Molecular and functional characterization of proteins interacting with the C-terminal domains of 5-HT2 receptors: emergence of 5-HT2 "receptosomes"

Many cellular functions are carried out by multiprotein complexes. The last five years of research have revealed that many G-protein coupled receptor (GPCR) functions that are not mediated by G proteins involve protein networks, which interact with their intracellular domains. This review focuses on one family of GPCRs activated by serotonin, the 5-HT(2) receptor family, which comprises three closely related subtypes, the 5-HT(2A), the 5-HT(2B) and the 5-HT(2c) receptors. These receptors still raise particular interest, because a large number of psychoactive drugs including hallucinogens, anti-psychotics, anxiolytics and anti-depressants, mediate their action, at least in part, through activation of 5-HT(2) receptors. Recent studies based on two-hybrid screens, proteomic, biochemical and cell biology approaches, have shown that the C-terminal domains of 5-HT(2) receptors interact with intracellular proteins. To date, the protein network associated with the C-terminus of the 5-HT(2C) receptor has been the most extensively characterized, using a proteomic approach combining affinity chromatography, mass spectrometry and immunoblotting. It includes scaffolding proteins containing one or several PDZ domains, signalling proteins and proteins of the cytoskeleton. Data indicating that the protein complexes interacting with 5-HT(2) receptor C-termini tightly control receptor trafficking and receptor-mediated signalling will also be reviewed.     

The serotonin 5-HT2A and 5-HT2C receptors interact with specific sets of PDZ proteins

The 5-hydroxytryptamine type 2A (5-HT(2A)) receptor and the 5-HT(2C) receptor are closely related members of the G-protein-coupled receptors activated by serotonin that share very similar pharmacological profiles and cellular signaling pathways. These receptors express a canonical class I PDZ ligand (SXV) at their C-terminal extremity. Here, we have identified proteins that interact with the PDZ ligand of the 5-HT(2A) and 5-HT(2C) receptors by a proteomic approach associating affinity chromatography using immobilized synthetic peptides encompassing the PDZ ligand and mass spectrometry. We report that both receptor C termini interact with specific sets of PDZ proteins in vitro. The 5-HT(2C) receptor but not the 5-HT(2A) receptor binds to the Veli-3.CASK.Mint1 ternary complex and to SAP102. In addition, the 5-HT(2C) receptor binds more strongly to PSD-95 and MPP-3 than the 5-HT(2A) receptor. In contrast, a robust interaction between the 5-HT(2A) receptor and the channel-interacting PDZ protein CIPP was found, whereas CIPP did not significantly associate with the 5-HT(2C) receptor. We also show that residues located at the -1 position and upstream the PDZ ligand in the C terminus of the 5-HT(2A) and 5-HT(2C) receptors are major determinants in their interaction with specific PDZ proteins. Immunofluorescence and electron microscopy studies strongly suggested that these specific interactions also take place in living cells and that the 5-HT(2) receptor-PDZ protein complexes occur in intracellular compartments. The interaction of the 5-HT(2A) and the 5-HT(2C) receptor with specific sets of PDZ proteins may contribute to their different signal transduction properties.     

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.     

Opposite effects of PSD-95 and MPP3 PDZ proteins on serotonin 5-hydroxytryptamine2C receptor desensitization and membrane stability

PSD-95/Disc large/Zonula occludens 1 (PDZ) domain-containing proteins (PDZ proteins) play an important role in the targeting and the trafficking of transmembrane proteins. Our previous studies identified a set of PDZ proteins that interact with the C terminus of the serotonin 5-hydroxytryptamine (5-HT)(2C) receptor. Here, we show that the prototypic scaffolding protein postsynaptic density-95 (PSD-95) and another membrane-associated guanylate kinase, MAGUK p55 subfamily member 3 (MPP3), oppositely regulate desensitization of the receptor response in both heterologous cells and mice cortical neurons in primary culture. PSD-95 increased desensitization of the 5-HT(2C) receptor-mediated Ca(2+) response, whereas MPP3 prevented desensitization of the Ca(2+) response. The effects of the PDZ proteins on the desensitization of the Ca(2+) response were correlated with a differential regulation of cell surface expression of the receptor. Additional experiments were performed to assess how PDZ proteins globally modulate desensitization of the 5-HT(2C) receptor response in neurons, by using a peptidyl mimetic of the 5-HT(2C) receptor C terminus fused to the human immunodeficiency virus type-1 Tat protein transduction domain, which disrupts interaction between the 5-HT(2C) receptor and PDZ proteins. Transduction of this peptide inhibitor into cultured cortical neurons increased the desensitization of the 5-HT(2C) receptor-mediated Ca(2+) response. This indicates that, overall, interaction of 5-HT(2C) receptors with PDZ proteins inhibits receptor desensitization in cortical neurons.     

