Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin.

NMDA receptors are linked to intracellular cytoskeletal and signaling molecules via the PSD-95 protein complex. We report a novel family of postsynaptic density (PSD) proteins, termed Shank, that binds via its PDZ domain to the C terminus of PSD-95-associated protein GKAP. A ternary complex of Shank/GKAP/PSD-95 assembles in heterologous cells and can be coimmunoprecipitated from rat brain. Synaptic localization of Shank in neurons is inhibited by a GKAP splice variant that lacks the Shank-binding C terminus. In addition to its PDZ domain, Shank contains a proline-rich region that binds to cortactin and a SAM domain that mediates multimerization. Shank may function as a scaffold protein in the PSD, potentially cross-linking NMDA receptor/PSD-95 complexes and coupling them to regulators of the actin cytoskeleton.     

Coupling of mGluR/Homer and PSD-95 complexes by the Shank family of postsynaptic density proteins.

Shank is a recently described family of postsynaptic proteins that function as part of the NMDA receptor-associated PSD-95 complex (Naisbitt et al., 1999 [this issue of Neuron]). Here, we report that Shank proteins also bind to Homer. Homer proteins form multivalent complexes that bind proline-rich motifs in group 1 metabotropic glutamate receptors and inositol trisphosphate receptors, thereby coupling these receptors in a signaling complex. A single Homer-binding site is identified in Shank, and Shank and Homer coimmunoprecipitate from brain and colocalize at postsynaptic densities. Moreover, Shank clusters mGluR5 in heterologous cells in the presence of Homer and mediates the coclustering of Homer with PSD-95/GKAP. Thus, Shank may cross-link Homer and PSD-95 complexes in the PSD and play a role in the signaling mechanisms of both mGluRs and NMDA receptors.     

SHN-1, a Shank homologue in C. elegans, affects defecation rhythm via the inositol-1,4,5-trisphosphate receptor

Protein localization in the postsynaptic density (PSD) of neurons is mediated by scaffolding proteins such as PSD-95 and Shank, which ensure proper function of receptors at the membrane. The Shank family of scaffolding proteins contain PDZ (PSD-95, Dlg, and ZO-1) domains and have been implicated in the localizations of many receptor proteins including glutamate receptors in mammals. We have identified and characterized shn-1, the only homologue of Shank in Caenorhabditis elegans. The shn-1 gene shows approximately 40% identity over 1000 amino acids to rat Shanks. SHN-1 protein is localized in various tissues including neurons, pharynx and intestine. RNAi suppression of SHN-1 did not cause lethality or developmental abnormality. However, suppression of SHN-1 in the itr-1 (sa73) mutant, which has a defective inositol-1,4,5-trisphosphate (IP(3)) receptor, resulted in animals with altered defecation rhythm. Our data suggest a possible role of SHN-1 in affecting function of IP(3) receptors in C. elegans.     

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.     

Interaction of brain somatostatin receptors with the PDZ domains of PSD-95

Using peptide affinity purification, we identified an interaction between somatostatin receptors SSTR4 and SSTR1 and PDZ domains 1 and 2 of the postsynaptic proteins postsynaptic density protein of 95kDa (PSD-95) and PSD-93. The existence of the SSTR4/PSD-95 complex was verified by coimmunoprecipitation from transfected cells and solubilized brain membranes. In neurons, dendritically localized SSTR4 partially colocalizes with postsynaptic PSD-95. As functional parameters of the receptor, such as coupling to potassium channels, are not affected by the interaction with PSD-95, the association may serve to localize the receptor to postsynaptic sites.     

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.     

The interaction of phospholipase C-beta3 with Shank2 regulates mGluR-mediated calcium signal

Phospholipase C-beta isozymes that are activated by G protein-coupled receptors (GPCR) and heterotrimeric G proteins carry a PSD-95/Dlg/ZO-1 (PDZ) domain binding motif at their C terminus. Through interactions with PDZ domains, this motif may endow the PLC-beta isozyme with specific roles in GPCR signaling events that occur in compartmentalized regions of the plasma membrane. In this study, we identified the interaction of PLC-beta3 with Shank2, a PDZ domain-containing multimodular scaffold in the postsynaptic density (PSD). The C terminus of PLC-beta3, but not other PLC-beta isotypes, specifically interacts with the PDZ domain of Shank2. Homer 1b, a Shank-interacting protein that is linked to group I metabotropic glutamate receptors and IP3 receptors, forms a multiple complex with Shank2 and PLC-beta3. Importantly, microinjection of a synthetic peptide specifically mimicking the C terminus of PLC-beta3 markedly reduces the mGluR-mediated intracellular calcium response. These results demonstrate that Shank2 brings PLC-beta3 closer to Homer 1b and constitutes an efficient mGluR-coupled signaling pathway in the PSD region of neuronal synapses.     

