Multiple association states between glycine receptors and gephyrin identified by SPT analysis

The scaffolding protein gephyrin is known to anchor glycine receptors (GlyR) at synapses and to participate in the dynamic equilibrium between synaptic and extrasynaptic GlyR in the neuronal membrane. Here we investigated the properties of this interaction in cells cotransfected with YFP-tagged gephyrin and GlyR subunits possessing an extracellular myc-tag. In HeLa cells and young neurons, single particle tracking was used to follow in real time individual GlyR, labeled with quantum dots, traveling into and out of gephyrin clusters. Analysis of the diffusion properties of two GlyR subunit types--able or unable to bind gephyrin--gave access to the association states of GlyR with its scaffolding protein. Our results indicated that an important portion of GlyR could be linked to a few molecules of gephyrin outside gephyrin clusters. This emphasizes the role of scaffolding proteins in the extrasynaptic membrane and supports the implication of gephyrin-gephyrin interactions in the stabilization of GlyR at synapses. The kinetic parameters controlling the equilibrium between GlyR inside and outside clusters were also characterized. Within clusters, we identified two subpopulations of GlyR with distinct degrees of stabilization between receptors and scaffolding proteins.     

Modeling synaptic dynamics driven by receptor lateral diffusion

The synaptic weight between a pre- and a postsynaptic neuron depends in part on the number of postsynaptic receptors. On the surface of neurons, receptors traffic by random motion in and out from a microstructure called the postsynaptic density (PSD). In the PSD, receptors can be stabilized at the membrane when they bind to scaffolding proteins. We propose a mathematical model to compute the postsynaptic counterpart of the synaptic weight based on receptor trafficking. We take into account the receptor fluxes at the PSD, which can be regulated by neuronal activity, and the interactions of receptors with the scaffolding molecules. Using a Markovian approach, we estimate the mean and the fluctuations of the number of bound receptors. When the number of receptors is large, a deterministic system is also derived. Moreover, these equations can be used, for example, to fit fluorescence-recovery-after-photobleaching experiments to determine, in living neurons, the chemical binding constants for the receptors/scaffolding molecules interaction at synapses.     

Glutamate receptor dynamics in dendritic microdomains

Among diverse factors regulating excitatory synaptic transmission, the abundance of postsynaptic glutamate receptors figures prominently in molecular memory and learning-related synaptic plasticity. To allow for both long-term maintenance of synaptic transmission and acute changes in synaptic strength, the relative rates of glutamate receptor insertion and removal must be tightly regulated. Interactions with scaffolding proteins control the targeting and signaling properties of glutamate receptors within the postsynaptic membrane. In addition, extrasynaptic receptor populations control the equilibrium of receptor exchange at synapses and activate distinct signaling pathways involved in plasticity. Here, we review recent findings that have shaped our current understanding of receptor mobility between synaptic and extrasynaptic compartments at glutamatergic synapses, focusing on AMPA and NMDA receptors. We also examine the cooperative relationship between intracellular trafficking and surface diffusion of glutamate receptors that underlies the expression of learning-related synaptic plasticity.     

AMPA and NMDA glutamate receptor trafficking: multiple roads for reaching and leaving the synapse

Glutamate receptor trafficking in and out of synapses is one of the core mechanisms for rapid changes in the number of functional receptors during synaptic plasticity. Recent data have shown that the fast gain and loss of receptors from synaptic sites are accounted for by endocytic/exocytic processes and by their lateral diffusion in the plane of the membrane. These events are interdependent and regulated by neuronal activity and interactions with scaffolding proteins. We review here the main cellular steps for AMPA and NMDA receptor synthesis, traffic within intracellular organelles, membrane exocytosis/endocytosis and surface trafficking. We focus on new findings that shed light on the regulation of receptor cycling events and surface trafficking and the way that this might reshape our thinking about the specific regulation of receptor accumulation at synapses.     

