Glutamate receptor IIA and Glutamate receptor IIB
Ionotropic glutamate receptors (iGluRs) mediate most excitatory synaptic signalling between neurons. Binding of the neurotransmitter glutamate causes a conformational change in these receptors that gates open a transmembrane pore through which ions can pass. The gating of iGluRs is crucially dependent on a conserved amino acid that was first identified in the 'lurcher' ataxic mouse. Through a screen for modifiers of iGluR function in a transgenic strain of C. elegans expressing a GLR-1 subunit containing the lurcher mutation, suppressor of lurcher (sol-1) was identified. This gene encodes a transmembrane protein that is predicted to contain four extracellular beta-barrel-forming domains known as CUB domains. SOL-1 and GLR-1 are colocalized at the cell surface and can be co-immunoprecipitated. By recording from neurons expressing GLR-1, it is shown that SOL-1 is an accessory protein that is selectively required for glutamate-gated currents. It is proposed that SOL-1 participates in the gating of non-NMDA iGluRs, thereby providing a previously unknown mechanism of regulation for this important class of neurotransmitter receptor (Zheng, 2004).
AMPA receptors (AMPARs) are a major subtype of ionotropic glutamate receptors (iGluRs) that mediate rapid excitatory synaptic transmission in the vertebrate brain. Putative AMPARs are also expressed in the nervous system of invertebrates. In C. elegans, the GLR-1 receptor subunit is expressed in neural circuits that mediate avoidance behaviors and is required for glutamate-gated current in the AVA and AVD interneurons. Glutamate-gated currents can be recorded from heterologous cells that express vertebrate AMPARs; however, when C. elegans GLR-1 is expressed in heterologous cells, little or no glutamate-gated current is detected. This finding suggests that other receptor subunits or auxiliary proteins are required for function. This study identifies Ce STG-1, a C. elegans stargazin-like protein, and shows that expression of Ce STG-1 together with GLR-1 and the CUB-domain protein SOL-1 reconstitutes glutamate-gated currents in Xenopus oocytes. Ce STG-1 and homologues cloned from Drosophila (Dro STG1; CG33670) and Apis mellifera (Apis STG1) have evolutionarily conserved functions and can partially substitute for one another to reconstitute glutamate-gated currents from rat, Drosophila, and C. elegans. Furthermore, Ce STG-1 and Apis STG1 are primarily required for function independent of possible roles in promoting the surface expression of invertebrate AMPARs (Walker, 2006a).
The neurotransmitter glutamate mediates excitatory synptic transmission by activating ionotropic glutamate receptors (iGluRs). In C. elegans, the GLR-1 receptor subunit is required for glutamate-gated current in a subset of interneurons that control avoidance behaviors. Current mediated by GLR-1-containing iGluRs depends on SOL-1, a transmembrane CUB-domain protein that immunoprecipitates with GLR-1. Reconstitution of glutamate-gated current in heterologous cells depends on three proteins, STG-1 (a C. elegans stargazin-like protein), SOL-1, and GLR-1. This study used genetic and pharmacological perturbations along with rapid perfusion electrophysiological techniques to demonstrate that SOL-1 functions to slow the rate and limit the extent of receptor desensitization as well as to enhance the recovery from desensitization. A SOL-1 homologue from Drosophila has been identified (CG31218) and it is shown that Dro SOL1 has a conserved function in promoting C. elegans glutamate-gated currents. SOL-1 homologues may play critical roles in regulating glutamatergic neurotransmission in more complex nervous systems (Walker, 2006b).
In Drosophila, a number of iGluRs have been cloned, including Dro GluRIA, which has significant identity with C. elegans GLR-1 and is expressed in the nervous system. However, only small kainate-gated and virtually no glutamate-gated currents can be recorded from oocytes that express Dro GluRIA. No glutamate-gated current was detected when Dro GluRIA was expressed alone. Whether the lack of current was secondary to a requirement for a stargazin-like auxiliary protein was tested. Unlike GLR-1-mediated currents, coexpression of Dro GluRIA with vertebrate stargazin, Ce STG-1, or SOL-1 and Ce STG-1 did not significantly increase glutamate-gated current. However, small currents were recorded with coexpression of Dro GluRIA and Dro STG1, and significantly larger currents with coexpression of Dro GluRIA and Apis STG1. The onset of the current observed with coexpression of Dro STG1 was very slow, indicating that the majority of the current is likely a consequence of secondary activation of an endogenous Ca2+-activated chloride conductance. Consistent with this idea, substitution of barium for calcium in the extracellular solution dramatically reduced the amplitude of currents in response to glutamate application. These results indicate that there are important functional differences between vertebrate, C. elegans, and insect stargazin molecules in their ability to promote Dro GluRIA-mediated current. The large glutamate-gated current observed with coexpression of Apis STG1 could not be explained by surface delivery of receptors; in fact, it was found that the fractional surface expression of Dro GluRIA alone was 2- to 3-fold higher than when coexpressed with Apis STG1 (Walker, 2006a).
Most rapid excitatory synaptic signaling in the brain is mediated by postsynaptic ionotropic glutamate receptors (iGluRs) that are gated open by the neurotransmitter glutamate. In Caenorhabditis elegans, sol-1 encodes a CUB-domain transmembrane protein that is required for currents that are mediated by the GLR-1 iGluR. Mutations in sol-1 do not affect GLR-1 expression, localization, membrane insertion, or stabilization at synapses, suggesting that SOL-1 is required for iGluR function. This study provides evidence that SOL-1 is an auxiliary subunit that modulates the gating of GLR-1 receptors. Mutant variants of GLR-1 with altered gating partially restore glutamate-gated current and GLR-1-dependent behaviors in sol-1 mutants. Domain analysis of SOL-1 indicates that extracellular CUB domain 3 is required for function and that a secreted variant partially restores glutamate-gated currents and behavior. Also, it is shown that endogenous glutamatergic synaptic currents are absent in sol-1 mutants. These data suggest that GLR-1 iGluRs are not simply stand-alone molecules and require the SOL-1 auxiliary protein to promote the open state of the receptor. This analysis presents the possibility that glutamatergic signaling in other organisms may be similarly modified by SOL-1-like transmembrane proteins (Zheng, 2006).
Stargazer, an ataxic and epileptic mutant mouse, lacks functional AMPA 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 (Chen, 2000).
Excitatory synapses in the central nervous system release
glutamate onto a number of receptor subtypes. The
principal ionotropic glutamate receptors include AMPA receptors and NMDARs. AMPARs mediate moment-to-moment signalling, whereas
NMDARs initiate synaptic plasticity. Recent studies
emphasize remarkable differences in synaptic expression of these
receptors. NMDARs are relatively fixed components of
the postsynaptic density (PSD), whereas AMPARs are more loosely
associated and their density at synapses is tightly
controlled by neuronal activity1-8. That the number of AMPARs at the
synapse is regulated independently of NMDARs
raises intriguing questions concerning mechanisms involved in
synaptic targeting of glutamate receptors and the role that
this plasticity plays in learning and memory (Chen, 2000).
This independent regulation of synaptic AMPARs versus NMDARs is
clearly manifested in the stargazer mutant mouse,
which exhibits seizures and cerebellar ataxia. Among the defects
identified in the cerebellum is the lack of functional
AMPARs on granule cells. The defective protein stargazin was
recently identified as a relative of the -1 subunit of the
skeletal muscle calcium channel. More recently, a family of
subunits has been identified, each with distinct
expression patterns in the brain. Why stargazin is necessary
for functional AMPARs in cerebellar granule cells has
remained mysterious and is the focus of this study (Chen, 2000).
Whether the defect in AMPARs in the stargazer
mouse results from abnormal cerebellar circuitry or
alternatively is an autonomous granule cell defect was examined. AMPARs were evaluated in granule cell cultures, which lack the
normal excitatory mossy fiber input, as well as Purkinje cells, the
primary target for granule cells. In +/stg cultures,
spontaneous rapid inward currents are routinely recorded from
individual granule cells, presumably generated by synapses
between granule cells. These events are largely mediated by
the action potential dependent release of glutamate
onto AMPARs, since their frequency is greatly reduced by tetrodotoxin
(TTX) and they are abolished by the AMPAR
antagonist CNQX. In striking contrast, stg/stg
neurons exhibit essentially no spontaneous activity (Chen, 2000).
The stg/stg neurons clearly form excitatory synaptic connections since
NMDAR currents can be recorded when Mg2+ is
removed from, and CNQX and glycine are added to, the solution. As expected for NMDARs, these currents are considerably slower than those mediated by AMPAR
currents and are blocked by the NMDAR
antagonist AP5. There was no difference in the
amplitude of NMDAR-mediated events in +/stg cells,
or in the frequency of these events (Chen, 2000).
In cerebellar cultures from +/stg mouse, the AMPAR subunit GluR4
forms discrete synaptic puncta that colocalize with
the presynaptic marker synaptophysin. By contrast, few
synaptic GluR4 puncta are evident in stg/stg cells.
Despite lacking synaptic GluR4 puncta, the cultured stg/stg granule
cells are decorated with presynaptic synaptophysin
puncta. To assess the synaptic localization of AMPAR subunits at
mossy fibre synapses in the intact cerebellar glomeruli,
post embedding immunogold electron-microscopic analysis was used.
