Phosphorylation of stargazin by protein kinase A regulates its interaction with PSD-95.

Stargazin is the first transmembrane protein known to associate with AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) glutamate receptors (AMPARs) and regulate their synaptic targeting by two distinct mechanisms, specifically via delivery of AMPARs to the surface membrane and synaptic targeting of these receptors by binding to PSD-95/SAP-90 and related PDZ proteins. However, it is not known whether and how this stargazin-mediated synaptic targeting of AMPARs is regulated. Stargazin interacts with the PDZ domains of PSD-95 through the C-terminal PDZ-binding motif. The stargazin C terminus contains a consensus sequence for phosphorylation by cAMP-dependent protein kinase A (PKA). Phosphorylation site-specific stargazin antibodies reveal that the stargazin C terminus is phosphorylated at the Thr-321 residue in heterologous cells and in vivo. Stargazin phosphorylation is enhanced by the catalytic subunit of PKA. Mutations mimicking stargazin phosphorylation (T321E and T321D) lead to elimination of yeast two-hybrid interactions, in vitro coimmunoprecipitation, and coclustering between stargazin and PSD-95. Phosphorylated stargazin shows a selective loss of coimmunoprecipitation with PSD-95 in heterologous cells and limited enrichment in postsynaptic density fractions of rat brain. These results suggest that phosphorylation of the stargazin C terminus by PKA regulates its interaction with PSD-95 and synaptic targeting of AMPARs.     

Biomimetic divalent ligands for the acute disruption of synaptic AMPAR stabilization

The interactions of the AMPA receptor (AMPAR) auxiliary subunit Stargazin with PDZ domain-containing scaffold proteins such as PSD-95 are critical for the synaptic stabilization of AMPARs. To investigate these interactions, we have developed biomimetic competing ligands that are assembled from two Stargazin-derived PSD-95/DLG/ZO-1 (PDZ) domain-binding motifs using 'click' chemistry. Characterization of the ligands in vitro and in a cellular FRET-based model revealed an enhanced affinity for the multiple PDZ domains of PSD-95 compared to monovalent peptides. In cultured neurons, the divalent ligands competed with transmembrane AMPAR regulatory protein (TARP) for the intracellular membrane-associated guanylate kinase resulting in increased lateral diffusion and endocytosis of surface AMPARs, while showing strong inhibition of synaptic AMPAR currents. This provides evidence for a model in which the TARP-containing AMPARs are stabilized at the synapse by engaging in multivalent interactions. In light of the prevalence of PDZ domain clusters, these new biomimetic chemical tools could find broad application for acutely perturbing multivalent complexes.     

Regulation of AMPA receptor surface diffusion by PSD-95 slots

Excitatory synaptic transmission is largely mediated by AMPA receptors (AMPARs) present at the postsynaptic density. Recent studies in single molecule tracking of AMPAR has revealed that extrasynaptic AMPARs are highly mobile and thus might serve as a readily available pool for their synaptic recruitment during synaptic plasticity events such as long-term potentiation (LTP). Because this hypothesis relies on the cell's ability to increase the number of diffusional traps or 'slots' at synapses during LTP, we will review a number of protein-protein interactions that might impact AMPARs lateral diffusion and thus potentially serve as slots. Recent studies have identified the interaction between the AMPAR-Stargazin complex and PSD-95 as the minimal components of the diffusional trapping slot. We will overview the molecular basis of this critical interaction, its activity-dependent regulation and its potential contribution to LTP.     

Regulated RalBP1 Binding to RalA and PSD-95 Controls AMPA Receptor Endocytosis and LTD

Long-term depression (LTD) is a long-lasting activity-dependent decrease in synaptic strength. NMDA receptor (NMDAR)–dependent LTD, an extensively studied form of LTD, involves the endocytosis of AMPA receptors (AMPARs) via protein dephosphorylation, but the underlying mechanism has remained unclear. We show here that a regulated interaction of the endocytic adaptor RalBP1 with two synaptic proteins, the small GTPase RalA and the postsynaptic scaffolding protein PSD-95, controls NMDAR-dependent AMPAR endocytosis during LTD. NMDAR activation stimulates RalA, which binds and translocates widespread RalBP1 to synapses. In addition, NMDAR activation dephosphorylates RalBP1, promoting the interaction of RalBP1 with PSD-95. These two regulated interactions are required for NMDAR-dependent AMPAR endocytosis and LTD and are sufficient to induce AMPAR endocytosis in the absence of NMDAR activation. RalA in the basal state, however, maintains surface AMPARs. We propose that NMDAR activation brings RalBP1 close to PSD-95 to promote the interaction of RalBP1-associated endocytic proteins with PSD-95-associated AMPARs. This suggests that scaffolding proteins at specialized cellular junctions can switch their function from maintenance to endocytosis of interacting membrane proteins in a regulated manner.     