Physiology of synapse: from molecular modules to retrograde modulation

Synapses are highly organized, specific structures assuring rapid and highly selective interactions between cells. Synaptic transmission involves the release of neurotransmitter from presynaptic neurons and its detection by specific ligand-gated ion channels at the surface membrane of postsynaptic neurons. The protenomic analysis shows that for self-formation and functioning of synapses nearly 2000 proteins are involved in mammalian brain. The core complex in excitatory synapses includes glutamate receptors, potassium channels, CaMKII, scaffolding protein and actin. These proteins exist as part of a highly organized protein complex known as the postsynaptic density (PSD). The coordinated functioning of the different PSD components determines the strength of signalling between the pre- and postsynaptic neurons. Synaptic plasticity is regulated by changes in the amount of receptors on the postsynaptic membrane, changes in the shape and size of dendritic spines, posttranslational modification of PSD components, modulation kinetics of synthesis and degradation of proteins. Integration of these processes leads to long-lasting changes in synaptic function and neuronal networks underlying learning-related plasticity, memory and information treatment in nervous system of multicellular organisms.     

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.     

ProSAP/Shank proteins - a family of higher order organizing molecules of the postsynaptic density with an emerging role in human neurological disease

The postsynaptic density (PSD) is a specialized electron-dense structure underneath the postsynaptic plasmamembrane of excitatory synapses. It is thought to anchor and cluster glutamate receptors exactly opposite to the presynaptic neurotransmitter release site. Various efforts to study the molecular structure of the PSD identified several new proteins including membrane receptors, cell adhesion molecules, components of signalling cascades, cytoskeletal elements and adaptor proteins with scaffolding functions to interconnect these PSD components. The characterization of a novel adaptor protein family, the ProSAPs or Shanks, sheds new light on the basic structural organization of the PSD. ProSAPs/Shanks are multidomain proteins that interact directly or indirectly with receptors of the postsynaptic membrane including NMDA-type and metabotropic glutamate receptors, and the actin-based cytoskeleton. These interactions suggest that ProSAP/Shanks may be important scaffolding molecules of the PSD with a crucial role in the assembly of the PSD during synaptogenesis, in synaptic plasticity and in the regulation of dendritic spine morphology. Moreover the analysis of a patient with 22q13.3 distal deletion syndrome revealed a balanced translocation with a breakpoint in the human ProSAP2/Shank3 gene. This ProSAP2/Shank3 haploinsufficiency may cause a syndrome that is characterized by severe expressive language delay, mild mental retardation and minor facial dysmorphisms.     

Scaffolding Proteins of the Post-synaptic Density Contribute to Synaptic Plasticity by Regulating Receptor Localization and Distribution: Relevance for Neuropsychiatric Diseases

Synaptic plasticity represents the long lasting activity-related strengthening or weakening of synaptic transmission, whose well-characterized types are the long term potentiation and depression. Despite this classical definition, however, the molecular mechanisms by which synaptic plasticity may occur appear to be extremely complex and various. The post-synaptic density (PSD) of glutamatergic synapses is a major site for synaptic plasticity processes and alterations of PSD members have been recently implicated in neuropsychiatric diseases where an impairment of synaptic plasticity has also been reported. Among PSD members, scaffolding proteins have been demonstrated to bridge surface receptors with their intracellular effectors and to regulate receptors distribution and localization both at surface membranes and within the PSD. This review will focus on the molecular physiology and pathophysiology of synaptic plasticity processes, which are tuned by scaffolding PSD proteins and their close related partners, through the modulation of receptor localization and distribution at post-synaptic sites. We suggest that, by regulating both the compartmentalization of receptors along surface membrane and their degradation as well as by modulating receptor trafficking into the PSD, postsynaptic scaffolding proteins may contribute to form distinct signaling micro-domains, whose efficacy in transmitting synaptic signals depends on the dynamic stability of the scaffold, which in turn is provided by relative amounts and post-translational modifications of scaffolding members. The putative relevance for neuropsychiatric diseases and possible pathophysiological mechanisms are discussed in the last part of this work.     