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.     

Synaptic removal of diacylglycerol by DGKζ and PSD‐95 regulates dendritic spine maintenance

Diacylglycerol (DAG) is an important lipid signalling molecule that exerts an effect on various effector proteins including protein kinase C. A main mechanism for DAG removal is to convert it to phosphatidic acid (PA) by DAG kinases (DGKs). However, it is not well understood how DGKs are targeted to specific subcellular sites and tightly regulates DAG levels. The neuronal synapse is a prominent site of DAG production. Here, we show that DGKζ is targeted to excitatory synapses through its direct interaction with the postsynaptic PDZ scaffold PSD‐95. Overexpression of DGKζ in cultured neurons increases the number of dendritic spines, which receive the majority of excitatory synaptic inputs, in a manner requiring its catalytic activity and PSD‐95 binding. Conversely, DGKζ knockdown reduces spine density. Mice deficient in DGKζ expression show reduced spine density and excitatory synaptic transmission. Time‐lapse imaging indicates that DGKζ is required for spine maintenance but not formation. We propose that PSD‐95 targets DGKζ to synaptic DAG‐producing receptors to tightly couple synaptic DAG production to its conversion to PA for the maintenance of spine density.     

G protein-coupled receptor cross-talk: pivotal roles of protein phosphorylation and protein-protein interactions

G protein-coupled receptors are dynamically regulated. Such regulation is frequently associated with covalent posttranslational modifications, such as phosphorylation, and with regulatory elements. G protein-coupled receptor kinases and casein kinase 1alpha play key roles in agonist-dependent receptor phosphorylations. Cross-talk between different receptors frequently involves second messenger-activated proteins, such as protein kinase C and protein kinase A. There is some evidence indicating that such kinases may not only turn off receptors but also switch their coupling to different G proteins. Receptor tyrosine kinases may phosphorylate and regulate G protein-coupled receptors and recent evidence indicates that other kinases, such as Akt/protein kinase B and phosphoinositide 3-kinase, may participate in such regulations as integrators of signalling.Recent approaches have shed new light on G protein-coupled receptor interactions that provide novel mechanisms of action and regulation. G protein-coupled receptor activities go beyond G proteins and receptors can be partners of exquisitely assembled signalling complexes through molecular bridges composed of multidomain proteins. The possibilities of interaction increase enormously through the diversity of structural and functional domains present in complex proteins, many of them just known as predicted sequences.     

The calcium-sensing receptor and its interacting proteins

Seven membrane-spanning, or G protein-coupled receptors were originally thought to act through het-erotrimeric G proteins that in turn activate intracellular enzymes or ion channels, creating relatively simple, linear signalling pathways. Although this basic model remains true in that this family does act via a relatively small number of G proteins, these signalling systems are considerably more complex because the receptors interact with or are located near additional proteins that are often unique to a receptor or subset of receptors. These additional proteins give receptors their unique signalling personalities. The extracellular Ca-sensing receptor (CaR) signals via Galpha(i), Galpha(q) and Galpha(12/13), but its effects in vivo demonstrate that the signalling pathways controlled by these subunits are not sufficient to explain all its biologic effects. Additional structural or signalling proteins that interact with the CaR may explain its behaviour more fully. Although the CaR is less well studied in this respect than other receptors, several CaR-interacting proteins such as filamin, a potential scaffolding protein, receptor activity modifying proteins (RAMPs) and potassium channels may contribute to the unique characteristics of the CaR. The CaR also appears to interact with additional proteins common to other G protein-coupled receptors such as arrestins, G protein receptor kinases, protein kinase C, caveolin and proteins in the ubiquitination pathway. These proteins probably represent a few initial members of CaR-based signalling complex. These and other proteins may not all be associated with the CaR in all tissues, but they form the basis for understanding the complete nature of CaR signalling.     