Granule cell synapses in stg/stg cerebellum are
virtually devoid of GluR2/3 labelling, whereas these synapses in
+/stg are labelled abundantly. Similar results
were obtained with an antibody to the GluR4 subunit; most synapses in
the +/stg mouse are GluR4-positive,
whereas synapses in the stg/stg mouse are rarely labelled.
A 'blind' quantitative analysis showed roughly a 10-fold decrease in the number of GluR2/3-reactive particles at stg/stg synapses (Chen, 2000).
In contrast to the defect in synaptic GluR 2/3 labelling, the NMDAR
subunit NR1 labelling was increased in the stg/stg
mutant. The cytoplasmic GluR2/3 labelling was low
in both +/stg and stg/stg granule cells, making quantification
difficult, but no obvious difference was noted. The presence
of cytoplasmic staining indicates that AMPARs are present in the
stg/stg granule cells, in agreement with Western blot
analysis. Finally, there was no obvious difference in the pre- or
post-synaptic morphology of the mossy fiber to granule
cell synapse between the stg/stg mice and +/stg mice (Chen, 2000).
Since stargazer granule cells lack AMPAR currents,
whether stargazin associates with GluR subunits was examined.
When co-transfected into COS cells, stargazin and GluR4
co-immunoprecipitate. Notably, stargazin also interacts
with co-transfected GluR1 or 2 subunits. These stargazin
interactions with GluR subunits appear specific, since
stargazin does not bind to NMDAR. In brain homogenates, stargazin is
enriched in Triton X-100 insoluble postsynaptic density fractions
together with GluR4, NR1 and postsynaptic density-95
(PSD-95). Although stargazin could not be co-immunoprecipitated with GluR from brain extracts, the harsh conditions required for solubilization of the PSD
disrupt many protein complexes and often preclude
detection of interactions (Chen, 2000).
Stargazin is predicted to contain four putative transmembrane domains
with intracellular amino- and carboxy-terminal
tails. Its C-terminal region contains a type I
PDZ-binding site for association with certain synaptic PDZ
proteins such as PSD-95. Stargazin was found to interact with
PSD-95 and SAP-97, as well as with PSD-93 and SAP-102. Deleting the final four amino acids of stargazin (stargazinC) disrupts interaction
with PSD-95, although stargazinC does retain binding to GluR4 (Chen, 2000).
Because PSD-95 can mediate clustering of ion channels, it was of interest to see if its interactions with stargazin might
cluster GluRs in heterologous cells. Co-expression of GluR4 with
PSD-95, or with stargazin results in diffuse, overlapping distributions for these proteins, which generally resemble the localization of GluR4 expressed
alone. However, transfecting the three together causes a remarkable redistribution of the proteins to patch-like clusters. This clustering requires interaction of stargazin with the PDZ domains from
PSD-95; clustering is not observed in co-transfections with
stargazinC. The stargazin/PSD-95-induced
clusters of GluR4 occur at the cell surface; they are labelled in
non-permeabilized cells by an antibody to the extracellular
green fluorescent protein (GFP) tag on GluR4 (Chen, 2000).
These findings suggest that stargazin has two distinct roles in
controlling AMPAR function: (1) stargazin regulates
delivery of AMPARs to the membrane surface and this function does not
require the PDZ-binding domain; (2) stargazin mediates synaptic targeting of AMPARs and this function
does require the PDZ-binding C terminus. Moreover,
it is proposed that stargazin and the related proteins mediate
AMPAR targeting throughout the brain. That
stargazin clusters at hippocampal synapses and that stargazinC acts
as a dominant-negative support a general role for
stargazin-like proteins in synaptic targeting of AMPARs. A class of
PDZ-domain-containing proteins, including GRIP
(glutamate receptor interacting protein) 1 and 2, ABP (AMPAR-binding
protein), PICK1 (protein interacting with C kinase
1) and SAP-97 (synapse-associated protein) have been implicated in
synaptic targeting/clustering of AMPARs. A recent study suggests that the association of GluR2 with ABP/GRIP is not essential for synaptic targeting, but is required for maintaining the synaptic surface accumulation of AMPARs.
Together, it seems that synaptic targeting/insertion and
synaptic stabilization of AMPARs may be mediated by several
mechanisms. Whereas the interaction between stargazin and
PSD-95 or related PDZ proteins is crucial for the initial synaptic
targeting of AMPARs, a different pathway involving the association of GluR2 with ABP/GRIP may be required for receptor stabilization (Chen, 2000).
Excitatory synapses in the brain exhibit a remarkable degree of functional plasticity, which largely reflects changes in the number of synaptic AMPA receptors. However, mechanisms involved in recruiting AMPA receptors to synapses are unknown. Hippocampal slice cultures and biolistic gene transfections have been used to study the targeting of AMPA receptors to synapses. AMPA receptors are localized to synapses through direct binding of the first two PDZ domains of synaptic PSD-95 to the AMPAR-associated protein, stargazin. Increasing the level of synaptic PSD-95 recruits new AMPA receptors to synapses without changing the number of surface AMPARs. At the same time, stargazin overexpression drastically increases the number of extra-synaptic AMPA receptors, but fails to alter synaptic currents if synaptic PSD-95 levels are kept constant. Finally, compensatory mutations were made to both PSD-95 and stargazin to demonstrate the central role of direct interactions between them in determining the number of synaptic AMPARs (Schnell, 2002).
Under standard conditions, cultured ventral spinal neurons cluster AMPA-type (but not NMDA-type) glutamate receptors at excitatory synapses on their dendritic shafts, in spite of abundant expression of the ubiquitous NMDA receptor subunit NR1. The NMDA receptor subunits NR2A and NR2B are not routinely expressed in cultured spinal neurons and transfection with NR2A or NR2B reconstitutes the synaptic targeting of NMDA receptors and confers on exogenous application of the immediate early gene product Narp the ability to cluster both AMPA and NMDA receptors. The use of dominant-negative mutants of GluR2 further shows that the synaptic targeting of NMDA receptors is dependent on the presence of synaptic AMPA receptors and that synaptic AMPA and NMDA receptors are linked by Stargazin and a MAGUK protein. This system of AMPA receptor-dependent synaptic NMDA receptor localization is preserved in hippocampal interneurons but reversed in hippocampal pyramidal neurons (Mi, 2004).
Accumulation of AMPA receptors at synapses is a fundamental feature of glutamatergic synaptic transmission. Stargazin, a member of the TARP family, is an AMPAR auxiliary subunit allowing interaction of the receptor with scaffold proteins of the postsynaptic density, such as PSD-95. How PSD-95 and Stargazin regulate AMPAR number in synaptic membranes remains elusive. Using single quantum dot and FRAP imaging in live hippocampal neurons, it has been shown that exchange of AMPAR by lateral diffusion between extrasynaptic and synaptic sites mostly depends on the interaction of Stargazin with PSD-95 and not upon the GluR2 AMPAR subunit C terminus. Disruption of interactions between Stargazin and PSD-95 strongly increases AMPAR surface diffusion, preventing AMPAR accumulation at postsynaptic sites. Furthermore, AMPARs and Stargazin diffuse as complexes in and out synapses. These results propose a model in which the Stargazin-PSD-95 interaction plays a key role to trap and transiently stabilize diffusing AMPARs in the postsynaptic density (Bats, 2007).
Endogenous polyamines profoundly affect the activity of various ion channels, including that of calcium-permeable AMPA-type glutamate receptors (CP-AMPARs). Stargazin, a transmembrane AMPAR regulatory protein (TARP) known to influence transport, gating and desensitization of AMPARs, greatly reduces block of CP-AMPARs by intracellular polyamines. By decreasing CP-AMPAR affinity for cytoplasmic polyamines, stargazin enhances the charge transfer following single glutamate applications and eliminates the frequency-dependent facilitation seen with repeated applications. In cerebellar stellate cells, which express both synaptic CP-AMPARs and stargazin, it was found that the rectification and unitary conductance of channels underlying excitatory postsynaptic currents were matched by those of recombinant AMPARs only when the latter were associated with stargazin. Taken together, these observations establish modulatory actions of stargazin that are specific to CP-AMPARs, and suggest that during synaptic transmission the activity of such receptors, and thus calcium influx, is fundamentally changed by TARPs (Soto, 2007).
Synaptic clustering of neurotransmitter receptors is crucial for efficient signal transduction and integration in neurons. PDZ
domain-containing proteins such as PSD-95/SAP90 interact with the intracellular C termini of a variety of receptors and are
thought to be important in the targeting and anchoring of receptors to specific synapses. PICK1
(protein interacting with C kinase), a PDZ domain-containing protein, interacts with the C termini of AMPA receptors in vitro and in vivo. In neurons, PICK1
specifically colocalizes with AMPA receptors at excitatory synapses. Furthermore, PICK1 induces clustering of AMPA
receptors in heterologous expression systems. These results suggest that PICK1 may play an important role in the
modulation of synaptic transmission by regulating the synaptic targeting of AMPA receptors (Xia, 1999).
AMPA receptor subunits interact with a PDZ domain-containing protein called PICK1, which is known to bind protein kinase
C alpha (PKC alpha). PICK1 interacts with sequences within the last ten amino acid residues containing a novel PDZ binding motif (E S V/I K I) of the short C-terminal
alternative splice variants of AMPA receptor subunits. No interaction occurs with the corresponding long splice variants which do not contain the E S V/I K I motif.