Acute knockdown of AMPA receptors reveals a trans-synaptic signal for presynaptic maturation

Newly formed glutamatergic synapses often lack postsynaptic AMPA-type glutamate receptors (AMPARs). Aside from 'unsilencing' the postsynaptic site, however, the significance of postsynaptic AMPAR insertion during synapse maturation remains unclear. To investigate the role of AMPAR in synapse maturation, we used RNA interference (RNAi) to knockdown AMPARs in cultured hippocampal neurons. Surprisingly, loss of postsynaptic AMPARs increased the occurrence of presynaptically inactive synapses without changing the release probability of the remaining active synapses. Additionally, heterologous synapses formed between axons and AMPAR-expressing HEK cells develop significantly fewer inactive presynaptic terminals. The extracellular domain of the AMPAR subunit GluA2 was sufficient to reproduce this effect at heterologous synapses. Indeed, the retrograde signalling by AMPARs is independent of their channel function as RNAi-resistant AMPARs restore synaptic transmission in neurons lacking AMPARs despite chronic receptor antagonist treatment. Our findings suggest that postsynaptic AMPARs perform an organizational function at synapses that exceeds their standard role as ionotropic receptors by conveying a retrograde trans-synaptic signal that increases the transmission efficacy at a synapse.     

Molecular dissociation of the role of PSD-95 in regulating synaptic strength and LTD

The postsynaptic density protein PSD-95 influences synaptic AMPA receptor (AMPAR) content and may play a critical role in LTD. Here we demonstrate that the effects of PSD-95 on AMPAR-mediated synaptic responses and LTD can be dissociated. Our findings suggest that N-terminal-domain-mediated dimerization is important for PSD-95's effect on basal synaptic AMPAR function, whereas the C-terminal SH(3)-GK domains are also necessary for localizing PSD-95 to synapses. We identify PSD-95 point mutants (Q15A, E17R) that maintain PSD-95's influence on basal AMPAR synaptic responses yet block LTD. These point mutants increase the proteolysis of PSD-95 within its N-terminal domain, resulting in a C-terminal fragment that functions as a dominant negative likely by scavenging critical signaling proteins required for LTD. Thus, the C-terminal portion of PSD-95 serves a dual function. It is required to localize PSD-95 at synapses and as a scaffold for signaling proteins that are required for LTD.     

PSD-95-like membrane associated guanylate kinases (PSD-MAGUKs) and synaptic plasticity

Activity-dependent modification of excitatory synaptic transmission is a fundamental mechanism for developmental plasticity of the neural circuits and experience-dependent plasticity. Synaptic glutamatergic receptors including AMPA receptors and NMDA receptors (AMPARs and NMDARs) are embedded in the postsynaptic density, a highly organized protein network. Overwhelming data have shown that PSD-95-like membrane associated guanylate kinases (PSD-MAGUKs), a major family of scaffold proteins at glutamatergic synapses, regulate basal synaptic AMPAR function and trafficking. It is now clear that PSD-MAGUKs have multifaceted functions in regulating both basal synaptic transmission and synaptic plasticity. Here we discuss recent advancements in understanding the roles of PSD-95 and other family members of PSD-MAGUKs in synaptic plasticity, both as an anchoring protein for synaptic AMPARs and as a signaling scaffold for mediating the interaction of the signaling complex and NMDARs.     

PKMζ is necessary and sufficient for synaptic clustering of PSD-95

The persistent activity of protein kinase Mzeta (PKMζ), a brain-specific, constitutively active protein kinase C isoform, maintains synaptic long-term potentiation (LTP). Structural remodeling of the postsynaptic density is believed to contribute to the expression of LTP. We therefore examined the role of PKMζ in reconfiguring PSD-95, the major postsynaptic scaffolding protein at excitatory synapses. In primary cultures of hippocampal neurons, PKMζ activity was critical for increasing the size of PSD-95 clusters during chemical LTP (cLTP). Increasing PKMζ activity by overexpressing the kinase in hippocampal neurons was sufficient to increase PSD-95 cluster size, spine size, and postsynaptic AMPAR subunit GluA2. Overexpression of an inactive mutant of PKMζ did not increase PSD-95 clustering, and applications of the ζ-pseudosubstrate inhibitor ZIP reversed the PKMζ-mediated increases in PSD-95 clustering, indicating that the activity of PKMζ is necessary to induce and maintain the increased size of PSD-95 clusters. Thus the persistent activity of PKMζ is both necessary and sufficient for maintaining increases of PSD-95 clusters, providing a unified mechanism for long-term functional and structural modifications of synapses.     