nArgBP2, a novel neural member of ponsin/ArgBP2/vinexin family that interacts with synapse-associated protein 90/postsynaptic density-95-associated protein (SAPAP).

Postsynaptic density (PSD)-95/synapse-associated protein (SAP) 90 and synaptic scaffolding molecule (S-SCAM) are synaptic membrane-associated guanylate kinases. Both the proteins interact with SAP90/PSD-95-associated protein (SAPAP) (also called guanylate kinase-associated protein/Dlg-associated protein). SAPAP is a protein highly enriched in the PSD fraction and may link PSD-95/SAP90 and S-SCAM to Triton X-100-insoluble structures. We found here a novel SAPAP-interacting protein, which was specifically expressed in neural tissue and was present in the postsynaptic density fraction in brain. This protein had a sorbin homology domain in the N terminus, a zinc finger motif in the middle region, and three src homology (SH) 3 domains in the C terminus and was homologous to the ponsin/ArgBP2/vinexin family proteins. We named this protein nArgBP2 because it was the most homologous to ArgBP2. nArgBP2 is a neural member of a growing family of SH3-containing proteins. nArgBP2 bound to the proline-rich region of SAPAP via its third SH3 domain and was coimmunoprecipitated with SAPAP from the extract of rat brain. Furthermore, nArgBP2 was colocalized with SAPAP at synapses in cerebellum. nArgBP2 bound to not only SAPAP but also vinculin and l-afadin, known to bind to ponsin and vinexin. nArgBP2 may be implicated in the protein network around SAPAP in the PSD.     

Scaffold proteins (MAGUK, Shank and Homer) in postsynaptic density in the central nervous system

The postsynaptic density (PSD) is a dynamic multi-protein complex attached to the postsynaptic membrane composed of several hundred proteins such as receptors and channels, scaffolding and adaptor proteins, cell-adhesion proteins, cytoskeletal proteins, G-proteins and their modulators and signaling molecules including kinases and phosphtases. This review focuses on the prominent PSD scaffolds proteins such as members of the MAGUK (membrane-associated guanylyl kinase), Shank (SH3 domain and ankyrin repeat-containing protein) and Homer families. These molecules interact simultaneously with different kinds of receptors and modulate their function by linking the receptors to downstream signaling events. For example PSD 95, a main member of MAGUK family, interacts directly with carboxyl termini of NMDA receptor subunits and clusters them to the postsynaptic membrane. In addition, PSD 95 is involved in binding and organizing proteins connected with NMDAR signaling. Based on the modular character and ability to form multiproteins interactions, MAGUK, Shank and Homer are perfectly suited to act as a major scaffold in postsynaptic density.     

Capping of the N‐terminus of PSD‐95 by calmodulin triggers its postsynaptic release

Postsynaptic density protein‐95 (PSD‐95) is a central element of the postsynaptic architecture of glutamatergic synapses. PSD‐95 mediates postsynaptic localization of AMPA receptors and NMDA receptors and plays an important role in synaptic plasticity. PSD‐95 is released from postsynaptic membranes in response to Ca²⁺ influx via NMDA receptors. Here, we show that Ca²⁺/calmodulin (CaM) binds at the N‐terminus of PSD‐95. Our NMR structure reveals that both lobes of CaM collapse onto a helical structure of PSD‐95 formed at its N‐terminus (residues 1–16). This N‐terminal capping of PSD‐95 by CaM blocks palmitoylation of C3 and C5, which is required for postsynaptic PSD‐95 targeting and the binding of CDKL5, a kinase important for synapse stability. CaM forms extensive hydrophobic contacts with Y12 of PSD‐95. The PSD‐95 mutant Y12E strongly impairs binding to CaM and Ca²⁺‐induced release of PSD‐95 from the postsynaptic membrane in dendritic spines. Our data indicate that CaM binding to PSD‐95 serves to block palmitoylation of PSD‐95, which in turn promotes Ca²⁺‐induced dissociation of PSD‐95 from the postsynaptic membrane.     

