Characterization of zebrafish PSD‐95 gene family members

The PSD‐95 family of membrane‐ associated guanylate kinases (MAGUKs) are thought to act as molecular scaffolds that regulate the assembly and function of the multiprotein signaling complex found at the postsynaptic density of excitatory synapses. Genetic analysis of PSD‐95 family members in the mammalian nervous system has so far been difficult, but the zebrafish is emerging as an ideal vertebrate system for studying the role of particular genes in the developing and mature nervous system. Here we describe the cloning of the zebrafish orthologs of PSD‐95, PSD‐93, and two isoforms of SAP‐97. Using in situ hybridization analysis we show that these zebrafish MAGUKs have overlapping but distinct patterns of expression in the developing nervous system and craniofacial skeleton. Using a pan‐MAGUK antibody we show that MAGUK proteins localize to neurons within the developing hindbrain, cerebellum, visual and olfactory systems, and to skin epithelial cells. In the olfactory and visual systems MAGUK proteins are expressed strongly in synaptic regions, and the onset of expression in these areas coincides with periods of synapse formation. These data are consistent with the idea that PSD‐95 family members are involved in synapse assembly and function, and provide a platform for future functional studies in vivo in a highly tractable model organism. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Plasma membrane Ca2+-atpase isoforms 2b and 4b interact promiscuously and selectively with members of the membrane-associated guanylate kinase family of PDZ (PSD95/Dlg/ZO-1) domain-containing proteins

Spatial and temporal regulation of intracellular Ca(2+) signaling depends on localized Ca(2+) microdomains containing the requisite molecular components for Ca(2+) influx, efflux, and signal transmission. Plasma membrane Ca(2+)-ATPase (PMCA) isoforms of the "b" splice type contain predicted PDZ (PSD95/Dlg/ZO-1) interaction domains. The COOH-terminal tail of PMCA2b isolated the membrane-associated guanylate kinase (MAGUK) protein SAP97/hDlg as a binding partner in a yeast two-hybrid screen. The related MAGUKs SAP90/PSD95, PSD93/chapsyn-110, SAP97, and SAP102 all bound to the COOH-terminal tail of PMCA4b, whereas only the first three bound to the tail of PMCA2b. Coimmunoprecipitations confirmed the interaction selectivity between PMCA4b and SAP102 as opposed to the promiscuity of PMCA2b and 4b in interacting with other SAPs. Confocal immunofluorescence microscopy revealed the exclusive presence and colocalization of PMCA4b and SAP97 in the basolateral membrane of polarized Madin-Darby canine kidney epithelial cells. In hippocampal neurons, PMCA2b was abundant throughout the somatodendritic compartment and often extended into the neck and head of individual spines where it colocalized with SAP90/PSD95. These data show that PMCA "b" splice forms interact promiscuously but also with specificity with different members of the PSD95 family of SAPs. PMCA-SAP interactions may play a role in the recruitment and maintenance of the PMCA at specific membrane domains involved in local Ca(2+) regulation.     

Corticotropin-Releasing Hormone Receptor Type 1 (CRHR1) Clustering with MAGUKs Is Mediated via Its C-Terminal PDZ Binding Motif

The corticotropin-releasing hormone receptor type 1 (CRHR1) plays an important role in orchestrating neuroendocrine, behavioral, and autonomic responses to stress. To identify molecules capable of directly modulating CRHR1 signaling, we performed a yeast-two-hybrid screen using the C-terminal intracellular tail of the receptor as bait. We identified several members of the membrane-associated guanylate kinase (MAGUK) family: postsynaptic density protein 95 (PSD95), synapse-associated protein 97 (SAP97), SAP102 and membrane associated guanylate kinase, WW and PDZ domain containing 2 (MAGI2). CRHR1 is co-expressed with the identified MAGUKs and with the additionally investigated PSD93 in neurons of the adult mouse brain and in primary hippocampal neurons, supporting the probability of a physiological interaction in vivo. The C-terminal PDZ (PSD-95, discs large, zona occludens 1) binding motif of CRHR1 is essential for its physical interaction with MAGUKs, as revealed by the CRHR1-STAVA mutant, which harbors a functionally impaired PDZ binding motif. The imitation of a phosphorylation at Thr413 within the PDZ binding motif also disrupted the interaction with MAGUKs. In contrast, distinct PDZ domains within the identified MAGUKs are involved in the interactions. Expression of CRHR1 in primary neurons demonstrated its localization throughout the neuronal plasma membrane, including the excitatory post synapse, where the receptor co-localized with PSD95 and SAP97. The co-expression of CRHR1 and respective interacting MAGUKs in HEK293 cells resulted in a clustered subcellular co-localization which required an intact PDZ binding motif. In conclusion, our study characterized the PDZ binding motif-mediated interaction of CRHR1 with multiple MAGUKs, which directly affects receptor function.     