Reciprocal stabilization of glycine receptors and gephyrin scaffold proteins at inhibitory synapses

Postsynaptic scaffold proteins immobilize neurotransmitter receptors in the synaptic membrane opposite to presynaptic vesicle release sites, thus ensuring efficient synaptic transmission. At inhibitory synapses in the spinal cord, the main scaffold protein gephyrin assembles in dense molecule clusters that provide binding sites for glycine receptors (GlyRs). Gephyrin and GlyRs can also interact outside of synapses, where they form receptor-scaffold complexes. Although several models for the formation of postsynaptic scaffold domains in the presence of receptor-scaffold interactions have been advanced, a clear picture of the coupled dynamics of receptors and scaffold proteins at synapses is lacking. To characterize the GlyR and gephyrin dynamics at inhibitory synapses, we performed fluorescence time-lapse imaging after photoconversion to directly visualize the exchange kinetics of recombinant Dendra2-gephyrin in cultured spinal cord neurons. Immuno-immobilization of endogenous GlyRs with specific antibodies abolished their lateral diffusion in the plasma membrane, as judged by the lack of fluorescence recovery after photobleaching. Moreover, the cross-linking of GlyRs significantly reduced the exchange of Dendra2-gephyrin compared with control conditions, suggesting that the kinetics of the synaptic gephyrin pool is strongly dependent on GlyR-gephyrin interactions. We did not observe any change in the total synaptic gephyrin levels after GlyR cross-linking, however, indicating that the number of gephyrin molecules at synapses is not primarily dependent on the exchange of GlyR-gephyrin complexes. We further show that our experimental data can be quantitatively accounted for by a model of receptor-scaffold dynamics that includes a tightly interacting receptor-scaffold domain, as well as more loosely bound receptor and scaffold populations that exchange with extrasynaptic pools. The model can make predictions for single-molecule data such as typical dwell times of synaptic proteins. Taken together, our data demonstrate the reciprocal stabilization of GlyRs and gephyrin at inhibitory synapses and provide a quantitative understanding of their dynamic organization.     

A receptor scaffold mediates stimulus-response coupling in bacterial chemotaxis

The mechanism of stimulus-response coupling in bacterial chemotaxis has emerged as a paradigm for understanding general features of intracellular signal transduction both in bacterial and eukaryotic cells. Until recently it was thought that the mechanism involved reversible stochastic interactions between dimeric receptors freely diffusing in the cytoplasmic membrane and several soluble signal transduction proteins within the cytoplasm. Recent results have shown that this view is an oversimplification. The receptors and most of the signal transduction proteins are organized together in a higher ordered structure at one pole of the bacterial cell. The scaffolding network within this structure appears to be composed of C-terminal alpha-helical extensions of the membrane chemoreceptor proteins held together in a lattice by tandem SH3-like domains. Results suggest that stimuli are detected through the perturbations they induce in scaffolding architecture.     

Diffusion barriers constrain receptors at synapses

The flux of neurotransmitter receptors in and out of synapses depends on receptor interaction with scaffolding molecules. However, the crowd of transmembrane proteins and the rich cytoskeletal environment may constitute obstacles to the diffusion of receptors within the synapse. To address this question, we studied the membrane diffusion of the γ-aminobutyric acid type A receptor (GABA(A)R) subunits clustered (γ2) or not (α5) at inhibitory synapses in rat hippocampal dissociated neurons. Relative to the extrasynaptic region, γ2 and α5 showed reduced diffusion and increased confinement at both inhibitory and excitatory synapses but they dwelled for a short time at excitatory synapses. In contrast, γ2 was ～3-fold more confined and dwelled ～3-fold longer in inhibitory synapses than α5, indicating faster synaptic escape of α5. Furthermore, using a gephyrin dominant-negative approach, we showed that the increased residency time of γ2 at inhibitory synapses was due to receptor-scaffold interactions. As shown for GABA(A)R, the excitatory glutamate receptor 2 subunit (GluA2) of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) had lower mobility in both excitatory and inhibitory synapses but a higher residency time at excitatory synapses. Therefore barriers impose significant diffusion constraints onto receptors at synapses where they accumulate or not. Our data further reveal that the confinement and the dwell time but not the diffusion coefficient report on the synapse specific sorting, trapping and accumulation of receptors.     