Sequential Delivery of Synaptic GluA1- and GluA4-containing AMPA Receptors (AMPARs) by SAP97 Anchored Protein Complexes in Classical Conditioning

Multiple signaling pathways are involved in AMPAR trafficking to synapses during synaptic plasticity and learning. The mechanisms for how these pathways are coordinated in parallel but maintain their functional specificity involves subcellular compartmentalization of kinase function by scaffolding proteins, but how this is accomplished is not well understood. Here, we focused on characterizing the molecular machinery that functions in the sequential synaptic delivery of GluA1- and GluA4-containing AMPARs using an in vitro model of eyeblink classical conditioning. We show that conditioning induces the interaction of selective protein complexes with the key structural protein SAP97, which tightly regulates the synaptic delivery of GluA1 and GluA4 AMPAR subunits. The results demonstrate that in the early stages of conditioning the initial activation of PKA stimulates the formation of a SAP97-AKAP/PKA-GluA1 protein complex leading to synaptic delivery of GluA1-containing AMPARs through a SAP97-PSD95 interaction. This is followed shortly thereafter by generation of a SAP97-KSR1/PKC-GluA4 complex for GluA4 AMPAR subunit delivery again through a SAP97-PSD95 interaction. These data suggest that SAP97 forms the molecular backbone of a protein scaffold critical for delivery of AMPARs to the PSD during conditioning. Together, the findings reveal a cooperative interaction of multiple scaffolding proteins for appropriately timed delivery of subunit-specific AMPARs to synapses and support a sequential two-stage model of AMPAR synaptic delivery during classical conditioning.     

KEL-8 is a substrate receptor for CUL3-dependent ubiquitin ligase that regulates synaptic glutamate receptor turnover

The regulated localization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors (AMPARs) to synapses is an important component of synaptic signaling and plasticity. Regulated ubiquitination and endocytosis determine the synaptic levels of AMPARs, but it is unclear which factors conduct these processes. To identify genes that regulate AMPAR synaptic abundance, we screened for mutants that accumulate high synaptic levels of the AMPAR subunit GLR-1 in Caenorhabditis elegans. GLR-1 is localized to postsynaptic clusters, and mutants for the BTB-Kelch protein KEL-8 have increased GLR-1 levels at clusters, whereas the levels and localization of other synaptic proteins seem normal. KEL-8 is a neuronal protein and is localized to sites adjacent to GLR-1 postsynaptic clusters along the ventral cord neurites. KEL-8 is required for the ubiquitin-mediated turnover of GLR-1 subunits, and kel-8 mutants show an increased frequency of spontaneous reversals in locomotion, suggesting increased levels of GLR-1 are present at synapses. KEL-8 binds to CUL-3, a Cullin 3 ubiquitin ligase subunit that we also find mediates GLR-1 turnover. Our findings indicate that KEL-8 is a substrate receptor for Cullin 3 ubiquitin ligases that is required for the proteolysis of GLR-1 receptors and suggest a novel postmitotic role in neurons for Kelch/CUL3 ubiquitin ligases.     

Substrate localization creates specificity in calcium/calmodulin-dependent protein kinase II signaling at synapses

Calcium/calmodulin-dependent protein kinase II (CaMKII), a major component of the postsynaptic density (PSD) of excitatory synapses, plays a key role in the regulation of synaptic function in the mammalian brain. Although many postsynaptic substrates for CaMKII have been characterized in vitro, relatively little is known about their phosphorylation in vivo. By tagging synaptic proteins with a peptide substrate specific for CaMKII and expressing them in cultured neurons, we have visualized substrate phosphorylation by CaMKII at intact synapses. All substrates tested were strongly phosphorylated by CaMKII in HEK293 cells. However, activity-dependent phosphorylation of substrates at synapses was highly selective in that the glutamate receptor subunits NR2B and GluR1 were poorly phosphorylated whereas PSD-95 and Stargazin, proteins implicated in the scaffolding and trafficking of AMPA receptors, were robustly phosphorylated. Phosphatase activity limited phosphorylation of Stargazin but not NR2B and GluR1. These results suggest that the unique molecular architecture of the PSD results in highly selective substrate discrimination by CaMKII.     