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.     

Ion channel clustering by membrane-associated guanylate kinases. Differential regulation by N-terminal lipid and metal binding motifs

The postsynaptic density protein PSD-95 and related membrane-associated guanylate kinase (MAGUK) proteins assemble signal transduction complexes at sites of cell-cell contact including synapses. Whereas PSD-95 and PSD-93 occur only at postsynaptic sites in hippocampal neurons, SAP-102 also occurs in axons. In heterologous cells, PSD-95 and PSD-93 mediate cell surface ion channel clustering, but SAP-102 and SAP-97 do not. This selective ion channel clustering activity by MAGUKs is explained by differential palmitoylation, as PSD-93 and PSD-95 are palmitoylated though SAP-97, and SAP-102 are not. Rather than being palmitoylated, we find that N-terminal cysteines from SAP-102 tightly bind to zinc. And, appending the N terminus of SAP-102 to PSD-95 results in localization of the chimera to both axons and dendrites. These data suggest that lipid modifications and heavy metal associations with the N termini of MAGUKs mediate differential functions and subcellular localizations of these synaptic scaffolds.     

Cloning and characterization of E-dlg, a novel splice variant of mouse homologue of the Drosophila discs large tumor suppressor binds preferentially to SAP102

Membrane-associated guanylate kinases (MAGUKs) act as scaffolds to coordinate signaling events through their multiple domains at the plasma membrane. The MAGUK SH3 domain is noncanonical and its function remains unclear. To identify potential binding partners of MAGUK SH3, the synapse-associated protein 102 (SAP102) SH3 domain was used as bait in a yeast two-hybrid screen of a mouse embryonic cDNA library. A mouse homologue of the Drosophila discs large tumor suppressor (Dlg, also known as SAP97) bound preferentially to SAP102 SH3. The 4347bp cDNA sequence encoded an 893 amino acid protein with 94% identity to mouse SAP97. A deleted region (33-aa) strongly suggests this is a novel splice variant, which we call Embryonic-dlg/SAP97 (E-dlg). The interaction of SAP102 and E-dlg was confirmed in mammalian cells. E-dlg can also bind to potassium channel Kv1.4 in a pull-down assay. E-dlg was highly expressed in embryonic and some adult mouse tissues, such as brain, kidney, and ovary. Furthermore, in situ hybridization showed that E-dlg was mostly expressed in olfactory bulb and cerebellum.     

Characterization of zebrafish PSD-95 gene family members

The PSD-95 family of membrane- associated guanylate kinases (MAGUKs) are thought to act as molecular scaffolds that regulate the assembly and function of the multiprotein signaling complex found at the postsynaptic density of excitatory synapses. Genetic analysis of PSD-95 family members in the mammalian nervous system has so far been difficult, but the zebrafish is emerging as an ideal vertebrate system for studying the role of particular genes in the developing and mature nervous system. Here we describe the cloning of the zebrafish orthologs of PSD-95, PSD-93, and two isoforms of SAP-97. Using in situ hybridization analysis we show that these zebrafish MAGUKs have overlapping but distinct patterns of expression in the developing nervous system and craniofacial skeleton. Using a pan-MAGUK antibody we show that MAGUK proteins localize to neurons within the developing hindbrain, cerebellum, visual and olfactory systems, and to skin epithelial cells. In the olfactory and visual systems MAGUK proteins are expressed strongly in synaptic regions, and the onset of expression in these areas coincides with periods of synapse formation. These data are consistent with the idea that PSD-95 family members are involved in synapse assembly and function, and provide a platform for future functional studies in vivo in a highly tractable model organism.     

Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules

Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated.     

Identification of multiple binding partners for the amino-terminal domain of synapse-associated protein 97

Multiprotein complexes mediate static and dynamic functions to establish and maintain cell polarity in both epithelial cells and neurons. Membrane-associated guanylate kinase (MAGUK) proteins are thought to be scaffolding molecules in these processes and bind multiple proteins via their obligate postsynaptic density (PSD)-95/Disc Large/Zona Occludens-1, Src homology 3, and guanylate kinase-like domains. Subsets of MAGUK proteins have additional protein-protein interaction domains. An additional domain we identified in SAP97 called the MAGUK recruitment (MRE) domain binds the LIN-2,7 amino-terminal (L27N) domain of mLIN-2/CASK, a MAGUK known to bind mLIN-7. Here we show that SAP97 binds two other mLIN-7 binding MAGUK proteins. One of these MAGUK proteins, DLG3, coimmunoprecipitates with SAP97 in lysates from rat brain and transfected Madin-Darby canine kidney cells. This interaction requires the MRE domain of SAP97 and surprisingly, both the L27N and L27 carboxyl-terminal (L27C) domains of DLG3. We also demonstrate that SAP97 can interact with the MAGUK protein, DLG2, but not the highly related protein, PALS2. The ability of SAP97 to interact with multiple MAGUK proteins is likely to be important for the targeting of specific protein complexes in polarized cells.     

SAP97 directs the localization of Kv4.2 to spines in hippocampal neurons: regulation by CaMKII

The pore-forming alpha-subunit Kv4.2 is a key constituent of the A-type channel and critically involved in the regulation of dendritic excitability and plasticity. Here we show that Kv4.2 is enriched in the postsynaptic density (PSD) fraction and specifically interacts with synapse-associated protein 97 (SAP97). This interaction requires an intact C terminus of Kv4.2 and occurs via the PDZ domains of SAP97. Pharmacologically induced translocation of SAP97 to spines also drives Kv4.2 to the PSD, whereas SAP97 lentivirally based RNA interference reduces Kv4.2 in the PSD. In addition, calcium/calmodulin-dependent protein kinase II (CaMKII)-dependent SAP97 phosphorylation regulates the subcellular localization of Kv4.2. These results show that SAP97-CaMKII pathway plays an important role for the trafficking of Kv4.2 to dendrites and spines.     

Selective binding of synapse-associated protein 97 to GluR-A alpha-amino-5-hydroxy-3-methyl-4-isoxazole propionate receptor subunit is determined by a novel sequence motif

A family of four closely related PDZ domain-containing membrane-associated guanylate kinase homologues (MAGUKs) is involved in the regulation of the amount and functional state of ionotropic glutamate receptors in excitatory synapses. To understand the mechanisms that determine the specificity of these interactions, we examined the structural basis of the highly selective association between the ionotropic GluR subunit GluR-A and synapse-associated protein 97 (SAP97). The C terminus of GluR-A bound to the PDZ domains of SAP97, but not to those of three related MAGUKs, PSD-93, PSD-95, and SAP102. Experiments with single PDZ domains indicated that the strongest contribution was by the second PDZ domain. Unexpectedly, mutation analysis of the GluR-A C terminus revealed that a tripeptide sequence SSG at position -9 to -11 plays an essential role in this binding, in addition to a C-terminal type I PDZ binding motif (leucine at C terminus and threonine at the -2 position). Analysis of the in vitro MAGUK-binding properties of a GluR-D mutant with a one-residue deletion at the C terminus provides further support for the view that an SSG sequence located N-terminally from a type I PDZ binding motif can mediate selective binding to SAP97 and suggest the existence of a novel variation of the PDZ domain-peptide interaction.     

