C-terminal synaptic targeting elements for postsynaptic density proteins ProSAP1/Shank2 and ProSAP2/Shank3

Synapses are specialized contact sites mediating communication between neurons. Synaptogenesis requires the specific assembly of protein clusters at both sides of the synaptic contact by mechanisms that are barely understood. We studied the synaptic targeting of multi-domain proteins of the ProSAP/Shank family thought to serve as master scaffolding molecules of the postsynaptic density. In contrast to Shank1, expression of green-fluorescent protein (GFP)-tagged ProSAP1/Shank2 and ProSAP2/Shank3 deletion constructs in hippocampal neurons revealed that their postsynaptic localization relies on the integrity of the C-termini. The shortest construct that was perfectly targeted to synaptic sites included the last 417 amino acids of ProSAP1/Shank2 and included the C-terminal sterile alpha motif (SAM) domain. Removal of 54 residues from the N-terminus of this construct resulted in a diffuse distribution in the cytoplasm. Altogether, our data delineate a hitherto unknown targeting signal in both ProSAP1/Shank2 and ProSAP2/Shank3 and provide evidence for an implication of these proteins and their close homologue, Shank1, in distinct molecular pathways.     

Concerted action of zinc and ProSAP/Shank in synaptogenesis and synapse maturation

Neuronal morphology and number of synapses is not static, but can change in response to a variety of factors, a process called synaptic plasticity. These structural and molecular changes are believed to represent the basis for learning and memory, thereby underling both the developmental and activity-dependent remodelling of excitatory synapses. Here, we report that Zn(2+) ions, which are highly enriched within the postsynaptic density (PSD), are able to influence the recruitment of ProSAP/Shank proteins to PSDs in a family member-specific manner during the course of synaptogenesis and synapse maturation. Through selectively overexpressing each family member at excitatory postsynapses and comparing this to shRNA-mediated knockdown, we could demonstrate that only the overexpression of zinc-sensitive ProSAP1/Shank2 or ProSAP2/Shank3 leads to increased synapse density, although all of them cause a decrease upon knockdown. Furthermore, depletion of synaptic Zn(2+) along with the knockdown of zinc-insensitive Shank1 causes the rapid disintegration of PSDs and the loss of several postsynaptic molecules including Homer1, PSD-95 and NMDA receptors. These findings lead to the model that the concerted action of ProSAP/Shank and Zn(2+) is essential for the structural integrity of PSDs and moreover that it is an important element of synapse formation, maturation and structural plasticity.     

Secretory granules of hypophyseal and pancreatic endocrine cells contain proteins of the neuronal postsynaptic density

The PDZ domain-containing protein Shank is a master scaffolding protein of the neuronal postsynaptic density and directly or indirectly links neurotransmitter receptors and cell adhesion molecules to the actin-based cytoskeleton. ProSAP/Shank proteins have recently also been detected in several non-neuronal cells in which they are mostly concentrated in the apical subplasmalemmal cytoplasm. In contrast, we have previously reported a more widespread cytoplasmic immunostaining pattern for the ProSAP1/Shank2 protein in endocrine cells at the light-microscopic level. Therefore, in the present study, we have determined the ultrastructural localization of ProSAP1/Shank2 and the ProSAP/Shank-interacting proteins ProSAPiP1 and IRSp53 in pancreatic islet and adenohypophyseal cells by using immunogold staining techniques. Dense immunolabeling of secretory granules including the granule core in cells such as hypophyseal somatotrophs and pancreatic B-cells indicates the unexpected presence of ProSAP/Shank and ProSAP/Shank-interacting proteins in the hormone-storing compartment of endocrine cells. Thus, ProSAP/Shank and certain ProSAP/Shank-interacting proteins exhibit distinct subcellular localizations in the different cell types, raising the possibility that the function of ProSAP/Shank proteins is more diverse than has been envisaged to date. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 497/B8 to J.B. and T.M.B.).     

ProSAP1 and membrane nanodomain-associated syndapin I promote postsynapse formation and function

Insights into mechanisms coordinating membrane remodeling, local actin nucleation, and postsynaptic scaffolding during postsynapse formation are important for understanding vertebrate brain function. Gene knockout and RNAi in individual neurons reveal that the F-BAR protein syndapin I is a crucial postsynaptic coordinator in formation of excitatory synapses. Syndapin I deficiency caused significant reductions of synapse and dendritic spine densities. These syndapin I functions reflected direct, SH3 domain–mediated associations and functional interactions with ProSAP1/Shank2. They furthermore required F-BAR domain-mediated membrane binding. Ultra-high-resolution imaging of specifically membrane-associated, endogenous syndapin I at membranes of freeze-fractured neurons revealed that membrane-bound syndapin I preferentially occurred in spines and formed clusters at distinct postsynaptic membrane subareas. Postsynaptic syndapin I deficiency led to reduced frequencies of miniature excitatory postsynaptic currents, i.e., to defects in synaptic transmission phenocopying ProSAP1/Shank2 knockout, and impairments in proper synaptic ProSAP1/Shank2 distribution. Syndapin I–enriched membrane nanodomains thus seem to be important spatial cues and organizing platforms, shaping dendritic membrane areas into synaptic compartments.     