Shape-Induced Asymmetric Diffusion in Dendritic Spines Allows Efficient Synaptic AMPA Receptor Trapping

Dendritic spines are the primary postsynaptic sites of excitatory neurotransmission in the brain. They exhibit a remarkable morphological variety, ranging from thin protrusions, to stubby shapes, to bulbous mushroom shapes. The remodeling of spines is thought to regulate the strength of the synaptic connection, which depends vitally on the number and the spatial distribution of AMPA-type glutamate receptors (AMPARs). We present numerical and analytical analyses demonstrating that this shape strongly affects AMPAR diffusion. We report a pronounced suppression of the receptor exit rate out of spines with decreasing neck radius. Thus, mushroomlike spines become highly effective at retaining receptors in the spine head. Moreover, we show that the postsynaptic density further enhances receptor trapping, particularly in mushroomlike spines local exocytosis in the spine head, in contrast to release at the base, provides rapid and specific regulatory control of AMPAR concentration at synapses.     

Insulin receptor substrate of 53 kDa links postsynaptic shank to PSD-95

The insulin receptor substrate of 53 kDa (IRSp53) is a target of the small GTPase cdc42 which is strongly enriched in the postsynaptic density of excitatory synapses. IRSp53 interacts with the postsynaptic shank1 scaffolding molecule in a cdc42 regulated manner. The functional significance of the cdc42/IRSp53 pathway in postsynaptic sites is however, unclear. Here we identify PSD-95 as a second synaptic interaction partner of IRSp53. Interaction is mediated by a C-terminal PDZ binding motif in IRSp53 and the second PDZ domain of PSD-95. In HEK cells, overexpressed IRSp53 induces filopodia and targets PSD-95 into these processes. Immunoprecipitation and immunocytochemistry experiments demonstrate that the interaction occurs at postsynaptic sites in the brain. By virtue of its PDZ-binding and SH3 domains, IRSp53 is capable of inducing the formation of a triple complex (shank1/IRSp53/PSD-95).     

Shape-Induced Asymmetric Diffusion in Dendritic Spines Allows Efficient Synaptic AMPA Receptor Trapping

Dendritic spines are the primary postsynaptic sites of excitatory neurotransmission in the brain. They exhibit a remarkable morphological variety, ranging from thin protrusions, to stubby shapes, to bulbous mushroom shapes. The remodeling of spines is thought to regulate the strength of the synaptic connection, which depends vitally on the number and the spatial distribution of AMPA-type glutamate receptors (AMPARs). We present numerical and analytical analyses demonstrating that this shape strongly affects AMPAR diffusion. We report a pronounced suppression of the receptor exit rate out of spines with decreasing neck radius. Thus, mushroomlike spines become highly effective at retaining receptors in the spine head. Moreover, we show that the postsynaptic density further enhances receptor trapping, particularly in mushroomlike spines local exocytosis in the spine head, in contrast to release at the base, provides rapid and specific regulatory control of AMPAR concentration at synapses.     

Quaternary structure, protein dynamics, and synaptic function of SAP97 controlled by L27 domain interactions

Single-particle electron microscopy (EM) combined with biochemical measurements revealed the molecular shape of SAP97 and a monomer-dimer transition that depended on the N-terminal L27 domain. Overexpression of SAP97 drove GluR1 to synapses, potentiated AMPA receptor (AMPAR) excitatory postsynaptic currents (EPSCs), and occluded LTP. Synaptic potentiation and GluR1 delivery were dissociable by L27 domain mutants that inhibit multimerization of SAP97. Loss of potentiation was correlated with faster turnover of monomeric SAP97 mutants in dendritic spines. We propose that L27-mediated interactions of SAP97 with itself or other proteins regulate the synaptic delivery of AMPARs. RNAi knockdown of endogenous PSD-95 depleted surface GluR1 and impaired AMPA EPSCs. In contrast, RNAi knockdown of endogenous SAP97 reduced surface expression of both GluR1 and GluR2 and inhibited both AMPA and NMDA EPSCs. Thus SAP97 has a broader role than its close relative, PSD-95, in the maintenance of synaptic function.     

Molecular constituents of neuronal AMPA receptors

Dynamic regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) underlies aspects of synaptic plasticity. Although numerous AMPAR-interacting proteins have been identified, their quantitative and relative contributions to native AMPAR complexes remain unclear. Here, we quantitated protein interactions with neuronal AMPARs by immunoprecipitation from brain extracts. We found that stargazin-like transmembrane AMPAR regulatory proteins (TARPs) copurified with neuronal AMPARs, but we found negligible binding to GRIP, PICK1, NSF, or SAP-97. To facilitate purification of neuronal AMPAR complexes, we generated a transgenic mouse expressing an epitope-tagged GluR2 subunit of AMPARs. Taking advantage of this powerful new tool, we isolated two populations of GluR2 containing AMPARs: an immature complex with the endoplasmic reticulum chaperone immunoglobulin-binding protein and a mature complex containing GluR1, TARPs, and PSD-95. These studies establish TARPs as the auxiliary components of neuronal AMPARs.     

