Ubiquitination regulates PSD-95 degradation and AMPA receptor surface expression

PSD-95 is a major scaffolding protein of the postsynaptic density, tethering NMDA- and AMPA-type glutamate receptors to signaling proteins and the neuronal cytoskeleton. Here we show that PSD-95 is regulated by the ubiquitin-proteasome pathway. PSD-95 interacts with and is ubiquitinated by the E3 ligase Mdm2. In response to NMDA receptor activation, PSD-95 is ubiquitinated and rapidly removed from synaptic sites by proteasome-dependent degradation. Mutations that block PSD-95 ubiquitination prevent NMDA-induced AMPA receptor endocytosis. Likewise, proteasome inhibitors prevent NMDA-induced AMPA receptor internalization and synaptically induced long-term depression. This is consistent with the notion that PSD-95 levels are an important determinant of AMPA receptor number at the synapse. These data suggest that ubiquitination of PSD-95 through an Mdm2-mediated pathway is critical in regulating AMPA receptor surface expression during synaptic plasticity.     

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.     

A GluR1-cGKII interaction regulates AMPA receptor trafficking

Trafficking of AMPA receptors (AMPARs) is regulated by specific interactions of the subunit intracellular C-terminal domains (CTDs) with other proteins, but the mechanisms involved in this process are still unclear. We have found that the GluR1 CTD binds to cGMP-dependent protein kinase II (cGKII) adjacent to the kinase catalytic site. Binding of GluR1 is increased when cGKII is activated by cGMP. cGKII and GluR1 form a complex in the brain, and cGKII in this complex phosphorylates GluR1 at S845, a site also phosphorylated by PKA. Activation of cGKII by cGMP increases the surface expression of AMPARs at extrasynaptic sites. Inhibition of cGKII activity blocks the surface increase of GluR1 during chemLTP and reduces LTP in the hippocampal slice. This work identifies a pathway, downstream from the NMDA receptor (NMDAR) and nitric oxide (NO), which stimulates GluR1 accumulation in the plasma membrane and plays an important role in synaptic plasticity.     

A direct interaction of PSD-95 with 5-HT2A serotonin receptors regulates receptor trafficking and signal transduction

The serotonin (5-hydroxytryptamine) 2A receptor (5-HT2A) is an important G protein-coupled receptor (GPCR) that mediates the effects of hallucinogens and is the target of a number of commonly prescribed medications including atypical antipsychotics, antidepressants, and anxiolytics. The 5-HT2A receptor possesses a canonical Type I PDZ-binding domain (X-Ser/Thr-X-Phi) at the carboxyl terminus and has been predicted, but never demonstrated, to interact with PDZ domain-containing proteins. We discovered that PSD-95, a prototypic PDZ domain-containing protein, directly associates with the 5-HT2A receptor and regulates 5-HT2A receptor-mediated signaling and trafficking in HEK-293 cells. Co-immunoprecipitation studies revealed that the native 5-HT2A receptor, but not a mutant lacking the PDZ-binding domain, interacted directly with PSD-95. The association with PSD-95 enhanced 5-HT2A receptor-mediated signal transduction, a novel action of PSD-95 on GPCRs. The augmentation of 5-HT2A receptor signaling by PSD-95 was not accompanied by alteration in the kinetics of 5-HT2A receptor desensitization but was associated with the inhibition of agonist-induced 5-HT2A receptor internalization. Additional studies demonstrated that 5-HT2A receptor and PSD-95 were co-localized in clusters on the cell surface of HEK-293 cells. Taken together, the present work elucidates novel roles for PSD-95 in regulating the functional activity and intracellular trafficking of 5-HT2A receptors and possibly other GPCRs.     

mPins modulates PSD-95 and SAP102 trafficking and influences NMDA receptor surface expression

Appropriate trafficking and targeting of glutamate receptors (GluRs) to the postsynaptic density is crucial for synaptic function. We show that mPins (mammalian homologue of Drosophila melanogaster partner of inscuteable) interacts with SAP102 and PSD-95 (two PDZ proteins present in neurons), and functions in the formation of the NMDAR-MAGUK (N-methyl-D-aspartate receptor-membrane-associated guanylate kinase) complex. mPins enhances trafficking of SAP102 and NMDARs to the plasma membrane in neurons. Expression of dominant-negative constructs and short-interfering RNA (siRNA)-mediated knockdown of mPins decreases SAP102 in dendrites and modifies surface expression of NMDARs. mPins changes the number and morphology of dendritic spines and these effects depend on its Galphai interaction domain, thus implicating G-protein signalling in the regulation of postsynaptic structure and trafficking of GluRs.     