Phosphorylation state of postsynaptic density proteins

The postsynaptic density (PSD) is an electron-dense structure located at the synaptic contacts between neurons. Its considerable complexity includes cytoskeletal and scaffold proteins, receptors, ion channels and signaling molecules, in line with the role of PSDs in signal transduction and processing. The phosphorylation state of components of the PSD is central to synaptic transmission and is known to play a role in synaptic plasticity, learning and memory. The presence of a range of kinases and phosphatases in the PSD defines potential key players in this context. However, the substrates that these enzymes target have not been fully identified to date. We analyzed the protein composition of purified PSD samples from adult mouse brains by strong cation exchange chromatography fractionation of a tryptic digest followed by nano-reverse phase liquid chromatography coupled with electrospray ionization-quadrupole time of flight tandem mass spectrometry. This led to the identification of 244 proteins. To gain an insight into the phosphoproteome of the PSD we then purified phosphorylated tryptic peptides by immobilized metal ion affinity chromatography. This approach for the specific enrichment of phosphopeptides resulted in the identification of 42 phosphoproteins in the PSD preparation, 39 of which are known PSD components. Here we present a total of 83 in vivo phosphorylation sites.     

Association of Shank 1A Scaffolding Protein with Cone Photoreceptor Terminals in the Mammalian Retina

Photoreceptor terminals contain post-synaptic density (PSD) proteins e.g., PSD-95/PSD-93, but their role at photoreceptor synapses is not known. PSDs are generally restricted to post-synaptic boutons in central neurons and form scaffolding with multiple proteins that have structural and functional roles in neuronal signaling. The Shank family of proteins (Shank 1–3) functions as putative anchoring proteins for PSDs and is involved in the organization of cytoskeletal/signaling complexes in neurons. Specifically, Shank 1 is restricted to neurons and interacts with both receptors and signaling molecules at central neurons to regulate plasticity. However, it is not known whether Shank 1 is expressed at photoreceptor terminals. In this study we have investigated Shank 1A localization in the outer retina at photoreceptor terminals. We find that Shank 1A is expressed presynaptically in cone pedicles, but not rod spherules, and it is absent from mice in which the Shank 1 gene is deleted. Shank 1A co-localizes with PSD-95, peanut agglutinin, a marker of cone terminals, and glycogen phosphorylase, a cone specific marker. These findings provide convincing evidence for Shank 1A expression in both the inner and outer plexiform layers, and indicate a potential role for PSD-95/Shank 1 complexes at cone synapses in the outer retina.     

Phosphoproteomic analysis of synaptosomes from human cerebral cortex

Protein phosphorylation is a crucial post-translational modification mechanism in the regulation of synaptic organization and function. Here, we analyzed synaptosome fractions from human cerebral cortex obtained during therapeutic surgery. To minimize changes in the phosphorylation state of proteins, the tissue was homogenized within two minutes of excision. Synaptosomal proteins were digested with trypsin and phosphopeptides were isolated by immobilized metal affinity chromatography and analyzed by liquid chromatography and tandem mass spectrometry. The method allowed the detection of residues on synaptic proteins that were presumably phosphorylated in the intact cell, including synapsin 1, syntaxin 1, and SNIP, PSD-93, NCAM, GABA-B receptor, chaperone molecules, and protein kinases. Some of the residues identified are the same or homologous to sites that had been previously described to be phosphorylated in mammals whereas others appear to be novel sites which, to our knowledge, have not been reported previously. The study shows that new phosphoproteomic strategies can be used to analyze subcellular fractions from small amounts of tissue for the identification of phosphorylated residues for research and potentially for diagnostic purposes.     

PDZ Ligand Binding-Induced Conformational Coupling of the PDZ–SH3–GK Tandems in PSD-95 Family MAGUKs

Discs large (DLG) MAGUKs are abundantly expressed in glutamatergic synapses, crucial for synaptic transmission, and plasticity by anchoring various postsynaptic components including glutamate receptors, downstream scaffold proteins and signaling enzymes. Different DLG members have shared structures and functions, but also contain unique features. How DLG family proteins function individually and cooperatively is largely unknown. Here, we report that PSD-95 PDZ3 directly couples with SH3–GK tandem in a PDZ ligand binding-dependent manner, and the coupling can promote PSD-95 dimerization and multimerization. Aided by sortase-mediated protein ligation and selectively labeling, we elucidated the PDZ3/SH3–GK conformational coupling mechanism using NMR spectroscopy. We further demonstrated that PSD-93, but not SAP102, can also undergo PDZ3 ligand binding-induced conformational coupling with SH3–GK and form homo-oligomers. Interestingly, PSD-95 and PSD-93 can also form ligand binding-induced hetero-oligomers, suggesting a cooperative assembly mechanism for the mega-N-methyl-d-aspartate receptor synaptic signaling complex. Finally, we provide evidence showing that ligand binding-induced conformational coupling between PDZ and SH3–GK is a common feature for other MAGUKs including CASK and PALS1.     