Moderate AMPA receptor clustering on the nanoscale can efficiently potentiate synaptic current

The prevailing view at present is that postsynaptic expression of the classical NMDA receptor-dependent long-term potentiation relies on an increase in the numbers of local AMPA receptors (AMPARs). This is thought to parallel an expansion of postsynaptic cell specializations, for instance dendritic spine heads, which accommodate synaptic receptor proteins. However, glutamate released into the synaptic cleft can normally activate only a hotspot of low-affinity AMPARs that occur in the vicinity of the release site. How the enlargement of the AMPAR pool is causally related to the potentiated AMPAR current remains therefore poorly understood. To understand possible scenarios of postsynaptic potentiation, here we explore a detailed Monte Carlo model of the typical small excitatory synapse. Simulations suggest that approximately 50% increase in the synaptic AMPAR current could be provided by expanding the existing AMPAR pool at the expense of 100–200% new AMPARs added at the same packing density. Alternatively, reducing the inter-receptor distances by only 30–35% could achieve a similar level of current potentiation without any changes in the receptor numbers. The NMDA receptor current also appears sensitive to the NMDA receptor crowding. Our observations provide a quantitative framework for understanding the ‘resource-efficient’ ways to enact use-dependent changes in the architecture of central synapses.     

Hippocampal LTD expression involves a pool of AMPARs regulated by the NSF-GluR2 interaction

We investigated whether the interaction between the N-ethyl-maleimide-sensitive fusion protein (NSF) and the AMPA receptor (AMPAR) subunit GluR2 is involved in synaptic plasticity in the CA1 region of the hippocampus. Blockade of the NSF-GluR2 interaction by a specific peptide (pep2m) introduced into neurons prevented homosynaptic, de novo long-term depression (LTD). Moreover, saturation of LTD prevented the pep2m-induced reduction in AMPAR-mediated excitatory postsynaptic currents (EPSCs). Minimal stimulation experiments indicated that both pep2m action and LTD were due to changes in quantal size and quantal content but were not associated with changes in AMPAR single-channel conductance or EPSC kinetics. These results suggest that there is a pool of AMPARs dependent on the NSF-GluR2 interaction and that LTD expression involves the removal of these receptors from synapses.     

Synaptic targeting of the postsynaptic density protein PSD-95 mediated by lipid and protein motifs

During synaptic development, proteins aggregate at specialized pre- and postsynaptic structures. Mechanisms that mediate protein clustering at these sites remain unknown. To investigate this process, we analyzed synaptic targeting of a postsynaptic density protein, PSD-95, by expressing green fluorescent protein- (GFP-) tagged PSD-95 in cultured hippocampal neurons. We find that postsynaptic clustering relies on three elements of PSD-95: N-terminal palmitoylation, the first two PDZ domains, and a C-terminal targeting motif. In contrast, disruptions of PDZ3, SH3, or guanylate kinase (GK) domains do not affect synaptic targeting. Palmitoylation is sufficient to target the diffusely expressed SAP-97 to synapses, and palmitoylation cannot be replaced with alternative membrane association motifs, suggesting that a specialized synaptic lipid environment mediates postsynaptic clustering. The requirements for PDZ domains and a C-terminal domain of PSD-95 indicate that protein-protein interactions cooperate with lipid interactions in synaptic targeting.     

S-nitrosylation and S-palmitoylation reciprocally regulate synaptic targeting of PSD-95

PSD-95, a principal scaffolding component of the postsynaptic density, is targeted to synapses by palmitoylation, where it couples NMDA receptor stimulation to production of nitric oxide (NO) by neuronal nitric oxide synthase (nNOS). Here, we show that PSD-95 is physiologically S-nitrosylated. We identify cysteines 3 and 5, which are palmitoylated, as sites of nitrosylation, suggesting a competition between these two modifications. In support of this hypothesis, physiologically produced NO inhibits PSD-95 palmitoylation in granule cells of the cerebellum, decreasing the number of PSD-95 clusters at synaptic sites. Further, decreased palmitoylation, as seen in heterologous cells treated with 2-bromopalmitate or in ZDHHC8 knockout mice deficient in a PSD-95 palmitoyltransferase, results in increased PSD-95 nitrosylation. These data support a model in which NMDA-mediated production of NO regulates targeting of PSD-95 to synapses via mutually competitive cysteine modifications. Thus, differential modification of cysteines may represent a general paradigm in signal transduction.