Interaction between SAP97 and PSD-95, two Maguk proteins involved in synaptic trafficking of AMPA receptors

Synapse-associated protein 97 (SAP97) and postsynaptic density 95 (PSD-95) are closely related membrane-associated guanylate kinase homologs (Maguks) implicated in the synaptic targeting and anchoring of alpha-amino-5-methyl-3-hydroxy-4-isoxazolepropionic acid (AMPA)-selective glutamate receptors. Prompted by accumulating evidence for an oligomeric nature of Maguks, we examined the potential of SAP97 and PSD-95 to form heteromeric complexes. SAP97 and PSD-95 coimmunoprecipitated from rat brain detergent extracts and subsequent glutathione S-transferase pull-down and immunoprecipitation experiments showed that the interaction is mediated by binding of the N-terminal segment of SAP97 (SAP97(NTD)) to the Src homology 3 domain of PSD-95 (PSD-95(SH3)). In cultured hippocampal neurons, expression of green fluorescent protein-tagged PSD-95 triggered accumulation of SAP97 in synaptic spines, which was totally inhibited by coexpression of PSD-95(SH3). Furthermore, overexpression of green fluorescent protein-PSD-95 induced dendritic clustering of GluR-A subunit-containing AMPA receptors, which was strongly inhibited by cotransfection with SAP97(NTD) and PSD-95(SH3) constructs. Our results demonstrated a direct interaction between SAP97 and PSD-95 and suggested that this association may play a functional role in the trafficking and clustering of AMPA receptors.     

Altered interaction between PSD-95 and the NMDA receptor following transient global ischemia

The postsynaptic density (PSD) is a cytoskeletal specialization involved in the anchoring of neurotransmitter receptors and in regulating the response of postsynaptic neurons to synaptic stimulation. The postsynaptic protein PSD-95 binds to NMDA receptor subunits NR2A and NR2B and to signaling molecules such as neuronal nitric oxide synthase and p135synGAP. We investigated the effects of transient cerebral ischemia on protein interactions involving PSD-95 and the NMDA receptor in the rat hippocampus. Ischemia followed by reperfusion resulted in a decrease in the solubility of the NMDA receptor and PSD-95 in 1% sodium deoxycholate, the decrease being greater in the vulnerable CA1 hippocampal subfield than in the less sensitive CA3/dentate gyrus regions. Solubilization of the kainic acid receptor GluR6/7 and the PSD-95 binding proteins, neuronal nitric oxide synthase and p135synGAP, also decreased following ischemia. The association between PSD-95 and NR2A and NR2B, as indicated by coimmunoprecipitation, was less in postischemic samples than in sham-operated controls. Ischemia also resulted in a decrease in the size of protein complexes containing PSD-95, but had only a small effect on the size distribution of complexes containing the NMDA receptor. The results indicate that molecular interactions involving PSD-95 and the NMDA receptor are modified by an ischemic challenge.     

Nedd4-mediated AMPA receptor ubiquitination regulates receptor turnover and trafficking

α-Amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptors (AMPARs) are the primary mediators of excitatory synaptic transmission in the brain. Alterations in AMPAR localization and turnover have been considered critical mechanisms underpinning synaptic plasticity and higher brain functions, but the molecular processes that control AMPAR trafficking and stability are still not fully understood. Here, we report that mammalian AMPARs are subject to ubiquitination in neurons and in transfected heterologous cells. Ubiquitination facilitates AMPAR endocytosis, leading to a reduction in AMPAR cell-surface localization and total receptor abundance. Mutation of lysine residues to arginine residues at the glutamate receptor subunit 1 (GluA1) C-terminus dramatically reduces GluA1 ubiquitination and abolishes ubiquitin-dependent GluA1 internalization and degradation, indicating that the lysine residues, particularly K868, are sites of ubiquitination. We also find that the E3 ligase neural precursor cell expressed, developmentally down-regulated 4 (Nedd4) is enriched in synaptosomes and co-localizes and associates with AMPARs in neurons. Nedd4 expression leads to AMPAR ubiquitination, leading to reduced AMPAR surface expression and suppressed excitatory synaptic transmission. Conversely, knockdown of Nedd4 by specific siRNAs abolishes AMPAR ubiquitination. These data indicate that Nedd4 is the E3 ubiquitin ligase responsible for AMPAR ubiquitination, a modification that regulates multiple aspects of AMPAR molecular biology including trafficking, localization and stability.     