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.     

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.     

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 Shank family of postsynaptic density proteins interacts with and promotes synaptic accumulation of the beta PIX guanine nucleotide exchange factor for Rac1 and Cdc42

The Shank/ProSAP family of multidomain proteins is known to play an important role in organizing synaptic multiprotein complexes. Here we report a novel interaction between Shank and beta PIX, a guanine nucleotide exchange factor for the Rac1 and Cdc42 small GTPases. This interaction is mediated by the PDZ domain of Shank and the C-terminal leucine zipper domain and the PDZ domain-binding motif at the extreme C terminus of beta PIX. Shank colocalizes with beta PIX at excitatory synaptic sites in cultured neurons. In brain, Shank forms a complex with beta PIX and beta PIX-associated signaling molecules including p21-associated kinase (PAK), an effector kinase of Rac1/Cdc42. Importantly, overexpression of Shank in cultured neurons promotes synaptic accumulation of beta PIX and PAK. Considering the involvement of Rac1 and PAK in spine dynamics, these results suggest that Shank recruits beta PIX and PAK to spines for the regulation of postsynaptic structure.     

Dynamin isoform-specific interaction with the shank/ProSAP scaffolding proteins of the postsynaptic density and actin cytoskeleton

Dynamin is a GTPase involved in endocytosis and other aspects of membrane trafficking. A critical function in the presynaptic compartment attributed to the brain-specific dynamin isoform, dynamin-1, is in synaptic vesicle recycling. We report that dynamin-2 specifically interacts with members of the Shank/ProSAP family of postsynaptic density scaffolding proteins and present evidence that dynamin-2 is specifically associated with the postsynaptic density. These data are consistent with a role for this otherwise broadly distributed form of dynamin in glutamate receptor down-regulation and other aspects of postsynaptic membrane turnover.     

Synaptogenesis of hippocampal neurons in primary cell culture

Hippocampal neurons in dissociated cell culture are one of the most extensively used model systems in the field of molecular and cellular neurobiology. Only limited data are however available on the normal time frame of synaptogenesis, synapse number and ultrastructure of excitatory synapses during early development in culture. Therefore, we analyzed the synaptic ultrastructure and morphology and the localization of presynaptic (Bassoon) and postsynaptic (ProSAP1/Shank2) marker proteins in cultures established from rat embryos at embryonic day 19, after 3, 7, 10, 14, and 21 days in culture. First excitatory synapses were identified at day 7 with a clearly defined postsynaptic density and presynaptically localized synaptic vesicles. Mature synapses on dendritic spines were seen from day 10 onward, and the number of synapses steeply increased in the third week. Fenestrated or multiple synapses were found after 14 or 21 days, respectively. So-called dense-core vesicles, responsible for the transport of proteins to the active zone of the presynaptic specialization, were seen on cultivation day 3 and 7 and could be detected in axons and especially in the presynaptic subcompartments. The expression and localization of the presynaptic protein Bassoon and of the postsynaptic molecule ProSAP1/Shank2 was found to correlate nicely with the ultrastructural results. This regular pattern of development and maturation of excitatory synapses in hippocampal culture starting from day 7 in culture should ease the comparison of synapse number and morphology of synaptic contacts in this widely used model system.     

ProSAP/Shank postsynaptic density proteins interact with insulin receptor tyrosine kinase substrate IRSp53

The ProSAP/Shank family of multidomain proteins of the postsynaptic density (PSD) can either directly or indirectly interact with NMDA-type and metabotropic glutamate receptors and the actin-based cytoskeleton. In a yeast two hybrid screen utilizing a proline-rich domain that is highly conserved among the ProSAP/Shank family members, we isolated several cDNA clones coding for the insulin receptor substrate IRSp53. The specificity of this interaction was confirmed in transfected COS cells. Co-immunoprecipitation of IRSp53 and ProSAP2 solubilized from rat brain membranes indicates that the interaction occurs in vivo. The C-terminal SH3 domain of IRSp53 is responsible for the interaction with a novel proline-rich consensus sequence of ProSAP/Shank that was characterized by mutational analysis. IRSp53 is a substrate for the insulin receptor in the brain and acts downstream of small GTPases of the Rho family. Binding of Cdc42Hs to IRSp53 induces actin filament assembly, reorganization and filopodia outgrowth in neuronal cell lines. Our data suggest that IRSp53 can be recruited to the PSD via its ProSAP/Shank interaction and may contribute to the morphological reorganization of spines and synapses after insulin receptor and/or Cdc42Hs activation.     