Identification of PSD-93 as a substrate for the Src family tyrosine kinase Fyn

In order to study the role of tyrosine kinase signaling in the post-synaptic density (PSD), tyrosine-phosphorylated proteins associated with the PSD-95/NMDA receptor complex were analyzed. The NMDA receptor complex from the mouse brain was successfully solubilized with deoxycholate and immunopurified with anti-PSD-95 or anti-phosphotyrosine antibody. Immunoblot analyses revealed that the predominantly tyrosine-phosphorylated proteins in the NMDA receptor complex are the NR2A/B subunits and a novel 120 kDa protein. Purification and microsequencing analysis showed that the 120 kDa protein is mouse PSD-93/Chapsyn-110. Recombinant PSD-93 was phosphorylated by Fyn in vitro, and Tyr-384 was identified as a major phosphorylation site. Tyrosine phosphorylation of PSD-93 was greatly reduced in brain tissue from Fyn-deficient mice compared with wild-type mice. Furthermore, an N-terminal palmitoylation signal of PSD-93 was found to be essential for its anchoring to the membrane, where Fyn is also localized. In COS7 cells, exogenously expressed PSD-93 was phosphorylated, dependent on its membrane localization. In addition, tyrosine-phosphorylated PSD-93 was able to bind to Csk, a negative regulator of Src family kinases, in vitro as well as in a brain lysate. These results suggest that PSD-93 serves as a membrane-anchored substrate of Fyn and plays a role in the regulation of Fyn-mediated modification of NMDA receptor function.     

Differential Distribution of Shank and GKAP at the Postsynaptic Density

Shank and GKAP are scaffold proteins and binding partners at the postsynaptic density (PSD). The distribution and dynamics of Shank and GKAP were studied in dissociated hippocampal cultures by pre-embedding immunogold electron microscopy. Antibodies against epitopes containing their respective mutual binding sites were used to verify the expected juxtapositioning of Shank and GKAP. If all Shank and GKAP molecules at the PSD were bound to each other, the distribution of label for the two proteins should coincide. However, labels for the mutual binding sites showed significant differences in distribution, with a narrow distribution for GKAP located close to the postsynaptic membrane, and a wider distribution for Shank extending deeper into the cytoplasm. Upon depolarization with high K⁺, neither the intensity nor distribution of label for GKAP changed, but labeling intensity for Shank at the PSD increased to ~150% of controls while the median distance of label from postsynaptic membrane increased by 7.5 nm. These results indicate a preferential recruitment of Shank to more distal parts of the PSD complex. Conversely, upon incubation in Ca²⁺-free medium containing EGTA, the labeling intensity of Shank at the PSD decreased to ~70% of controls and the median distance of label from postsynaptic membrane decreased by 9 nm, indicating a preferential loss of Shank molecules in more distal parts of the PSD complex. These observations identify two pools of Shank at the PSD complex, one relatively stable pool, closer to the postsynaptic membrane that can bind to GKAP, and another more dynamic pool at a location too far away to bind to GKAP.     

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.     

Flavonoids from Radix Scutellariae as potential stroke therapeutic agents by targeting the second postsynaptic density 95 (PSD-95)/disc large/zonula occludens-1 (PDZ) domain of PSD-95.

Excessive activation of N-methyl-D-aspartate receptors (NMDARs) and subsequent production of nitric oxide by neuronal nitric oxide synthase (nNOS) contribute to neuronal damage resulting from hypoxic and ischemic insults. NMDARs and nNOS are coupled together at the postsynaptic membrane through their interaction with postsynaptic density protein (PSD) 95 via PSD-95/disc large/zonula occludens-1 (PDZ) domains. We used NMR (nuclear magnetic resonance) spectroscopy to screen medicinal herbs used in traditional Chinese medicine (TCM) stroke therapy for compounds binding to the second PDZ domain (PDZ2) of PSD-95, the domain linking nNOS and PSD-95. Aqueous extract of Huangqin, the root of Scutellaria baicalensis Georgi (Labiatae), showed significant binding to PDZ2 of PSD-95. The binding site of the active components in the extract overlapped with the nNOS/NR2B-binding pocket of PDZ2 of PSD-95. Four flavones, baicalin, norwogonoside, oroxylin A-glucuronide (oroxyloside), and wogonoside were isolated and found to account for the PDZ-binding activity of the extract. NMR titration experiments showed that baicalin and norwogonoside displayed the highest PDZ2 binding affinity, while oroxylin A-glucuronide and wogonoside showed 4-5 fold less potency in binding to the PDZ domain. Identification of the PDZ binding activity of these compounds will allow investigating whether or not it contributes to the observed clinical effects of Radix Scutellariae. Furthermore, these molecules might provide leads for the development of drugs targeting the signaling pathways mediated by PDZ domains.     