Targeting of PKA to glutamate receptors through a MAGUK-AKAP complex

Compartmentalization of glutamate receptors with the signaling enzymes that regulate their activity supports synaptic transmission. Two classes of binding proteins organize these complexes: the MAGUK proteins that cluster glutamate receptors and AKAPs that anchor kinases and phosphatases. In this report, we demonstrate that glutamate receptors and PKA are recruited into a macromolecular signaling complex through direct interaction between the MAGUK proteins, PSD-95 and SAP97, and AKAP79/150. The SH3 and GK regions of the MAGUKs mediate binding to the AKAP. Cell-based studies indicate that phosphorylation of AMPA receptors is enhanced by a SAP97-AKAP79 complex that directs PKA to GluR1 via a PDZ domain interaction. As AMPA receptor phosphorylation is implicated in regulating synaptic plasticity, these data suggest that a MAGUK-AKAP complex may be centrally involved.     

Selective reduction of a PDZ protein,SAP-97, in the prefrontal cortex of patients with chronic schizophrenia

Many postsynaptic density proteins carrying postsynaptic density-95/discs large/zone occludens-1 (PDZ) domain(s) interact with glutamate receptors to control receptor dynamics and synaptic plasticity. Here we examined the expression of PDZ proteins, synapse-associated protein (SAP) 97, postsynaptic density (PSD)-95, chapsyn-110, GRIP1 and SAP102, in post-mortem brains of schizophrenic patients and control subjects, and evaluated their contribution to schizophrenic pathology. Among these PDZ proteins, SAP97 exhibited the most marked change: SAP97 protein levels were decreased to less than half that of the control levels specifically in the prefrontal cortex of schizophrenic patients. In parallel, its binding partner, GluR1, similarly decreased in the same brain region. The correlation between SAP97 and GluR1 levels in control subjects was, however, altered in schizophrenic patients. SAP102 levels were also significantly reduced in the hippocampus of schizophrenic patients, but this reduction was correlated with sample storage time and post-mortem interval. There were no changes in the levels of the other PDZ proteins in any of the regions examined. In addition, neuroleptic treatment failed to mimic the SAP97 change. These findings suggest that a phenotypic loss of SAP97 is associated with the postsynaptic impairment in prefrontal excitatory circuits of schizophrenic patients.     

Distribution of the scaffolding proteins PSD-95, PSD-93, and SAP97 in isolated PSDs

We compared the distribution of three scaffolding proteins, all belonging to a family of membrane-associated guanylate kinases, thought to have key roles in the organization of the postsynaptic density (PSD). Isolated PSDs readily adhered to treated glass coverslips where they were labeled with immunogold and rotary shadowed for analysis by EM. The distribution of proteins within individual PSDs were measured by counting and mapping individual immunogold particles. PSD-95, as previously described, is distributed evenly throughout the PSD. We find here that PSD-93 has a nearly identical distribution suggesting that PSD-95 and PSD-93 could perform similar roles. SAP97, in contrast, is concentrated near edges of cleft sides of the PSDs, and in small clumps on their cytoplasmic sides. The homogenous distribution of PSD-95 and PSD-93 throughout the PSD is consistent with their being part of a backbone that stabilizes their various binding partners within the PSD. The distribution of SAP97 confirms that this protein is actually an integral component of the PSD, and suggests that it may have a role in inserting or stabilizing its main binding partner, Glu-R1, at the edge of the PSD.     

Regulation of dendritic spine morphology by SPAR, a PSD-95-associated RapGAP

The PSD-95/SAP90 family of scaffold proteins organizes the postsynaptic density (PSD) and regulates NMDA receptor signaling at excitatory synapses. We report that SPAR, a Rap-specific GTPase-activating protein (RapGAP), interacts with the guanylate kinase-like domain of PSD-95 and forms a complex with PSD-95 and NMDA receptors in brain. In heterologous cells, SPAR reorganizes the actin cytoskeleton and recruits PSD-95 to F-actin. In hippocampal neurons, SPAR localizes to dendritic spines and causes enlargement of spine heads, many of which adopt an irregular appearance with putative multiple synapses. Dominant negative SPAR constructs cause narrowing and elongation of spines. The effects of SPAR on spine morphology depend on the RapGAP and actin-interacting domains, implicating Rap signaling in the regulation of postsynaptic structure.     