The PDZ domain of PICK1 is required for the interaction; the mutation of a single amino acid in this region (Lys-27 to Glu) prevents interaction between PICK1
and GluR2 in the yeast two-hybrid assay. A similar mutation has been reported to prevent the binding of PICK1 to PKC alpha, indicating that the same domain of
PICK1 binds both PKC alpha and GluRs. Flag-tagged PICK1 is retained by a glutathione S-transferase (GST) fusion of the C-terminal of GluR2 (GST-ct-GluR2;
short splice variant) but not by GST-ct-GluR1 (long splice variant). Recombinant full length GluR2 is coimmunoprecipitated with flag-PICK1 using an anti-flag
antibody and flag-PICK1 is coimmunoprecipitated with an N-terminal directed anti-GluR2 antibody. Transient expression of both proteins in COS cells reveals
colocalization and an altered pattern of distribution for each protein, in comparison to the expression patterns when expressed individually. This novel interaction provides a possible regulatory
mechanism to specifically modulate distinct splice variants and may be involved in targeting the phosphorylation of short form GluRs by PKC alpha (Dev, 1999).
AMPA receptor-binding protein
(ABP) is a postsynaptic density (PSD) protein related to glutamate receptor-interacting protein (GRIP: see Drosophila Grip). ABP has two sets of three
PDZ domains, which bind the GluR2/3 AMPA receptor subunits. ABP exhibits widespread CNS expression and is found
at the postsynaptic membrane. The protein interactions of the ABP/GRIP family differ from the PSD-95
family, which bind N-methyl-D-aspartate (NMDA) receptors. ABP binds to the GluR2/3 C-terminal VKI-COOH motif via
class II hydrophobic PDZ interactions, distinct from the class I PSD-95-NMDA receptor interaction. ABP and GRIP also
form homo- and hetero-multimers through PDZ-PDZ interactions but do not bind PSD-95. It is suggested that the ABP/GRIP
and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors (Srivastava, 1998).
The molecular mechanisms underlying the targeting and localization of glutamate receptors at postsynaptic sites is poorly
understood. A PDZ domain-containing protein, glutamate receptor-interacting protein 1 (GRIP1), has been identified
that specifically binds to the C termini of AMPA receptor subunits and may be involved in the synaptic targeting of these
receptors. The cloning of GRIP2, a homolog of GRIP1, is reported along with the characterization of the GRIP1 and GRIP2
proteins in the rat CNS. Recently, a GluR2/3 binding protein homologous to
GRIP1, AMPA receptor-binding protein (ABP), has been described (Srivastava, 1998). ABP is apparently a short splice variant of GRIP2 that lacks
the N terminus and the seventh PDZ domain of GRIP2.
Similar to GRIP1, GRIP2 contains seven PDZ domains that are very homologous to GRIP1 within the PDZ domains (64%-93% identity) but has little sequence
similarity in the linker regions between the PDZ domains. GRIP1 and GRIP2 are ~130 kDa proteins that are highly enriched in brain. GRIP1 and GRIP2 are
widely expressed in brain, with the highest levels found in the cerebral cortex, hippocampus, and olfactory bulb. Biochemical studies show that GRIP1 and GRIP2
are enriched in synaptic plasma membrane and postsynaptic density fractions. GRIP1 is expressed early in embryonic development before the expression of AMPA
receptors and peaks in expression at postnatal day 8-10. In contrast, GRIP2 is expressed relatively late in development and parallels the expression of AMPA
receptors. Immunohistochemistry using the GRIP1 antibodies demonstrates that GRIP1 is expressed in neurons in a somatodendritic staining pattern. At the
ultrastructural level GRIP1 is enriched in dendritic spines near the postsynaptic density and is
expressed in dendritic shafts and in peri-Golgi regions in the neuronal soma. GRIP1 appears to be clustered at both glutamatergic and GABAergic synapses. These
results suggest that GRIP1 and GRIP2 are AMPA receptor binding proteins potentially involved in the targeting of AMPA receptors to synapses. GRIP1 also may
play functional roles at both excitatory and inhibitory synapses, as well as in early neuronal development (Dong, 1999).
The NMDA and AMPA classes of ionotropic glutamate receptors are concentrated at postsynaptic sites in excitatory synapses.
NMDA receptors interact via their NR2 subunits with PSD-95/SAP90 family proteins, whereas AMPA receptors bind via their
GluR2/3 subunits to glutamate receptor-interacting protein (GRIP), AMPA receptor-binding protein (ABP), and protein interacting
with C kinase 1 (PICK1). A novel cDNA (termed ABP-L/GRIP2) is described that is virtually identical to ABP except for
additional GRIP-like sequences at the N-terminal and C-terminal ends. Like GRIP (here termed GRIP1), ABP-L/GRIP2
contains a seventh PDZ domain at its C terminus. Using antibodies that recognize both these proteins, the subcellular
localization of GRIP1 and ABP-L/GRIP2 (collectively termed GRIP) and their biochemical association with AMPA receptors are reported.
GRIP is present at excitatory synapses and also at nonsynaptic membranes and
within intracellular compartments. The association of native GRIP and AMPA receptors was confirmed biochemically by
coimmunoprecipitation from rat brain extracts. A majority of detergent-extractable GluR2/3 is complexed with GRIP in the
brain. However, only approximately half of GRIP is associated with AMPA receptors. Unexpectedly, immunocytochemistry of
cultured hippocampal neurons and rat brain at the light microscopic level shows enrichment of GRIP in GABAergic neurons and
in GABAergic nerve terminals. Thus GRIP is associated with inhibitory as well as excitatory synapses. Collectively, these findings
support a role for GRIP in the synaptic anchoring of AMPA receptors but also suggest that GRIP has additional functions unrelated
to the binding of AMPA receptors (Wyszynski, 1999).
The PDZ domain-containing proteins, such as PSD-95 and GRIP, have been suggested to be involved in the targeting of glutamate receptors, a process that plays a
critical role in the efficiency of synaptic transmission and plasticity. To address the molecular mechanisms underlying AMPA receptor synaptic localization, several GRIP-associated proteins (GRASPs) have been identified that bind to distinct PDZ domains within GRIP. GRASP-1 is a neuronal rasGEF associated with GRIP and
AMPA receptors in vivo. Overexpression of GRASP-1 in cultured neurons specifically reduces the synaptic targeting of AMPA receptors. In addition, the
subcellular distribution of both AMPA receptors and GRASP-1 is rapidly regulated by the activation of NMDA receptors. These results suggest that GRASP-1
may regulate neuronal ras signaling and contribute to the regulation of AMPA receptor distribution by NMDA receptor activity (Ye, 2000).
LTP and LTD have been proposed to be mediated, in part, by changes in AMPA receptor function. Increases in AMPA receptor responses have been observed
during the expression of LTP. Recently, it has been shown that a high
proportion of synapses in hippocampal CA1 region contains only NMDA receptors and acquires AMPA receptors only after the induction of LTP. This emergence of AMPA receptor current seems due to the appearance of synaptic AMPA receptors. Moreover, NMDA receptor-dependent LTD in cultured neurons has recently been observed to correlate with a decrease in the levels of synaptic
AMPA receptors. Previous studies have suggested that AMPA receptor-associated proteins, such as GRIP, are involved in the synaptic
targeting of AMPA receptors. In this study, GRASP-1 has been added to this complex and evidence is provided that GRASP-1 may
also be important in regulation of AMPA receptor function and may play a role in AMPA receptor synaptic targeting. Overexpression of GRASP-1 in neurons
downregulates synaptic AMPA receptor clusters, while it has no effect on synaptic NMDA receptor synaptic targeting. Both the rasGEF catalytic domain and the
C-terminal 'regulatory' domain were required for this activity. Activation of NMDA receptors dramatically induces the redistribution of both GRASP-1 and AMPA
receptors from punctate membrane structures to a more diffuse pattern. Together with the GRASP-1 overexpression data, these results suggest that the overall
spatial distribution of GRASP-1, as well as the absolute levels, may be important for AMPA receptor targeting. These results suggest that GRASP-1 and possibly
ras signaling may play a role in the regulation of AMPA receptor synaptic targeting and its regulation by NMDA receptor activity (Ye, 2000).
Several proteins have been shown to interact specifically with the C termini of the GluR2 and GluR3 AMPA receptor subunits. These include three PDZ
proteins, ABP (AMPA Receptor Binding protein), GRIP (Glutamate Receptor Interacting Protein), and PICK1
(Protein Interacting with C Kinase). The two splice forms of ABP that contain either 6 or 7 PDZ domains, and the 7 PDZ domain GRIP are members of a novel sequence-related protein family. GRIP PDZ4-5 domains and ABP PDZ5 domain show the highest
affinity for the GluR2/3 C terminus. The remaining PDZ domains of GRIP and ABP are likely to mediate additional
interactions, possibly anchoring the AMPA receptor to cytoskeletal proteins or coupling the receptor to intracellular enzymes. PICK1, which was cloned as a
PKC-interacting protein, contains a single N-terminal PDZ domain. When coexpressed with GluR2 in heterologous cells, PICK1
induces GluR2 surface clustering and intracellular redistribution (Osten, 2000 and references therein).