Disassembly of Shank and Homer Synaptic Clusters Is Driven by Soluble β-Amyloid1-40 through Divergent NMDAR-Dependent Signalling Pathways

Disruption of the postsynaptic density (PSD), a network of scaffold proteins located in dendritic spines, is thought to be responsible for synaptic dysfunction and loss in early-stage Alzheimer''s disease (AD). Extending our previous demonstration that derangement of the PSD by soluble amyloid-β (Aβ) involves proteasomal degradation of PSD-95, a protein important for ionotropic glutamate receptor trafficking, we now show that Aβ also disrupts two other scaffold proteins, Homer1b and Shank1, that couple PSD-95 with ionotropic and metabotropic glutamate receptors. Treatment of fronto-cortical neurons with soluble Aβ results in rapid (within 1 h) and significant thinning of the PSD, decreased synaptic levels of Homer1b and Shank1, and reduced synaptic mGluR1 levels. We show that de novo protein synthesis is required for the declustering effects of Aβ on Homer1b (but not Shank1) and that, in contrast to PSD-95, Aβ-induced Homer1b and Shank1 cluster disassembly does not depend on proteasome activity. The regulation of Homer1b and Shank1 by Aβ diverges in two other respects: i) whereas the activity of both NMDAR and VDCC is required for Aβ-induced declustering of Homer1b, Aβ-induced declustering of Shank1 only requires NMDAR activity; and ii) whereas the effects of Aβ on Homer1b involve engagement of the PI-3K pathway and calcineurin phosphatase (PP2B) activity, those on Shank1 involve activation of the ERK pathway. In summary, soluble Aβ recruits discrete signalling pathways to rapidly reduce the synaptic localization of major components of the PSD and to regulate the availability of mGluR1 in the synapse.     

ProSAPiP2, a novel postsynaptic density protein that interacts with ProSAP2/Shank3

The postsynaptic density (PSD) is a highly specialized structure that is located juxtaposed to the presynaptic active zone of excitatory synapses. It is composed of a variety of proteins that include receptors, signaling molecules, cytoskeletal components and scaffolding proteins. ProSAP/Shank proteins are large multidomain proteins that facilitate multiple functions within the PSD. They build large scaffolds that are the structural basis for the direct and/or indirect connection between receptor proteins and the actin based cytoskeleton. Here, we characterize a novel interaction partner of ProSAP2/Shank3, named ProSAP interacting protein 2 (ProSAPiP2) that does not show any close homology to other known proteins. It binds to the PDZ domain of ProSAP2/Shank3 and is highly expressed in the neuronal system. ProSAPiP2 is located in dendrites and spines, is enriched in the PSD and interacts with actin. Therefore ProSAPiP2 could be involved in the linkage between molecules of the PSD and the cytoskeleton.     

DGKι regulates presynaptic release during mGluR-dependent LTD

Diacylglycerol (DAG) is an important lipid second messenger. DAG signalling is terminated by conversion of DAG to phosphatidic acid (PA) by diacylglycerol kinases (DGKs). The neuronal synapse is a major site of DAG production and action; however, how DGKs are targeted to subcellular sites of DAG generation is largely unknown. We report here that postsynaptic density (PSD)-95 family proteins interact with and promote synaptic localization of DGKι. In addition, we establish that DGKι acts presynaptically, a function that contrasts with the known postsynaptic function of DGKζ, a close relative of DGKι. Deficiency of DGKι in mice does not affect dendritic spines, but leads to a small increase in presynaptic release probability. In addition, DGKι-/- synapses show a reduction in metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) at neonatal (～2 weeks) stages that involve suppression of a decrease in presynaptic release probability. Inhibition of protein kinase C normalizes presynaptic release probability and mGluR-LTD at DGKι-/- synapses. These results suggest that DGKι requires PSD-95 family proteins for synaptic localization and regulates presynaptic DAG signalling and neurotransmitter release during mGluR-LTD.     

