive K+ channel (K(ATP)) trafficking reveal an essential quality control function for a trafficking motif present in each of the alpha (Kir6.1/2) and beta (SUR1) subunits of the K(ATP) complex. We show that this novel motif for endoplasmic reticulum (ER) retention/retrieval is required at multiple stages of K(ATP) assembly to restrict surface expression to fully assembled and correctly regulated octameric channels. We conclude that exposure of a three amino acid motif (RKR) can explain how assembly of an ion channel complex is coupled to intracellular trafficking.
Subcellular localization of the NMDA receptor NR1 splice forms was studied by expressing individual splice variants and their epitope-tagged derivatives in mouse fibroblasts and in hippocampal neurons. When NR1 splice variants were expressed in fibroblasts, the amount of NR1 molecules expressed on the cell surface varied among forms with different C-terminal cytoplasmic domains. The splice forms with the longest C-terminal cytoplasmic tail (NR1-1a and NR1-1b) showed the lowest amount of cell surface expression, and the splice forms with the shortest C-terminal cytoplasmic tail (NR1-4a and NR1-4b) showed the highest cell surface expression. Cell surface expression of NR1 was enhanced by the coexpression of the NR2 subunit. We measured the glutamate-induced increase of calcium concentration in fibroblasts expressing one of the NR1 splice forms and the NR2B subunit. The increase of calcium concentration after glutamate application had a positive correlation with the amount of NR1 splice forms expressed on the cell surface. When epitope-tagged NR1 splice variants were expressed in primary hippocampal neurons using recombinant adenoviruses, we also observed the differential expression on the cell surface between splice variants. These results suggest that the splicing of the C-terminal domain of the NR1 subunit regulates the cell surface expression of the functional NMDA receptors.
Compounds known to disrupt exocytosis or endocytosis were introduced into CA1 pyramidal cells while monitoring excitatory postsynaptic currents (EPSCs). Disrupting exocytosis or the interaction of GluR2 with NSF caused a gradual reduction in the AMPAR EPSC, while inhibition of endocytosis caused a gradual increase in the AMPAR EPSC. These manipulations had no effect on the NMDAR EPSC but prevented the subsequent induction of LTD. These results suggest that AMPARs, but not NMDARs, cycle into and out of the synaptic membrane at a rapid rate and that certain forms of synaptic plasticity may utilize this dynamic process.
A variety of mutant polypeptides that are associated with human disease are targeted for degradation by an endoplasmic reticulum (ER) quality control system. In addition, physiological signals and viral gene products can target the degradation of several ER resident proteins and secreted proteins passing through the ER. Although the mechanism of protein quality control and the site of degradation were obscure, recent data indicate that degradation requires the cytosolic proteasome. Biochemical and genetic analyses have indicated that both lumenal and integral membrane proteins are selected for proteolysis and exported to the cytosol by a process that in several cases requires ER associated molecular chaperones.
Surface expression of GABA(B) receptors requires heterodimerization of GB1 and GB2 subunits, but little is known about mechanisms that ensure efficient heterodimer assembly. We found that expression of the GB1 subunit on the cell surface is prevented through a C-terminal retention motif RXR(R); this sequence is reminiscent of the ER retention/retrieval motif RKR identified in subunits of the ATP-sensitive K+ channel. Interaction of GB1 and GB2 through their C-terminal coiled-coil alpha helices masks the retention signal in GB1, allowing the plasma membrane expression of the assembled complexes. Because individual GABA(B) receptor subunits and improperly assembled receptor complexes are not functional even if expressed on the cell surface, we conclude that a trafficking checkpoint ensures efficient assembly of functional GABA(B) receptors.
The NMDA receptor NR1 subunit has four splice variants that differ in their C-terminal, cytoplasmic domain. We investigated the contribution of the C-terminal cassettes, C0, C1, C2, and C2', to trafficking of NR1 in heterologous cells and neurons. We identified an ER retention signal (RRR) in the C1 cassette of NR1, which is similar to the RXR motif in ATP-sensitive K(+) channels (Zerangue et al., 1999). We found that surface expression of NR1-3, which contains C1, is due to a site on the C2' cassette, which includes the terminal 4 amino acid PDZ-interacting domain. This site suppresses ER retention of the C1 cassette and leads to surface expression. These findings suggest a role for PDZ proteins in facilitating the transition of receptors from an intracellular pool to the surface of the neuron.
As in the case of many ligand-gated ion channels, the biochemical and electrophysiological properties of the ionotropic glutamate receptors have been studied extensively. Nevertheless, we still do not understand the molecular mechanisms that harness the free energy of agonist binding, first to drive channel opening, and then to allow the channel to close (desensitize) even though agonist remains bound. Recent crystallographic analyses of the ligand-binding domains of these receptors have identified conformational changes associated with agonist binding, yielding a working hypothesis of channel function. This opens the way to determining how the domains and subunits are assembled into an oligomeric channel, how the domains are connected, how the channel is formed, and where it is located relative to the ligand-binding domains, all of which govern the processes of channel activation and desensitization.
Channels and receptors on the cell surface mediate neuronal signaling. It is therefore important to understand how their surface density is controlled. Recent studies on the trafficking of potassium channels and neurotransmitter receptors have revealed unexpected complexity in the regulation of transport from the endoplasmic reticulum to the Golgi apparatus, raising the possibility that the surface composition of channels and receptors may be adjusted by controlling their export from the endoplasmic reticulum.
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