The roles of GRIP, ABP, and PICK1 in GluR2 AMPA receptor trafficking have been studied. An epitope-tagged MycGluR2 subunit, when expressed in
hippocampal cultured neurons, is specifically targeted to the synaptic surface. With the mutant MycGluR2delta1-10, which lacks the PDZ binding site, the
overall dendritic intracellular transport and the synaptic surface targeting are not affected. However, over time, MycGluR2delta1-10 accumulates at
synapses significantly less than MycGluR2. Notably, a single residue substitution, S880A, which blocks binding to ABP/GRIP but not to PICK1, reduces synaptic
accumulation to the same extent as the PDZ site truncation. It is concluded that the association of GluR2 with ABP and/or GRIP but not PICK1 is essential for
maintaining the synaptic surface accumulation of the receptor, possibly by limiting its endocytotic rate (Osten, 2000).
N-ethylmaleimide-sensitive fusion protein (NSF) interacts directly and selectively with the intracellular
C-terminal domain of the GluR2 subunit of AMPA receptors. The interaction requires all three domains of NSF but occurs
between residues Lys-844 and Gln-853 of rat GluR2, with Asn-851 playing a critical role. Loading of decapeptides
corresponding to the NSF-binding domain of GluR2 into rat hippocampal CA1 pyramidal neurons results in a marked,
progressive decrement of AMPA receptor-mediated synaptic transmission. This reduction in synaptic transmission is also
observed when an anti-NSF monoclonal antibody (mAb) is loaded into CA1 neurons. These results demonstrate a
previously unsuspected direct interaction in the postsynaptic neuron between two major proteins involved in synaptic
transmission and suggest a rapid NSF-dependent modulation of AMPA receptor function (Nishimune, 1998).
Specific interaction is demonstrated between the GluR2 AMPA receptor subunit C-terminal peptide with an ATPase N-ethylmaleimide-sensitive fusion protein (NSF) and alpha-
and beta-soluble NSF attachment proteins (SNAPs). These proteins are colocated in dendrites. The assembly of
the GluR2-NSF-SNAP complex is ATP hydrolysis reversible and resembles the binding of NSF and SNAP with the
SNAP receptor (SNARE) membrane fusion apparatus. Evidence that the molar ratio of NSF to SNAP in the
GluR2-NSF-SNAP complex is similar to that of the t-SNARE syntaxin-NSF-SNAP complex. NSF is known to
disassemble the SNARE protein complex in a chaperone-like interaction driven by ATP hydrolysis. A model is proposed in
which NSF functions as a chaperone in the molecular processing of the AMPA receptor (Osten, 1998).
Glutamate receptors mediate the majority of rapid excitatory synaptic transmission in the central nervous system (CNS) and
play important roles in synaptic plasticity and neuronal development. Recently, protein-protein interactions with the
C-terminal domain of glutamate receptor subunits have been shown to be involved in the modulation of receptor function
and clustering at excitatory synapses. The N-ethylmaleimide-sensitive factor (NSF), a
protein involved in membrane fusion events, specifically interacts with the C terminus of the GluR2 and GluR4c subunits
of AMPA receptors in vitro and in vivo. Moreover, intracellular perfusion of neurons with a synthetic peptide that competes
with the interaction of NSF and AMPA receptor subunits rapidly decreases the amplitude of miniature excitatory
postsynaptic currents (mEPSCs), suggesting that NSF regulates AMPA receptor function (Song, 1998).
Disruption of N-ethylmaleimide-sensitive fusion protein- (NSF-) GluR2 interaction by infusion into cultured hippocampal neurons of a
blocking peptide (pep2m) causes a rapid decrease in the frequency but no change in the amplitude of AMPA receptor-mediated miniature excitatory
postsynaptic currents (mEPSCs). NMDA receptor-mediated mEPSCs were not changed. Viral expression of pep2m reduces the
surface expression of AMPA receptors, whereas NMDA receptor surface expression in
the same living cells is unchanged. In permeabilized neurons, the total amount of GluR2 immunoreactivity is unchanged, and a punctate distribution of
GluR2 is observed throughout the dendritic tree. These data suggest that the NSF-GluR2 interaction is required for the surface expression of GluR2-containing AMPA receptors and that disruption of the interaction leads to the functional elimination of AMPA receptors at synapses. Based on these findings and the known properties of NSF, a model is favored in which the
interaction between NSF and GluR2 is involved in the part of the cycling process that is necessary for the insertion and/or stabilization of AMPA receptors
at the postsynaptic membrane. By analogy with its known presynaptic functions, NSF could act at the AMPA receptor complex by stripping the receptors
of associated proteins. Candidate proteins interacting with GluR2 include the PDZ-containing proteins GRIP, ABP, and PICK1. Removal of associated proteins could prime or "reset" the
AMPA receptor complex to a naive state, thereby allowing insertion into the postsynaptic membrane. If the action of NSF is
prevented, for example, by peptide block, the receptors cannot be appropriately processed, and insertion/reinsertion of the reconfigured receptors into the
postsynaptic membrane cannot occur (Noel, 1999).
Narp (neuronal activity-regulated pentraxin) is a secreted immediate-early gene (IEG) regulated by synaptic activity in brain. Narp was originally
identified by a novel subtractive cloning strategy from stimulated hippocampus and is a member of the newly recognized subfamily of 'long
pentraxins' that includes neuronal pentraxin 1 and 2, which are found in the brain; TSG-14, a tumor necrosis
factor-inducible acute phase reactant; and apexin, which is localized to the acrosome of mature sperm. These molecules are similar in
structure in that they possess a C-terminal pentraxin domain and a 200 amino acid unique N terminus whose function up to this point is unknown. The pentraxin domain on Narp is similar to the mammalian protein C-reactive protein (CRP) and to mammalian serum amyloid protein (SAP), as
well as highly conserved homologs from species as distant as Limulus. Pentraxins are secreted proteins that self-multimerize to form
pentamers and may further dimerize to form decamers. A crystal structure of SAP shows that the pentraxin sugar-binding motif is
remarkably homologous in secondary and tertiary structure to the plant lectin concanavalin A, a feature that is conserved in Narp. The physiological roles of pentraxins have remained obscure, although CRP has been postulated to play a role in nonantibody-mediated
immune responses by binding and aggregating bacteria and other pathogens (O'Brien, 1999 and references).
Narp
possesses several properties that make it likely to play a key role in excitatory synaptogenesis. Narp is shown to be selectively enriched at excitatory synapses on
neurons from both the hippocampus and spinal cord. Overexpression of recombinant Narp increases the number of excitatory but not inhibitory synapses in cultured
spinal neurons. In transfected HEK 293T cells, Narp interacts with itself, forming large surface clusters that coaggregate AMPA receptor subunits. Moreover,
Narp-expressing HEK 293T cells can induce the aggregation of neuronal AMPA receptors. These studies support a model in which Narp functions as an
extracellular aggregating factor for AMPA receptors (O'Brien, 1999).
The neuronal IEG Narp is selectively expressed at the majority of excitatory, axodendritic shaft synapses on aspiny spinal cord and
hippocampal neurons in vitro. In addition, a small number of spine-bearing neurons express Narp at their excitatory synapses in culture. In vivo,
Narp to be present at both pre- and post-synaptic sites of spiny and aspiny synapses. The prominent presynaptic localization of Narp in mossy fiber terminals is
associated with synaptic vesicles. Because Narp is dramatically upregulated in neurons in response to patterned synaptic activity and is
expressed at relatively high levels in developing and adult brain, these studies suggest that Narp may play a critical role in linking activity with
the development and plasticity of excitatory synapses.
The family of long pentraxins, of which Narp is a member, has several characteristics that might play a role in promoting excitatory synapse formation. Included
among these are the ability to form side-to-side and head-to-head multimeric aggregates and the ability to bind other proteins via a lectin-like domain. The ability of Narp to
cluster AMPA receptors would not have been predicted from a knowledge of the family of pentraxins, since the association of Narp with AMPA receptor subunits in
the presence of tunicamycin suggests that it is not the lectin component of Narp that mediates this interaction. Indeed, the specificity of the interaction (GluR1-GluR3
but not GluR4, GluR6, or NR1) would also argue against a nonspecific interaction such as that mediated by a lectin (O'Brien, 1999).
Another notable functional property of Narp-expressing cells is their ability to cluster AMPA receptors on apposing cells, even when the contacted cell does not
express Narp. In view of the documented physical interaction between Narp and AMPA receptors when these proteins are expressed in the same cells, it seems
likely that the intercellular clustering activity also involves their physical interaction. The transcellular clustering activity of Narp is further enhanced when the apposing
cell coexpresses Narp with AMPA receptors, suggesting that a Narp-Narp interaction may also contribute to transcellular clustering. In this regard, Narp may
potentially display similarities with cadherins, which self associate and participate in synaptogenesis from both the pre- and post-synaptic surfaces. Unlike the family of cadherins, however, Narp appears to be completely extracellular, with no
transmembrane domain (O'Brien, 1999).