Contrasting localizations of MALS/LIN-7 PDZ proteins in brain and molecular compensation in knockout mice

Proteins containing PDZ (postsynaptic density-95, discs large, zonula occludens) domains play a general role in recruiting receptors and enzymes to specific synaptic sites. In Caenorhabditis elegans, a complex of three PDZ proteins, LIN-2/7/10, mediates basolateral targeting of a receptor tyrosine kinase. Homologs of these LIN proteins have also been identified in higher organisms, and here we analyze the MALS/Veli (mammalian LIN-7/vertebrate homolog of LIN-7) proteins in brain. Immunohistochemical staining and in situ hybridization show that MALS occur differentially in discrete populations of neurons throughout the brain. Most neurons express only one MALS protein, although some cells contain two or even all three MALS isoforms. At the subcellular level, MALS proteins are found in both dendritic and axonal locations, suggesting that they may regulate processes at both pre- and postsynaptic sites. Targeted disruption of MALS-1 and MALS-2 does not yield a detectable phenotype, and hippocampal synaptic function and plasticity are intact in the MALS-1/2 double knockouts. Interestingly, MALS-3 protein is dramatically induced in the MALS-1/2 double knockouts, implying that dynamic changes in protein expression may play an important regulatory role for this family of synaptic PDZ proteins.     

Composition of the synaptic PSD-95 complex

Postsynaptic density protein 95 (PSD-95), a specialized scaffold protein with multiple protein interaction domains, forms the backbone of an extensive postsynaptic protein complex that organizes receptors and signal transduction molecules at the synaptic contact zone. Large, detergent-insoluble PSD-95-based postsynaptic complexes can be affinity-purified from conventional PSD fractions using magnetic beads coated with a PSD-95 antibody. In the present study purified PSD-95 complexes were analyzed by LC/MS/MS. A semiquantitative measure of the relative abundances of proteins in the purified PSD-95 complexes and the parent PSD fraction was estimated based on the cumulative ion current intensities of corresponding peptides. The affinity-purified preparation was largely depleted of presynaptic proteins, spectrin, intermediate filaments, and other contaminants prominent in the parent PSD fraction. We identified 525 of the proteins previously reported in parent PSD fractions, but only 288 of these were detected after affinity purification. We discuss 26 proteins that are major components in the PSD-95 complex based upon abundance ranking and affinity co-purification with PSD-95. This subset represents a minimal list of constituent proteins of the PSD-95 complex and includes, in addition to the specialized scaffolds and N-methyl-d-aspartate (NMDA) receptors, an abundance of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, small G-protein regulators, cell adhesion molecules, and hypothetical proteins. The identification of two Arf regulators, BRAG1 and BRAG2b, as co-purifying components of the complex implies pivotal functions in spine plasticity such as the reorganization of the actin cytoskeleton and insertion and retrieval of proteins to and from the plasma membrane. Another co-purifying protein (Q8BZM2) with two sterile alpha motif domains may represent a novel structural core element of the PSD.     

Caveolin-1 interacts with 5-HT2A serotonin receptors and profoundly modulates the signaling of selected Galphaq-coupled protein receptors

5-Hydroxytryptamine 2A (5-HT(2A)) serotonin receptors are important for a variety of functions including vascular smooth muscle contraction, platelet aggregation, and the modulation of perception, cognition, and emotion. In a search for 5-HT(2A) receptor-interacting proteins, we discovered that caveolin-1 (Cav-1), a scaffolding protein enriched in caveolae, complexes with 5-HT(2A) receptors in a number of cell types including C6 glioma cells, transfected HEK-293 cells, and rat brain synaptic membrane preparations. To address the functional significance of this interaction, we performed RNA interference-mediated knockdown of Cav-1 in C6 glioma cells, a cell type that endogenously expresses both 5-HT(2A) receptors and Cav-1. We discovered that the in vitro knockdown of Cav-1 in C6 glioma cells nearly abolished 5-HT(2A) receptor-mediated signal transduction as measured by calcium flux assays. RNA interference-mediated knockdown of Cav-1 also greatly attenuated endogenous Galpha(q)-coupled P2Y purinergic receptor-mediated signaling without altering the signaling of PAR-1 thrombin receptors. Cav-1 appeared to modulate 5-HT(2A) signaling by facilitating the interaction of 5-HT(2A) receptors with Galpha(q). These studies provide compelling evidence for a prominent role of Cav-1 in regulating the functional activity of not only 5-HT(2A) serotonin receptors but also selected Galpha(q)-coupled receptors.     