Regulation of G protein‐coupled receptor signaling by plasma membrane organization and endocytosis

The trafficking of G protein coupled‐receptors (GPCRs) is one of the most exciting areas in cell biology because of recent advances demonstrating that GPCR signaling is spatially encoded. GPCRs, acting in a diverse array of physiological systems, can have differential signaling consequences depending on their subcellular localization. At the plasma membrane, GPCR organization could fine‐tune the initial stages of receptor signaling by determining the magnitude of signaling and the type of effectors to which receptors can couple. This organization is mediated by the lipid composition of the plasma membrane, receptor‐receptor interactions, and receptor interactions with intracellular scaffolding proteins. GPCR organization is subsequently changed by ligand binding and the regulated endocytosis of these receptors. Activated GPCRs can modulate the dynamics of their own endocytosis through changing clathrin‐coated pit dynamics, and through the scaffolding adaptor protein β‐arrestin. This endocytic regulation has signaling consequences, predominantly through modulation of the MAPK cascade. This review explores what is known about receptor sorting at the plasma membrane, protein partners that control receptor endocytosis, and the ways in which receptor sorting at the plasma membrane regulates downstream trafficking and signaling.      

The Role of Synaptopodin in Membrane Protein Diffusion in the Dendritic Spine Neck

The dynamic exchange of neurotransmitter receptors at synapses relies on their lateral diffusion in the plasma membrane. At synapses located on dendritic spines this process is limited by the geometry of the spine neck that restricts the passage of membrane proteins. Biochemical compartmentalisation of the spine is believed to underlie the input-specificity of excitatory synapses and to set the scale on which functional changes can occur. Synaptopodin is located predominantly in the neck of dendritic spines, and is thus ideally placed to regulate the exchange of synaptic membrane proteins. The central aim of our study was to assess whether the presence of synaptopodin influences the mobility of membrane proteins in the spine neck and to characterise whether this was due to direct molecular interactions or to spatial constraints that are related to the structural organisation of the neck. Using single particle tracking we have identified a specific effect of synaptopodin on the diffusion of metabotropic mGluR5 receptors in the spine neck. However, super-resolution STORM/PALM imaging showed that this was not due to direct interactions between the two proteins, but that the presence of synaptopodin is associated with an altered local organisation of the F-actin cytoskeleton, that in turn could restrict the diffusion of membrane proteins with large intracellular domains through the spine neck. This study contributes new data on the way in which the spine neck compartmentalises excitatory synapses. Our data complement models that consider the impact of the spine neck as a function of its shape, by showing that the internal organisation of the neck imposes additional physical barriers to membrane protein diffusion.     

Posttranslational Modifications Regulate the Postsynaptic Localization of PSD-95

The postsynaptic density (PSD) consists of a lattice-like array of interacting proteins that organizes and stabilizes synaptic receptors, ion channels, structural proteins, and signaling molecules required for normal synaptic transmission and synaptic function. The scaffolding and hub protein postsynaptic density protein-95 (PSD-95) is a major element of central chemical synapses and interacts with glutamate receptors, cell adhesion molecules, and cytoskeletal elements. In fact, PSD-95 can regulate basal synaptic stability as well as the activity-dependent structural plasticity of the PSD and, therefore, of the excitatory chemical synapse. Several studies have shown that PSD-95 is highly enriched at excitatory synapses and have identified multiple protein structural domains and protein-protein interactions that mediate PSD-95 function and trafficking to the postsynaptic region. PSD-95 is also a target of several signaling pathways that induce posttranslational modifications, including palmitoylation, phosphorylation, ubiquitination, nitrosylation, and neddylation; these modifications determine the synaptic stability and function of PSD-95 and thus regulate the fates of individual dendritic spines in the nervous system. In the present work, we review the posttranslational modifications that regulate the synaptic localization of PSD-95 and describe their functional consequences. We also explore the signaling pathways that induce such changes.     

A role for zinc in postsynaptic density asSAMbly and plasticity?

Chemical synapses are asymmetric cell junctions that mediate communication between neurons. Multidomain scaffolding proteins of the Shank family act as major organizing elements of the "postsynaptic density"--that is, the cytoskeletal protein matrix associated with the postsynaptic membrane. A recent study has shown that the C-terminal sterile alpha-motif or "SAM domain" of Shank3 (also known as ProSAP2) can form two-dimensional sheets of helical fibers. Assembly and packaging of these fibers are markedly enhanced by the presence of Zn2+ ions. Zn2+ can be released together with glutamate from synaptic vesicles and can enter the postsynaptic cell through specific ionotropic receptors. Based on these observations, we propose a new model of synaptic plasticity in which Zn2+ influx directly and instantly modulates the structure and function of the postsynaptic density.     