A model is proposed in which Narp-Narp interactions between pre- and post-synaptic cells contribute to excitatory synapse formation by a secondary clustering of
synaptic AMPA receptors due to Narp-AMPA receptor interactions. In support of a 'presynaptic' effect of Narp, it is noted that Narp expressed on heterologous
cells induces AMPA receptor clusters on neurons. By contrast, a 'postsynaptic' effect of Narp is suggested by the observation that when Narp expression is
upregulated by transfection, it results in a 2-fold increase in the number of excitatory synapses, with no change in the number of inhibitory synapses. Since only a small
percentage of cells is transfected in this experiment, the increase in the number of excitatory synapses on transfected cells as compared with nontransfected cells in
the same dish argues strongly for a postsynaptic action of Narp. The synaptogenic effect of upregulated Narp expression in neurons would be a manifestation of the
potentiating effect of Narp on the transcellular cluster formation seen in heterologous cells when Narp is coexpressed with AMPA receptor subunits. It is also
possible that excess Narp in the postsynaptic neuron promotes Narp-Narp intercellular interactions with presynaptic elements and makes it more favorable for the
overexpressing cell to attract new synapses. This would favor excitatory synapses over inhibitory synapses, since inhibitory axons do not appear to express Narp (O'Brien, 1999).
Silent synapses form between some primary sensory afferents and dorsal horn neurons in the spinal cord. Molecular mechanisms for activation or conversion of silent synapses to conducting synapses are unknown. Serotonin can trigger activation of silent synapses in dorsal horn neurons by recruiting AMPA receptors. AMPA-receptor subunits GluR2 and GluR3 interact via their cytoplasmic C termini with PDZ-domain-containing proteins such as GRIP (glutamate receptor interacting protein), but the functional significance of these interactions is unclear. Protein interactions involving the GluR2/3 C terminus are important for serotonin-induced activation of silent synapses in the spinal cord. Furthermore, PKC is a necessary and sufficient trigger for this activation. These results implicate AMPA receptor-PDZ interactions in mechanisms underlying sensory synaptic potentiation and provide insights into the pathogenesis of chronic pain mediated by the sensitization of spinal dorsal horn neurons to input from primary afferent fibers (Li, 1999).
Compartmentalization of glutamate receptors with the signaling enzymes that regulate their activity supports synaptic transmission. Two classes of binding proteins
organize these complexes: the MAGUK proteins that cluster glutamate receptors and AKAPs that anchor kinases and phosphatases. Glutamate receptors and PKA are recruited into a macromolecular signaling complex through direct interaction between the MAGUK proteins, PSD-95 and
SAP97, and AKAP79/150. The SH3 and GK regions of the MAGUKs mediate binding to the AKAP. Cell-based studies indicate that phosphorylation of AMPA
receptors is enhanced by a SAP97-AKAP79 complex that directs PKA to GluR1 via a PDZ domain interaction. Since AMPA receptor phosphorylation is implicated
in regulating synaptic plasticity, these data suggest that a MAGUK-AKAP complex may be centrally involved (Colledge, 2000).
Phosphorylation of glutamate receptors is a critical regulatory event in the control of synaptic function and plasticity. Evidence is provided for the existence of a macromolecular transduction unit in which PKA is targeted to glutamate receptors through the direct interaction of
two distinct sets of synaptic organizing molecules -- the MAGUK proteins and AKAP79/150. These interactions increase the complexity of signaling networks at
excitatory synapses and may provide a structural framework that permits preferential targeting of kinases to glutamate receptors. Presumably, such a highly organized
kinase-substrate complex ensures rapid and efficient phosphorylation of ion channels in response to local synaptic signals (Colledge, 2000).
The MAGUK proteins provide the central scaffold upon which the complex is assembled. The N-terminal PDZ domains of PSD-95 and SAP97, two members of
the MAGUK protein family, bind to the tails of NMDA and AMPA receptor subunits, respectively. AKAP79/150 and its associated kinases can be recruited to these glutamate receptor complexes via interaction with the C-terminal SH3 and GK
domains of the MAGUKs. The demonstration that two independent sites of contact mediate interaction between AKAP79/150 and MAGUK proteins is interesting
in light of other mapping studies that have defined linear sequences of 4-6 amino acids as ligands for PDZ, SH2, and SH3 domains. Certainly, multiple sites of contact are not unprecedented and are likely to provide additional stability to a given protein complex. For example, the KA2 subunit of
the kainate receptor, like AKAP79, binds to both the SH3 and GK domains of PSD-95. In addition, AKAP79/150 appears to bind to the beta2 adrenergic
receptor through sites in both the third intracellular loop and the C-terminal tail. Interestingly, mutations in the SH3 and GK domains of the
Drosophila MAGUK Discs large produce severe phenotypes, suggesting that these modules mediate interactions that are critical for regulating MAGUK function. Furthermore, deletion of these regions of PSD-95 in mice produces defects in synaptic plasticity that have been attributed to altered
downstream signaling events. A potential explanation for these observations is that the AKAP79/150 signaling scaffold no longer can be
recruited to glutamate receptors through interaction with MAGUK proteins (Colledge, 2000 and references therein).
AKAP79/150 previously has been shown to provide a scaffold for three signaling enzymes: PKA, PKC, and calcineurin. Interestingly, PSD-95 competes with calcineurin for binding to AKAP79/150 in vitro. Preliminary mapping experiments suggest that the two proteins do not share
the same binding site on the AKAP, since a deletion mutant that does not bind to calcineurin still binds to PSD-95. Thus, a more likely explanation is that PSD-95 binding to AKAP79/150 sterically hinders interaction with calcineurin. These data support the
notion that, when bound to MAGUKs, AKAP79/150 may preferentially target kinases but not phosphatases to certain glutamate receptors at the PSD. This could
provide a mechanism to favor ion channel phosphorylation through preferential recruitment of regulatory kinases (Colledge, 2000).
While these results clearly argue for a role for anchored PKA in receptor phosphorylation, targeted phosphatases are also certain to participate in receptor
dephosphorylation. However, interaction of AKAP79 with MAGUKs appears to exclude the phosphatase calcineurin from the complex. One
possibility is that phosphatases may be recruited to AMPA receptors through anchoring proteins other than AKAP79/150. In fact, recent reports suggest that the
phosphatase PP1 may be targeted to AMPA receptor complexes through its association with spinophilin. Kinase-phosphatase targeting to
some NMDA receptors may be more direct. Through interaction with the NR1-1A splice variant, the anchoring protein yotiao targets both PKA and active PP1 to
NMDA receptor complexes, conferring bidirectional regulation of NMDA receptor activity. When considered in light of the present data,
this raises the intriguing possibility that signaling enzymes may be recruited to certain NMDA receptors through simultaneous association with two anchoring proteins:
yotiao and AKAP79 (Colledge, 2000 and references therein).
Phosphorylation of the cytoplasmic tail of GluR1 potentiates receptor function. CaMKII increases the unitary channel
conductance via phosphorylation of Ser-831, while PKA phosphorylation of Ser-845 increases the peak open
probability. Phosphorylation-dependent changes in AMPA receptor activity have been proposed to underlie some aspects of LTP and LTD. For example, CamKII phosphorylation appears to be essential for the induction of hippocampal LTP, while recent studies have implicated a role for PKA in LTD. The present results suggest that AKAP79/150 functions as an important player in the postsynaptic regulation of excitatory transmission by targeting
PKA to AMPA receptors. Specifically, cAMP-dependent phosphorylation of Ser-845, a known PKA site in GluR1, is enhanced when the
kinase is targeted to the channel via a SAP97-AKAP79 complex. This enhancement in phosphorylation is significantly reduced when a PKA anchoring-defective
form of AKAP79 is substituted in the complex. Furthermore, a mutation in the PDZ binding site in the tail of GluR1, which uncouples the receptor from SAP97,
reduces the basal level of phosphorylation of Ser-845 compared to wild-type GluR1. Together, these results suggest that phosphorylation of Ser-845 is mediated
through a SAP97-AKAP79 complex that targets PKA to GluR1 via a PDZ domain interaction. This is particularly interesting in light of recent evidence
implicating a GluR1-PDZ domain interaction in the delivery of AMPA receptors into synapses. These data suggest that recruitment of a
SAP97-AKAP79-PKA complex may play a role in this process. Manipulation of these protein-protein interactions in animals should provide models to study the
role of this synaptic signaling unit in regulating glutamate receptor function in vivo (Colledge, 2000 and references therein).
AMPA receptor (AMPAR) trafficking is crucial for synaptic plasticity, which may be important for learning and memory. NSF and PICK1 bind the AMPAR GluR2 subunit and are involved in trafficking of AMPARs. GluR2, PICK1, NSF,
and alpha-/beta-SNAPs form a complex in the presence of ATPgammaS. Similar to
SNARE complex disassembly, NSF ATPase activity disrupts PICK1-GluR2 interactions in this complex. Alpha- and beta-SNAP have differential effects on this reaction. SNAP overexpression in hippocampal neurons leads to corresponding changes in AMPAR trafficking by acting on GluR2-PICK1 complexes. This demonstrates that the previously reported synaptic stabilization of AMPARs by NSF involves disruption of GluR2-PICK1 interactions (Hanley, 2002).