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.     

Glutamatergic Postsynaptic Density Protein Dysfunctions in Synaptic Plasticity and Dendritic Spines Morphology: Relevance to Schizophrenia and Other Behavioral Disorders Pathophysiology,and Implications for Novel Therapeutic Approaches

Emerging researches point to a relevant role of postsynaptic density (PSD) proteins, such as PSD-95, Homer, Shank, and DISC-1, in the pathophysiology of schizophrenia and autism spectrum disorders. The PSD is a thickness, detectable at electronic microscopy, localized at the postsynaptic membrane of glutamatergic synapses, and made by scaffolding proteins, receptors, and effector proteins; it is considered a structural and functional crossroad where multiple neurotransmitter systems converge, including the dopaminergic, serotonergic, and glutamatergic ones, which are all implicated in the pathophysiology of psychosis. Decreased PSD-95 protein levels have been reported in postmortem brains of schizophrenia patients. Variants of Homer1, a key PSD protein for glutamate signaling, have been associated with schizophrenia symptoms severity and therapeutic response. Mutations in Shank gene have been recognized in autism spectrum disorder patients, as well as reported to be associated to behaviors reminiscent of schizophrenia symptoms when expressed in genetically engineered mice. Here, we provide a critical appraisal of PSD proteins role in the pathophysiology of schizophrenia and autism spectrum disorders. Then, we discuss how antipsychotics may affect PSD proteins in brain regions relevant to psychosis pathophysiology, possibly by controlling synaptic plasticity and dendritic spine rearrangements through the modulation of glutamate-related targets. We finally provide a framework that may explain how PSD proteins might be useful candidates to develop new therapeutic approaches for schizophrenia and related disorders in which there is a need for new biological treatments, especially against some symptom domains, such as negative symptoms, that are poorly affected by current antipsychotics.     

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

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

Proteomic analysis revealed a novel synaptic proline-rich membrane protein (PRR7) associated with PSD-95 and NMDA receptor

Proteomic analyses have revealed a novel synaptic proline-rich membrane protein: PRR7 (proline rich 7), in the postsynaptic density (PSD) fraction of rat forebrain. PRR7 is 269 amino acid residues long, and displays a unique architecture, composed of a very short N-terminal extracellular region, a single membrane spanning domain, and a cytoplasmic domain possessing a proline-rich sequence and a C-terminal type-1 PDZ binding motif. A fraction of PRR7 accumulates in spines along with synapse maturation, and colocalizes with PSD-95 in a punctate pattern in rat hippocampal neural cultures. Immunoprecipitation and GST pull-down assays demonstrated that PRR7 binds to the third PDZ domain of PSD-95. In addition, the NMDA receptor subunits, NR1 and NR2B, specifically co-immunoprecipitated with PRR7. These results suggest that PRR7 is involved in modulating neural activities via interactions with the NMDA receptor and PSD-95, and PSD core formation.     

Phosphorylation of a PDZ domain extension modulates binding affinity and interdomain interactions in postsynaptic density-95 (PSD-95) protein, a membrane-associated guanylate kinase (MAGUK)

Postsynaptic density-95 is a multidomain scaffolding protein that recruits glutamate receptors to postsynaptic sites and facilitates signal processing and connection to the cytoskeleton. It is the leading member of the membrane-associated guanylate kinase family of proteins, which are defined by the PSD-95/Discs large/ZO-1 (PDZ)-Src homology 3 (SH3)-guanylate kinase domain sequence. We used NMR to show that phosphorylation of conserved tyrosine 397, which occurs in vivo and is located in an atypical helical extension (α3), initiates a rapid equilibrium of docked and undocked conformations. Undocking reduced ligand binding affinity allosterically and weakened the interaction of PDZ3 with SH3 even though these domains are separated by a ~25-residue linker. Additional phosphorylation at two linker sites further disrupted the interaction, implicating α3 and the linker in tuning interdomain communication. These experiments revealed a novel mode of regulation by a detachable PDZ element and offer a first glimpse at the dynamic interaction of PDZ and SH3-guanylate kinase domains in membrane-associated guanylate kinases.