The scaffolding protein PSD-95 interacts with the glycine transporter GLYT1 and impairs its internalization

Recent evidence indicates that the glycine transporter-1 (GLYT1) plays a role in regulation of NMDA receptor function through tight control of glycine concentration in its surrounding medium. Immunohistochemical studies have demonstrated that, as well as being found in glial cells, GLYT1 is also associated with the pre- and postsynaptic aspects of glutamatergic synapses. In this article, we describe the interaction between GLYT1 and PSD-95 in the rat brain, PSD-95 being a scaffolding protein that participates in the organization of glutamatergic synapses. Mutational analysis reveals that the C-terminal sequence of GLYT1 (-SRI) is necessary for the transporter to interact with the PDZ domains I and II of PSD-95. This C-terminal tripeptide motif also seems to be involved in the trafficking of GLYT1 to the membrane, although this process does not involve PDZ proteins. GLYT1 is able to recruit PSD-95 to the plasma membrane, but it does not affect its clustering. However, the interaction stabilizes this transporter at the plasma membrane, blocking its internalization and producing a significant increase in the V(max) of glycine uptake. We hypothesize that PSD-95 might act as a scaffold for GLYT1 and NMDA receptors, allowing GLYT1 to regulate the concentrations of glycine in the micro-environment of NMDA receptors.     

Neto2 Interacts with the Scaffolding Protein GRIP and Regulates Synaptic Abundance of Kainate Receptors

Kainate receptors (KARs) are a class of ionotropic glutamate receptors that are expressed throughout the central nervous system. The function and subcellular localization of KARs are tightly regulated by accessory proteins. We have previously identified the single-pass transmembrane proteins, Neto1 and Neto2, to be associated with native KARs. In the hippocampus, Neto1, but not Neto2, controls the abundance and modulates the kinetics of postsynaptic KARs. Here we evaluated whether Neto2 regulates synaptic KAR levels in the cerebellum where Neto1 expression is limited to the deep cerebellar nuclei. In the cerebellum, where Neto2 is present abundantly, we found a ∼40% decrease in GluK2-KARs at the postsynaptic density (PSD) of Neto2-null mice. No change, however, was observed in total level of GluK2-KARs, thereby suggesting a critical role of Neto2 on the synaptic localization of cerebellar KARs. The presence of a putative class II PDZ binding motif on Neto2 led us to also investigate whether it interacts with PDZ domain-containing proteins previously implicated in regulating synaptic abundance of KARs. We identified a PDZ-dependent interaction between Neto2 and the scaffolding protein GRIP. Furthermore, coexpression of Neto2 significantly increased the amount of GRIP associated with GluK2, suggesting that Neto2 may promote and/or stabilize GluK2:GRIP interactions. Our results demonstrate that Neto2, like Neto1, is an important auxiliary protein for modulating the synaptic levels of KARs. Moreover, we propose that the interactions of Neto1/2 with various scaffolding proteins is a critical mechanism by which KARs are stabilized at diverse synapses.     

ProSAP-interacting protein 1 (ProSAPiP1), a novel protein of the postsynaptic density that links the spine-associated Rap-Gap (SPAR) to the scaffolding protein ProSAP2/Shank3

ProSAPs/Shanks are a family of proteins that have a major scaffolding function for components of the postsynaptic density (PSD) of excitatory brain synapses. Members of the family harbor a variety of domains for protein-protein interactions, one of which is a unique PDZ domain that differs significantly from those of other proteins. We have identified a novel binding partner for this PDZ domain, termed ProSAPiP1, that is highly enriched in the PSD and shares significant sequence homology with the PSD protein PSD-Zip70. Both molecules code for a Fez1 domain that can be found in a total of four related proteins. ProSAPiP1 is widely expressed in rat brain and co-localizes with ProSAP2/Shank3 in excitatory spines and synapses. ProSAP2/Shank3 co-immunoprecipitates with ProSAPiP1 but not with PSD-Zip70. Both proteins, however, bind and recruit SPAR to synapses with a central coiled-coil region that harbors a leucine zipper motif. This region is also responsible for homo- and heteromultimerization of ProSAPiP1 and PSD-Zip70. Thus, ProSAPiP1 and PSD-Zip70 are founders of a novel family of scaffolding proteins, the "Fezzins," which adds further complexity to the organization of the PSD protein network.