AMPAR trafficking is thought to involve constitutive cycling of receptors by endocytosis/exocytosis, as well as regulated events as part of LTD (endocytosis) and LTP (exocytosis). AMPAR endocytosis during some forms of LTD is dependent upon GluR2 phosphorylation and regulation of accessory protein binding. The NSF-mediated disassembly of the GluR2-PICK1 complex described in this study is therefore likely to be crucial in limiting endocytosis of AMPARs to maintain constitutive cycling at a constant rate and hence maintain a constant level of receptors at the synaptic membrane. From this baseline, LTD could be induced (in conjunction with phosphorylation events) by reducing the activity of NSF, possibly by modulation of SNAP-PICK1 binding, to stabilize GluR2-PICK1 interactions, and consequently enhance receptor endocytosis. This study has identified the molecular mechanism for the activity of NSF in AMPA receptor trafficking, and has demonstrated that NSF can function as a disassembling molecular chaperone in a protein complex other than the 20S SNARE complex. As additional NSF binding partners are identified, it is possible that this ATPase, previously thought to be faithful to the SNARE complex, will show more promiscuous chaperone behavior (Hanley, 2002).
Four PDZ domain-containing proteins, syntenin, PICK1, GRIP, and PSD95, have been identified as interactors with the kainate receptor (KAR) subunits GluR52b, GluR52c, and GluR6. Of these, it is shown that both GRIP and PICK1 interactions are required to maintain KAR-mediated synaptic function at mossy fiber-CA3 synapses. In addition, PKCalpha can phosphorylate ct-GluR52b at residues S880 and S886, and PKC activity is required to maintain KAR-mediated synaptic responses. It is proposed that PICK1 targets PKCalpha to phosphorylate KARs, causing their stabilization at the synapse by an interaction with GRIP. Importantly, this mechanism is not involved in the constitutive recycling of AMPA receptors since blockade of PDZ interactions can simultaneously increase AMPAR- and decrease KAR-mediated synaptic transmission at the same population of synapses (Hirbec, 2003).
The finding that KARs and AMPARs can bind to a common pool of PDZ proteins suggests that these proteins may play important general roles in the regulation of glutamatergic synapses. Based on the present findings and previous work on AMPARs, it is possible to speculate on the molecular mechanisms that mediate the differential regulation of AMPARs and KARs by these PDZ proteins. In this scheme, AMPARs are secured in intracellular pools via association of the GluR2 subunit with GRIP and/or ABP. These 'gripped' receptors are immobile over the time course of the electrophysiology experiments. PICK1 exchanges for GRIP and targets PKCalpha, which then phosphorylates S880 of GluR2, thereby preventing the rebinding of GRIP. The S880-phosphorylated AMPARs are mobile and available for surface expression. It is proposed that KARs are also 'gripped' by GRIP, but in this case, PICK1-targetted, PKC-dependent phosphorylation stabilizes the GRIP interaction with GluR5/6 and anchors the receptors at the postsynaptic membrane. These data are entirely consistent with the observations that blockade of either GRIP or PICK1 binding, or inhibition of PKC, results in a rapid decrease in KAR-mediated synaptic currents. It is speculated that, whereas phosphorylation of S880 of GluR2 prevents GRIP binding, phosphorylation of S880 and/or S886 of GluR52b (and/or equivalent residues of GluR6) stabilizes GRIP binding and anchors the receptors at the synapse (Hirbec, 2003).
These differences in the molecular consequences of PKC-mediated phosphorylation of AMPARs and KARs can explain the differential regulation in opposite directions of the functional synaptic responses. The results showing that, at the same population of synapses, disruption of PDZ protein interactions results in an increase in EPSCA
and a simultaneous decrease in EPSCK
suggests that these proteins may act to regulate the relative proportions of AMPARs and KARs at synapses. Physiologically, given the distinct biophysical and functional profiles of AMPARs and KARs, the dynamic regulation of these interactions will play important roles in the modulation of basal glutamatergic synaptic transmission. Furthermore, it has been reported previously that some forms of developmental and activity-dependent synaptic plasticity involve a switch from functionally expressed KARs to AMPARs. The differential effects of PDZ-interacting proteins demonstrated here on these two receptor types provide an attractive molecular mechanism to account for these developmental and activity-dependent changes in the AMPAR and KAR complement at synapses (Hirbec, 2003).
A clone has been isolated from a rat brain cDNA library corresponding to a 2779-bp cDNA encoding a novel splice form of the glutamate receptor interacting protein-1 (GRIP1). This 696-amino acid has been termed splice form GRIP1c 4-7 to differentiate it from longer splice forms of GRIP1a/b containing seven PDZ domains. The four PDZ domains of GRIP1c 4-7 are identical to PDZ domains 4-7 of GRIP1a/b. GRIP1c 4-7 also contains 35 amino acids at the N terminus and 12 amino acids at the C terminus that are different from GRIP1a/b. In transfected HEK293 cells, a majority of GRIP1c 4-7 was associated with the plasma membrane. GRIP1c 4-7 interacts with GluR2/3 subunits of the AMPA receptor. In low density hippocampal cultures, GRIP1c 4-7 clusters colocalized with GABAergic (where GABA is gamma-aminobutyric acid) and glutamatergic synapses, although a higher percentage of GRIP1c 4-7 clusters colocalized with gamma-aminobutyric acid, type A, receptor [GABA(A)R] clusters than with AMPA receptor clusters. Transfection of hippocampal neurons with hemagglutinin-tagged GRIP1c 4-7 showed that it targets to the postsynaptic complex of GABAergic synapses colocalizing with GABA(A)R clusters. GRIP1c 4-7-specific antibodies, which did not recognize previously described splice forms of GRIP1, recognized a 75-kDa protein that is enriched in a postsynaptic density fraction isolated from rat brain. EM immunocytochemistry experiments show that in intact brain GRIP1c 4-7 concentrates at postsynaptic complexes of both type I glutamatergic and type II GABAergic synapses although it is also presynaptically localized. These results indicate that GRIP1c 4-7 plays a role not only in glutamatergic synapses but also in GABAergic synapses (Charych, 2004).
Single-particle electron microscopy (EM) combined with biochemical measurements revealed the molecular shape of SAP97 (also known as hDlg) and a monomer-dimer transition that depends on the N-terminal L27 domain. Overexpression of SAP97 drives GluR1 to synapses, potentiates AMPA receptor (AMPAR) excitatory postsynaptic currents (EPSCs), and occludes LTP. Synaptic potentiation and GluR1 delivery are dissociable by L27 domain mutants that inhibit multimerization of SAP97. Loss of potentiation is correlated with faster turnover of monomeric SAP97 mutants in dendritic spines. It is proposed that L27-mediated interactions of SAP97 with itself or other proteins regulate the synaptic delivery of AMPARs. RNAi knockdown of endogenous PSD-95 depletes surface GluR1 and impaires AMPA EPSCs. In contrast, RNAi knockdown of endogenous SAP97 reduces surface expression of both GluR1 and GluR2 and inhibits both AMPA and NMDA EPSCs. Thus SAP97 has a broader role than its close relative, PSD-95, in the maintenance of synaptic function (Nakagawa, 2004).
A novel rat gene, tanc (GenBank Accession No. AB098072), has been cloned that encodes a protein containing three tetratricopeptide repeats (TPRs), ten ankyrin repeats and a coiled-coil region, and is possibly a rat homolog of Drosophila rolling pebbles. The tanc gene is expressed widely in the adult rat brain. Subcellular distribution, immunohistochemical study of the brain and immunocytochemical studies of cultured neuronal cells indicate the postsynaptic localization of TANC protein of 200 kDa. Pull-down experiments have shown that TANC protein binds PSD-95, SAP97, and Homer via its C-terminal PDZ-binding motif, -ESNV, and fodrin via both its ankyrin repeats and the TPRs together with the coiled-coil domain. TANC also binds the alpha subunit of Ca2+/calmodulin-dependent protein kinase II. An immunoprecipitation study shows TANC association with various postsynaptic proteins, including guanylate kinase-associated protein (GKAP), alpha-internexin, and N-methyl-D-aspartate (NMDA)-type glutamate receptor 2B and AMPA-type glutamate receptor (GluR1) subunits. These results suggest that TANC protein may work as a postsynaptic scaffold component by forming a multiprotein complex with various postsynaptic density proteins (Suzuki, 2004).
At many excitatory central synapses, activity produces a lasting change in the synaptic response by modifying postsynaptic AMPA receptors (AMPARs). Although much is known about proteins involved in the trafficking of Ca2+-impermeable (GluR2-containing) AMPARs, little is known about protein partners that regulate subunit trafficking and plasticity of Ca2+-permeable (GluR2-lacking) AMPARs. At cerebellar parallel fiber-stellate cell synapses, activity triggers a novel type of plasticity: Ca2+ influx through GluR2-lacking synaptic AMPARs drives incorporation of GluR2-containing AMPARs, generating rapid, lasting changes in excitatory postsynaptic current properties. This study examined how GRIP and protein interacting with C-kinase-1 (PICK) regulate subunit trafficking and plasticity. It was found that repetitive synaptic activity triggers loss of synaptic GluR2-lacking AMPARs by selectively disrupting their interaction with GRIP and that PICK drives activity-dependent delivery of GluR2-containing receptors. This dynamic regulation of AMPARs provides a feedback mechanism for controlling Ca2+ permeability of synaptic receptors (Liu, 2005).