Stargazin promotes closure of the AMPA receptor ligand-binding domain

Transmembrane AMPA receptor (AMPAR) regulatory proteins (TARPs) markedly enhance AMPAR function, altering ligand efficacy and receptor gating kinetics and thereby shaping the postsynaptic response. The structural mechanism underlying TARP effects on gating, however, is unknown. Here we find that the prototypical member of the TARP family, stargazin or γ-2, rescues gating deficits in AMPARs carrying mutations that destabilize the closed-cleft states of the ligand-binding domain (LBD), suggesting that stargazin reverses the effects of these mutations and likely stabilizes closed LBD states. Furthermore, stargazin promotes a more closed conformation of the LBD, as indicated by reduced accessibility to the large antagonist NBQX. Consistent with the functional studies, luminescence resonance energy transfer experiments directly demonstrate that the AMPAR LBD is on average more closed in the presence of stargazin, in both the apo and agonist-bound states. The additional cleft closure and/or stabilization of the more closed-cleft states of the LBD is expected to translate to higher agonist efficacy and could contribute to the structural mechanism for stargazin modulation of AMPAR function.     

AMPA receptor biogenesis and trafficking

AMPA-type glutamate receptors mediate the majority of fast excitatory transmission in the central nervous system. The trafficking of AMPA receptors to and from synapses alters synaptic strength and has been recognized as a central mechanism underlying various forms of synaptic plasticity. Both secretory and endocytic trafficking events seem to be driven by the subunit composition of AMPA receptor tetramers. Moreover, recent work suggests that synapses employ different tetramer combinations in response to altered synaptic input, suggesting the existence of signalling pathways that mediate remodelling of AMPA receptors. These latest developments and recent progress in elucidating the mechanisms that underlie channel assembly and trafficking are the subject of this review.     

G protein-coupled receptor kinase 5 regulates beta 1-adrenergic receptor association with PSD-95.

We previously reported that the beta(1)-adrenergic receptor (beta(1)AR) associates with PSD-95 through a PDZ domain-mediated interaction, by which PSD-95 modulates beta(1)AR function and facilitates the physical association of beta(1)AR with other synaptic proteins such as N-methyl-d-aspartate receptors. Here we demonstrate that beta(1)AR association with PSD-95 is regulated by G protein-coupled receptor kinase 5 (GRK5). When beta(1)AR and PSD-95 were coexpressed with either GRK2 or GRK5 in COS-7 cells, GRK5 alone dramatically decreased the association of beta(1)AR with PSD-95, although GRK2 and GRK5 both could be co-immunoprecipitated with beta(1)AR and both could enhance receptor phosphorylation in vivo. Increasing expression of GRK5 in the cells led to further decreased beta(1)AR association with PSD-95. Stimulation with the beta(1)AR agonist isoproterenol further decreased PSD-95 binding to beta(1)AR. In addition, GRK5 protein kinase activity was required for this regulatory effect since a kinase-inactive GRK5 mutant had no effect on PSD-95 binding to beta(1)AR. Moreover, the regulatory effect of GRK5 on beta(1)AR association with PSD-95 was observed only when GRK5 was expressed together with the receptor, but not when GRK5 was coexpressed with PSD-95. Thus, we propose that GRK5 regulates beta(1)AR association with PSD-95 through phosphorylation of beta(1)AR. Regulation of protein association through receptor phosphorylation may be a general mechanism used by G protein-coupled receptors that associate via PDZ domain-mediated protein/protein interactions.     

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.     

AMPA receptor trafficking and long-term potentiation

Activity-dependent changes in synaptic function are believed to underlie the formation of memories. A prominent example is long-term potentiation (LTP), whose mechanisms have been the subject of considerable scrutiny over the past few decades. I review studies from our laboratory that support a critical role for AMPA receptor trafficking in LTP and experience-dependent plasticity.     