Spinophilin: from partners to functions

Spinophilin/neurabin 2 has been isolated independently by two laboratories as a protein interacting with protein phosphatase 1 (PP1) and F-actin. Gene analysis and biochemical approaches have contributed to define a number of distinct modular domains in spinophilin that govern protein-protein interactions such as two F-actin-, three potential Src homology 3 (SH3)-, a receptor- and a PP1-binding domains, a PSD95/DLG/zo-1 (PDZ) and three coiled-coil domains, and a potential leucine/isoleucine zipper (LIZ) motif. More than 30 partner proteins of spinophilin have been discovered, including cytoskeletal and cell adhesion molecules, enzymes, guanine nucleotide exchange factors (GEF) and regulator of G-protein signalling protein, membrane receptors, ion channels and others proteins like the tumour suppressor ARF. The physiological relevance of some of these interactions remains to be demonstrated. However, spinophilin structure suggests that the protein is a multifunctional protein scaffold that regulates both membrane and cytoskeletal functions. Spinophilin plays important functions in the nervous system where it is implicated in spine morphology and density regulation, synaptic plasticity and neuronal migration. Spinophilin regulates also seven-transmembrane receptor signalling and may provide a link between some of these receptors and intracellular mitogenic signalling events dependent on p70(S6) kinase and Rac G protein-GEF. Strikingly a role for spinophilin in cell growth was demonstrated and this effect was enhanced by its interaction with ARF. Here we review the current knowledge of the protein partners of spinophilin and present the available data that are contributing to the appreciation of spinophilin functions.     

Differential interaction of NMDA receptor subtypes with the post-synaptic density-95 family of membrane associated guanylate kinase proteins

NMDA receptors are a subclass of ionotropic glutamate receptors. They are trafficked and/or clustered at synapses by the post-synaptic density (PSD)-95 membrane associated guanylate kinase (MAGUK) family of scaffolding proteins that associate with NMDA receptor NR2 subunits via their C-terminal glutamate serine (aspartate/glutamate) valine motifs. We have carried out a systematic study investigating in a heterologous expression system, the association of the four major NMDA receptor subtypes with the PSD-95 family of MAGUK proteins, chapsyn-110, PSD-95, synapse associated protein (SAP) 97 and SAP102. We report that although each PSD-95 MAGUK was shown to co-immunoprecipitate with NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D receptor subtypes, they elicited differential effects with regard to the enhancement of total NR2 subunit expression which then results in an increased cell surface expression of NMDA receptor subtypes. PSD-95 and chapsyn-110 enhanced NR2A and NR2B total expression which resulted in increased NR1/NR2A and NR1/NR2B receptor cell surface expression whereas SAP97 and SAP102 had no effect on total or cell surface expression of these subtypes. PSD-95, chapsyn-110, SAP97 and SAP102 had no effect on either total NR2C and NR2D subunit expression or cell surface NR1/NR2C and NR1/NR2D expression. A comparison of PSD-95α, PSD-95β and PSD-95α^C3S,C5S showed that PSD-95-enhanced cell surface expression of NR1/NR2A receptors was dependent upon the PSD-95 N-terminal C3,C5 cysteines. These observations support differential interaction of NMDA receptor subtypes with different PSD-95 MAGUK scaffolding proteins. This has implications for the stabilisation, turnover and compartmentalisation of NMDA receptor subtypes in neurones during development and in the mature brain.