The targeting and surface expression of membrane proteins are critical to their functions. In neurons, synaptic targeting and surface expression of AMPA-type glutamate receptors were found to be critical for synaptic plasticity such as long-term potentiation and long-term depression (LTD). PICK1 (protein interacting with C kinase 1) is a cytosolic protein that interacts with many membrane proteins, including AMPA receptors via its PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain. Its interactions with membrane proteins regulate their subcellular targeting and surface expression. However, the mechanism by which PICK1 regulates protein trafficking has not been fully elucidated. This study shows that PICK1 directly binds to lipids, mainly phosphoinositides, via its BAR (Bin/amphiphysin/Rvs) domain. Lipid binding of the PICK1 BAR domain is positively regulated by its PDZ domain and negatively regulated by its C-terminal acidic domain. Mutation of critical residues of the PICK1 BAR domain eliminates its lipid-binding capability. Lipid binding of PICK1 controls the subcellular localization of the protein, because BAR domain mutant of PICK1 has diminished synaptic targeting compared with wild-type PICK1. In addition, the BAR domain mutant of PICK1 does not cluster AMPA receptors. Moreover, wild-type PICK1 enhances synaptic targeting of AMPA receptors, whereas the BAR domain mutant of PICK1 fails to do so. The BAR domain mutant of PICK1 loses its ability to regulate surface expression of the AMPA receptors and impairs expression of LTD in hippocampal neurons. Together, these findings indicate that the lipid binding of the PICK1 BAR domain is important for its synaptic targeting, AMPA receptor trafficking, and synaptic plasticity (Jin, 2006).
Via its extracellular N-terminal domain (NTD), the AMPA receptor subunit GluR2 promotes the formation and growth of dendritic spines in cultured hippocampal neurons. The first N-terminal 92 amino acids of the extracellular domain are necessary and sufficient for GluR2's spine-promoting activity. Moreover, overexpression of this extracellular domain increases the frequency of miniature excitatory postsynaptic currents (mEPSCs). Biochemically, the NTD of GluR2 can interact directly with the cell adhesion molecule N-cadherin, in cis or in trans. N-cadherin-coated beads recruit GluR2 on the surface of hippocampal neurons, and N-cadherin immobilization decreases GluR2 lateral diffusion on the neuronal surface. RNAi knockdown of N-cadherin prevents the enhancing effect of GluR2 on spine morphogenesis and mEPSC frequency. These data indicate that in hippocampal neurons N-cadherin and GluR2 form a synaptic complex that stimulates presynaptic development and function as well as promoting dendritic spine formation (Saglietti, 2007).
AMPA-type glutamate receptors (GluRs) play major roles in excitatory synaptic transmission. Neuronal AMPA receptors comprise GluR subunits and transmembrane AMPA receptor regulatory proteins (TARPs). Previous studies have identified five mammalian TARPs, γ-2 (or stargazin), γ-3, γ-4, γ-7, and γ-8, that enhance AMPA receptor function. This study classifies γ-5 as a distinct class of TARP that modulates specific GluR2-containing AMPA receptors and displays properties entirely dissimilar from canonical TARPs. γ-5 increases peak currents and decreases the steady-state currents selectively from GluR2-containing AMPA receptors. Furthermore, γ-5 increases rates of GluR2 deactivation and desensitization and decreases glutamate potency. Remarkably, all effects of γ-5 require editing of GluR2 mRNA. Unlike other TARPs, γ-5 modulates GluR2 without promoting receptor trafficking. γ-7 regulation of GluR2 is dictated by mRNA editing. These data establish γ-5 and γ-7 as a separate family of 'type II TARPs' that impart distinct physiological features to specific AMPA receptors (Kato, 2008).
At synapses, cell adhesion molecules (CAMs) provide the molecular framework for coordinating signaling events across the synaptic cleft. Among synaptic CAMs, the integrins, receptors for extracellular matrix proteins and counterreceptors on adjacent cells, are implicated in synapse maturation and plasticity and memory formation. However, little is known about the molecular mechanisms of integrin action at central synapses. This study reports that postsynaptic β3 integrins control synaptic strength by regulating AMPA receptors (AMPARs) in a subunit-specific manner. Pharmacological perturbation targeting β3 integrins promotes endocytosis of GluR2-containing AMPARs via Rap1 signaling, and expression of β3 integrins produces robust changes in the abundance and composition of synaptic AMPARs without affecting dendritic spine structure. Importantly, homeostatic synaptic scaling induced by activity deprivation elevates surface expression of β3 integrins, and in turn, β3 integrins are required for synaptic scaling. These findings demonstrate a key role for integrins in the feedback regulation of excitatory synaptic strength (Cingolani, 2008).
Oligophrenin-1 (OPHN1) encodes a Rho-GTPase-activating protein (Rho-GAP) whose loss of function has been associated with X-linked mental retardation (MR). The pathophysiological role of OPHN1, however, remains poorly understood. This study shows that OPHN1 through its Rho-GAP activity plays a critical role in the activity-dependent maturation and plasticity of excitatory synapses by controlling their structural and functional stability. Synaptic activity through NMDA receptor activation drives OPHN1 into dendritic spines, where it forms a complex with AMPA receptors, and selectively enhances AMPA-receptor-mediated synaptic transmission and spine size by stabilizing synaptic AMPA receptors. Consequently, decreased or defective OPHN1 signaling prevents glutamatergic synapse maturation and causes loss of synaptic structure, function, and plasticity. These results imply that normal activity-driven glutamatergic synapse development is impaired by perturbation of OPHN1 function. Thus, these findings link genetic deficits in OPHN1 to glutamatergic dysfunction and suggest that defects in early circuitry development are an important contributory factor to this form of MR (Nadif Kasri, 2009).
Src-family protein tyrosine kinases (PTKs) transduce signals to regulate neuronal development and synaptic plasticity.
However, the nature of their activators and the molecular mechanisms underlying these neural processes are unknown. Brain-derived neurotrophic factor (BDNF) and platelet-derived growth factor enhance expression of
AMPA-type glutamate receptor 1 and 2/3 proteins in rodent
neocortical neurons via the Src-family PTK(s). The increase in AMPA receptor levels is blocked in cultured neocortical
neurons by the addition of a Src-family-selective PTK inhibitor. Accordingly, neocortical cultures from Fyn-knockout mice
fail to respond to BDNF, whereas those from wild-type mice do respond. Moreover, the neocortex of young Fyn mutants
exhibits a significant in vivo reduction in these AMPA receptor proteins but not in their mRNA levels. In vitro kinase
assay reveals that BDNF can indeed activate the Fyn kinase: it enhances tyrosine phosphorylation of Fyn as well as that of exogenously
supplemented enolase. All of these results suggest that the Src-family kinase Fyn, activated by the growth
factors, plays a crucial role in modulating AMPA receptor expression during brain development (Narisawa-Saito, 1999).
The regulation of AMPA receptor channels by serotonin signaling in pyramidal neurons of prefrontal cortex (PFC) was studied. Application of serotonin reduced the amplitude of AMPA-evoked currents, an effect mimicked by 5-HT1A receptor agonists (see Drosophila Serotonin receptor 1A) and blocked by 5-HT1A antagonists, indicating the mediation by 5-HT1A receptors. The serotonergic modulation of AMPA receptor currents was blocked by protein kinase A (PKA) activators and occluded by PKA inhibitors. Inhibiting the catalytic activity of protein phosphatase 1 (PP1) also eliminated the effect of serotonin on AMPA currents. Furthermore, the serotonergic modulation of AMPA currents was occluded by application of the Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibitors and blocked by intracellular injection of calmodulin or recombinant CaMKII. Application of serotonin or 5-HT1A agonists to PFC slices reduced CaMKII activity and the phosphorylation of AMPA receptor subunit GluR1 at the CaMKII site in a PP1-dependent manner. It is concluded that serotonin, by activating 5-HT1A receptors, suppress glutamatergic signaling through the inhibition of CaMKII, which is achieved by the inhibition of PKA and ensuing activation of PP1. This modulation demonstrates the critical role of CaMKII in serotonergic regulation of PFC neuronal activity, which may explain the neuropsychiatric behavioral phenotypes seen in CaMKII knockout mice (Cai, 2002).
Extracellular signal-regulated kinase (ERK) signaling is important for neuronal synaptic plasticity. The protein kinase ribosomal S6 kinase (RSK2; see Drosophila RSK), a downstream target of ERK, uses a C-terminal motif to bind several PDZ domain proteins in heterologous systems and in vivo. Different RSK isoforms display distinct specificities in their interactions with PDZ domain proteins. Mutation of the RSK2 PDZ ligand does not inhibit RSK2 activation in intact cells or phosphorylation of peptide substrates by RSK2 in vitro but greatly reduces RSK2 phosphorylation of PDZ domain proteins of the Shank family in heterologous cells. In primary neurons, NMDA receptor (NMDA-R) activation leads to ERK and RSK2 activation and RSK-dependent phosphorylation of transfected Shank3. RSK2-PDZ domain interactions are functionally important for synaptic transmission because neurons expressing kinase-dead RSK2 display a dramatic reduction in frequency of AMPA-type glutamate receptor-mediated miniature excitatory postsynaptic currents, an effect dependent on the PDZ ligand. These results suggest that binding of RSK2 to PDZ domain proteins and phosphorylation of these proteins or their binding partners regulates excitatory synaptic transmission (Thomas, 2005).