Alternative N-terminal domains of PSD-95 and SAP97 govern activity-dependent regulation of synaptic AMPA receptor function

PSD-95 and SAP97 are scaffolding proteins that have been implicated in regulating AMPA receptor incorporation and function at synapses. Gain- and loss-of-function approaches, however, have generated conflicting results. To minimize adaptations during development and potential dominant-negative effects of overexpression, we have combined silencing of endogenous PSD-95 in mature neurons with heterologous expression of specific SAP97 or PSD-95 isoforms. We find that both PSD-95 and SAP97 contain alternative N termini expressing either double cysteines that normally are palmitoylated (alpha-isoforms) or an L27 domain (beta-isoforms). Whereas alpha-isoforms of PSD-95 and SAP97 influence AMPA receptor-mediated synaptic strength independent of activity, the effects of beta-isoforms are regulated by activity in a CaMKII-dependent manner. Importantly, the synaptic effects of the beta-isoforms are masked by the endogenous alpha-isoform of PSD-95. These results demonstrate that the different N termini of the predominant endogenous forms of PSD-95 (alpha-isoform) and SAP97 (beta-isoform) govern their role in regulating synaptic function.     

Acute Inactivation of PSD-95 Destabilizes AMPA Receptors at Hippocampal Synapses

Postsynatptic density protein (PSD-95) is a 95 kDa scaffolding protein that assembles signaling complexes at synapses. Over-expression of PSD-95 in primary hippocampal neurons selectively increases synaptic localization of AMPA receptors; however, mice lacking PSD-95 display grossly normal glutamatergic transmission in hippocampus. To further study the scaffolding role of PSD-95 at excitatory synapses, we generated a recombinant PSD-95-4c containing a tetracysteine motif, which specifically binds a fluorescein derivative and allows for acute and permanent inactivation of PSD-95. Interestingly, acute inactivation of PSD-95 in rat hippocampal cultures rapidly reduced surface AMPA receptor immunostaining, but did not affected NMDA or transferrin receptor localization. Acute photoinactivation of PSD-95 in dissociated neurons causes ∼80% decrease in GluR2 surface staining observed by live-cell microscopy within 15 minutes of PSD-95-4c ablation. These results confirm that PSD-95 stabilizes AMPA receptors at postsynaptic sites and provides insight into the dynamic interplay between PSD-95 and AMPA receptors in live neurons.     

The interaction between PSD-95 and Ca2+/calmodulin is enhanced by PDZ-binding proteins

In this study, we evaluate the interaction between the postsynaptic scaffolding protein, PSD-95, and calmodulin. Surface plasmon resonance spectroscopy was used to characterize the binding of PSD-95 to calmodulin that had been immobilized on a sensor chip. Additionally, soluble calmodulin was found to inhibit the binding of PSD-95 to immobilized calmodulin. The HOOK region of PSD-95, which is located between the src homology 3 domain and the guanylate kinase-like domain, was determined to be involved in the binding of PSD-95 to calmodulin. We also found that C-terminal peptides from proteins such as CRIPT and the N-methyl-d-aspartate receptor NR2B subunit, which associate with the PDZ domain of PSD-95, enhanced the affinity of PSD-95 for calmodulin. The binding of ligands to the PDZ domain may change the conformation of PSD-95 and affect the interaction between PSD-95 and calmodulin.     

TARPs and the AMPA receptor trafficking paradox

AMPA receptors (AMPARs) conduct fast, excitatory currents that depolarize neurons and trigger action potentials. AMPARs took on new importance when it was shown that AMPAR transport can increase or decrease the number of AMPARs at synapses and give rise to synapse plasticity, including long-term potentiation (LTP) and long-term depression (LTD). This review considers how transmembrane AMPAR regulatory proteins (TARPs), a novel family of AMPAR auxiliary subunits, have changed our view of AMPAR transport and raised some perplexing questions.     

AMPA receptor trafficking at excitatory synapses

Excitatory synapses in the CNS release glutamate, which acts primarily on two sides of ionotropic receptors: AMPA receptors and NMDA receptors. AMPA receptors mediate the postsynaptic depolarization that initiates neuronal firing, whereas NMDA receptors initiate synaptic plasticity. Recent studies have emphasized that distinct mechanisms control synaptic expression of these two receptor classes. Whereas NMDA receptor proteins are relatively fixed, AMPA receptors cycle synaptic membranes on and off. A large family of interacting proteins regulates AMPA receptor turnover at synapses and thereby influences synaptic strength. Furthermore, neuronal activity controls synaptic AMPA receptor trafficking, and this dynamic process plays a key role in the synaptic plasticity that is thought to underlie aspects of learning and memory.