Intramolecular interactions regulate SAP97 binding to GKAP

Membrane-associated guanylate kinase homologs (MAGUKs) are multidomain proteins found to be central organizers of cellular junctions. In this study, we examined the molecular mechanisms that regulate the interaction of the MAGUK SAP97 with its GUK domain binding partner GKAP (GUK-associated protein). The GKAP-GUK interaction is regulated by a series of intramolecular interactions. Specifically, the association of the Src homology 3 (SH3) domain and sequences situated between the SH3 and GUK domains with the GUK domain was found to interfere with GKAP binding. In contrast, N-terminal sequences that precede the first PDZ domain in SAP97, facilitated GKAP binding via its association with the SH3 domain. Utilizing crystal structure data available for PDZ, SH3 and GUK domains, molecular models of SAP97 were generated. These models revealed that SAP97 can exist in a compact U-shaped conformation in which the N-terminal domain folds back and interacts with the SH3 and GUK domains. These models support the biochemical data and provide new insights into how intramolecular interactions may regulate the association of SAP97 with its binding partners.     

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.     

FRET-FLIM Investigation of PSD95-NMDA Receptor Interaction in Dendritic Spines; Control by Calpain,CaMKII and Src Family Kinase

Little is known about the changes in protein interactions inside synapses during synaptic remodeling, as their live monitoring in spines has been limited. We used a FRET-FLIM approach in developing cultured rat hippocampal neurons expressing fluorescently tagged NMDA receptor (NMDAR) and PSD95, two essential proteins in synaptic plasticity, to examine the regulation of their interaction. NMDAR stimulation caused a transient decrease in FRET between the NMDAR and PSD95 in spines of young and mature neurons. The activity of both CaMKII and calpain were essential for this effect in both developmental stages. Meanwhile, inhibition of Src family kinase (SFK) had opposing impacts on this decrease in FRET in young versus mature neurons. Our data suggest concerted roles for CaMKII, SFK and calpain activity in regulating activity-dependent separation of PSD95 from GluN2A or GluN2B. Finally, we found that calpain inhibition reduced spine growth that was caused by NMDAR activity, supporting the hypothesis that PSD95-NMDAR separation is implicated in synaptic remodeling.     

Subunit-specific Regulation of N-Methyl-d-aspartate (NMDA) Receptor Trafficking by SAP102 Protein Splice Variants

Synapse-associated protein 102 (SAP102) is a scaffolding protein abundantly expressed early in development that mediates glutamate receptor trafficking during synaptogenesis. Mutations in human SAP102 have been reported to cause intellectual disability, which is consistent with its important role during early postnatal development. SAP102 contains PDZ, SH3, and guanylate kinase (GK)-like domains, which mediate specific protein-protein interactions. SAP102 binds directly to N-methyl-d-aspartate receptors (NMDARs), anchors receptors at synapses, and facilitates transduction of NMDAR signals. Proper localization of SAP102 at the postsynaptic density is essential to these functions. However, how SAP102 is targeted to synapses is unclear. In the current study we find that synaptic localization of SAP102 is regulated by alternative splicing. The SAP102 splice variant that possesses a C-terminal insert (I2) between the SH3 and GK domains is highly enriched at dendritic spines. We also show that there is an intramolecular interaction between the SH3 and GK domains in SAP102 but that the I2 splicing does not influence SH3-GK interaction. Previously, we have shown that SAP102 expression promotes spine lengthening. We now find that the spine lengthening effect is independent of the C-terminal alternative splicing of SAP102. In addition, expression of I2-containing SAP102 isoforms is regulated developmentally. Knockdown of endogenous I2-containing SAP102 isoforms differentially affect NMDAR surface expression in a subunit-specific manner. These data shed new light on the role of SAP102 in the regulation of NMDAR trafficking.