Neuregulin-1 (NRG1) signaling participates in numerous neurodevelopmental processes. Through linkage analysis, nrg1 has been associated with schizophrenia, although its pathophysiological role is not understood. The prevailing models of schizophrenia invoke hypofunction of the glutamatergic synapse and defects in early development of hippocampal-cortical circuitry. This study shows that the erbB4 receptor, as a postsynaptic target of NRG1, plays a key role in activity-dependent maturation and plasticity of excitatory synaptic structure and function. Synaptic activity leads to the activation and recruitment of erbB4 into the synapse. Overexpressed erbB4 selectively enhances AMPA synaptic currents and increases dendritic spine size. Preventing NRG1/erbB4 signaling destabilizes synaptic AMPA receptors and leads to loss of synaptic NMDA currents and spines. These results indicate that normal activity-driven glutamatergic synapse development is impaired by genetic deficits in NRG1/erbB4 signaling leading to glutamatergic hypofunction. These findings link proposed effectors in schizophrenia: NRG1/erbB4 signaling perturbation, neurodevelopmental deficit, and glutamatergic hypofunction (Li, 2007).
Mouse mutants were generated with targeted AMPA receptor (AMPAR) GluR-B subunit alleles, functionally expressed at
different levels and deficient in Q/R-site editing. All mutant lines have increased AMPAR calcium permeabilities in pyramidal
neurons, and one shows elevated macroscopic conductances of these channels. The AMPAR-mediated calcium influx
induces NMDA-receptor-independent long-term potentiation (LTP) in hippocampal pyramidal cell connections.
Calcium-triggered neuronal death is not observed, but mutants have mild to severe neurological dysfunctions, including
epilepsy and deficits in dendritic architecture. The seizure-prone phenotype correlate with an increase in the macroscopic
conductance, as independently revealed by the effect of a transgene for a Q/R-site-altered GluR-B subunit. Thus, changes
in GluR-B gene expression and Q/R site editing can affect critical architectural and functional aspects of excitatory principal
neurons (Feldmeyer, 1999).
Desensitization of AMPA receptors is thought to shape the
synaptic response and act as a neuroprotective mechanism at central synapses, but the molecular mechanism underlying
desensitization is poorly understood. Replacing the glutamate binding domain S1 of GluR3 (an AMPA
receptor) with S1 of GluR6 (a kainate receptor) results in a fully active but completely nondesensitizing receptor. Smaller
substitutions within S1 identify (in addition to two additional modulatory regions) a single exchange, L507Y, that is required and
sufficient for the block of desensitization. This phenotype is specific for AMPA receptors and requires an aromatic
residue at this position. L507 lies between two residues (T504 and R509) that form part of the glutamate binding site. The
physical proximity of these residues, which are involved in binding and gating, suggests they may form part of the link
between these two events (Stern-Bach, 1998).
Although GluR1o and GluR3o are
homologous at the amino acid level, GluR3o desensitizes
approximately threefold faster than GluR1o. By creating
chimeras of GluR1o and GluR3o and point amino acid exchanges in their S2 regions, two residues have been identified as
critical for GluR1o desensitization: Y716 and the
R/G RNA-edited site, R757. Intronic elements determine a codon switch in the primary transcripts at the R/G site that immediately precedes the flip/flop region. With creation of the double-point mutant (Y716F, R757G)GluR1o, complete exchange of the desensitization rate of GluR1o to that of
GluR3o is obtained. In addition, both the potency and
affinity of the subtype-selective agonist bromohomoibotenic acid are
exchanged by the Y716F mutation. A model is proposed of the AMPA
receptor binding site whereby a hydrogen-bonding matrix of water
molecules plays an important role in determining both ligand affinity
and receptor desensitization properties. Residues Y716 in GluR1 and F728 in GluR3 differentially interact with this matrix to affect the
binding affinity of some ligands, providing the possibility of
developing subtype-selective compounds (Banke, 2001).
AMPA receptor (AMPAR)-mediated ionic currents that govern gene expression, synaptic strength, and plasticity also can trigger
excitotoxicity. However, native AMPARs exhibit heterogeneous pharmacological, biochemical, and ionic permeability
characteristics, which are governed partly by receptor subunit composition.
AMPARs exhibit heterogeneous macroscopic ionic current properties and ionic permeability
characteristics. Their biophysical and pharmacological properties are governed by four genes (GluR1 to GluR4 or GluR-A to GluR-D) that encode heteromeric
receptors with high AMPA affinities that are permeable to Na+ and K+ ions. However, the relative expression of these
genes, as well as the splicing and editing of their mRNAs, imparts a diversity of pharmacological properties, gating characteristics, and Ca2+ permeability between
cells. Specifically, impermeability to Ca2+ is determined by the presence of the GluR2 subunit, which has a positively charged arginine at
position 586 of transmembrane segment 2 (Q/R site) instead of a neutral glutamine. Thus permeability to Ca2+ ions is highest in AMPARs that lack GluR2.
However, the GluR2 subunit governs more than just Ca2+ permeability. GluR subunits display sequence divergence within the C-terminal (CT) cytoplasmic tail, and
this region has been shown to mediate subunit-specific interactions with various cytoplasmic proteins. These AMPAR CT-protein interactions may govern the pharmacological properties of the receptor, receptor turnover at synapses, clustering, synaptic transmission,
efficacy, and plasticity. Thus the influence of GluR2 subunits on neuronal function
and vulnerability to excitotoxicity may occur by mechanisms other than solely those attributable to the effects of GluR2 on ionic permeability profiles (Iihara, 2001 and references therein).
GluR2 is expressed widely in mammalian neurons. For example, in cultured dissociated cortical neurons, a preparation that commonly is used to study excitotoxicity,
only 8%-15% of neurons express AMPA channels lacking GluR2. In vivo, GluR2 is
expressed widely in hippocampal pyramidal and granule neurons and in cortical neurons that
frequently are damaged by ischemia. Thus the relative abundance and, yet, heterogeneity of GluR2 expression have made it more difficult to define its role in
AMPAR-mediated excitotoxicity. In this paper mutant
mice lacking the AMPAR subunit GluR2 were used to study AMPAR-mediated excitotoxicity in cultured cortical neurons and in hippocampal neurons in vivo. It was hypothesized that in these mice the level of GluR2 expression governs the vulnerability of neurons to excitotoxicity; also examined were the ionic mechanisms involved. In cortical neuronal cultures AMPAR-mediated neurotoxicity parallels the magnitude of kainate-evoked AMPAR-mediated currents, which are
increased in neurons lacking GluR2. Ca2+ permeability, although elevated in GluR2-deficient neurons, do not correlate with excitotoxicity. However, toxicity is
reduced by removal of extracellular Na+, the main charge carrier of AMPAR-mediated currents. In vivo, the vulnerability of CA1 hippocampal neurons to stereotactic kainate injections and of CA3 neurons to intraperitoneal kainate administration is independent of GluR2 level. Neurons lacking the GluR2 subunit do not demonstrate compensatory changes in the distribution, expression, or function of AMPARs or of Ca2+-buffering proteins. Thus GluR2 level may influence excitotoxicity by effects additional to those on Ca2+ permeability, such as effects on agonist potency, ionic currents, and synaptic reorganization (Iihara, 2001).
Ionotropic glutamate receptors are tetramers, the isolated ligand binding cores that assemble as dimers. Previous work on nondesensitizing AMPA receptor mutants, which combined crystallography, ultracentrifugation, and patch-clamp recording, show that dimer formation by the ligand binding cores is required for activation of ion channel gating by agonists. To define the mechanisms responsible for stabilization of dimer assembly in native AMPA receptors, contacts between the adjacent ligand binding cores were individually targeted by amino acid substitutions, using the GluR2 crystal structure as a guide to design mutants. Disruption of a salt bridge, hydrogen bond network, and intermolecular van der Waals contacts between helices D and J in adjacent ligand binding cores greatly accelerates desensitization. Conservation of these contacts in AMPA and kainate receptors indicates that they are important determinants of dimer stability and that the dimer interface is a key structural element in the gating mechanism of these glutamate receptor families (Horning, 2004).
CA1 pyramidal neurons degenerate after transient global ischemia, whereas neurons in other regions of the hippocampus remain intact. A step in this selective injury is Ca2+ and/or Zn2+ entry through Ca2+-permeable AMPA receptor channels; reducing Ca2+ permeability of AMPA receptors via expression of Ca2+-impermeable GluR2(R) channels or activation of CRE transcription in the hippocampus of adult rats in vivo using shutoff-deficient pSFV-based vectors rescues vulnerable CA1 pyramidal neurons from forebrain ischemic injury. Conversely, the induction of Ca2+ and/or Zn2+ influx through AMPA receptors by expressing functional Ca2+-permeable GluR2(Q) channels causes the postischemic degeneration of hippocampal granule neurons that otherwise are insensitive to ischemic insult. Thus, the AMPA receptor subunit GluR2 gates entry of Ca2+ and/or Zn2+ that leads to cell death following transient forebrain ischemia (Liu, 2004).
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Glutamate receptor IIA and Glutamate receptor IIB:
Biological Overview
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| Developmental Biology
| Effects of Mutation
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