The postsynaptic density

Glutamatergic synapses in the central nervous system are characterized by an electron-dense web underneath the postsynaptic membrane; this web is called the postsynaptic density (PSD). PSDs are composed of a dense network of several hundred proteins, creating a macromolecular complex that serves a wide range of functions. Prominent PSD proteins such as members of the MaGuk or ProSAP/Shank family build up a dense scaffold that creates an interface between clustered membrane-bound receptors, cell adhesion molecules and the actin-based cytoskeleton. Moreover, kinases, phosphatases and several proteins of different signalling pathways are specifically localized within the spine/PSD compartment. Small GTPases and regulating proteins are also enriched in PSDs being the molecular basis for regulated structural changes of cytoskeletal components within the synapse in response to external or internal stimuli, e.g. synaptic activation. This synaptic rearrangement (structural plasticity) is a rapid process and is believed to underlie learning and memory formation. The characterization of synapse/PSD proteins is especially important in the light of recent data suggesting that several mental disorders have their molecular defect at the synapse/PSD level.The work of former and current colleagues in my laboratory and the support with respect to research on components of the PSD network by the DFG (SFB497/B8, Bo1718/2-2) and by the Land Baden-Württemberg (1423/74) are gratefully acknowledged.     

Loss of COMMD1 and copper overload disrupt zinc homeostasis and influence an autism-associated pathway at glutamatergic synapses

Recent studies suggest that synaptic pathology in autism spectrum disorder (ASD) might be caused by the disruption of a signaling pathway at excitatory glutamatergic synapses, which can be influenced by environmental factors. Some factors, such as prenatal zinc deficiency, dysfunction of metallothioneins as well as deletion of COMMD1, all affect brain metal-ion homeostasis and have been associated with ASD. Given that COMMD1 regulates copper levels and that copper and zinc have antagonistic properties, here, we followed the idea that copper overload might induce a local zinc deficiency affecting key players of a putative ASD pathway such as ProSAP/Shank proteins as reported before. Our results show that increased copper levels indeed interfere with intracellular zinc concentrations and affect synaptic ProSAP/Shank levels, which similarly are altered by manipulation of copper and zinc levels through overexpression and knockdown of COMMD1. In line with this, acute and prenatal copper overload lead to local zinc deficiencies in mice. Pups exposed to prenatal copper overload furthermore show a reduction in ProSAP/Shank protein levels in the brain as well as a decreased NMDAR subunit 1 concentration. Thus, it might be likely that brain metal ion status influences a distinct pathway in excitatory synapses associated with genetic forms of ASD.     

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.     

Local sharing as a predominant determinant of synaptic matrix molecular dynamics

Recent studies suggest that central nervous system synapses can persist for weeks, months, perhaps lifetimes, yet little is known as to how synapses maintain their structural and functional characteristics for so long. As a step toward a better understanding of synaptic maintenance we examined the loss, redistribution, reincorporation, and replenishment dynamics of Synapsin I and ProSAP2/Shank3, prominent presynaptic and postsynaptic matrix molecules, respectively. Fluorescence recovery after photobleaching and photoactivation experiments revealed that both molecules are continuously lost from, redistributed among, and reincorporated into synaptic structures at time-scales of minutes to hours. Exchange rates were not affected by inhibiting protein synthesis or proteasome-mediated protein degradation, were accelerated by stimulation, and greatly exceeded rates of replenishment from somatic sources. These findings indicate that the dynamics of key synaptic matrix molecules may be dominated by local protein exchange and redistribution, whereas protein synthesis and degradation serve to maintain and regulate the sizes of local, shared pools of these proteins.     

The neuronal scaffold protein Shank3 mediates signaling and biological function of the receptor tyrosine kinase Ret in epithelial cells

Shank proteins, initially also described as ProSAP proteins, are scaffolding adaptors that have been previously shown to integrate neurotransmitter receptors into the cortical cytoskeleton at postsynaptic densities. We show here that Shank proteins are also crucial in receptor tyrosine kinase signaling. The PDZ domain-containing Shank3 protein was found to represent a novel interaction partner of the receptor tyrosine kinase Ret, which binds specifically to a PDZ-binding motif present in the Ret9 but not in the Ret51 isoform. Furthermore, we show that Ret9 but not Ret51 induces epithelial cells to form branched tubular structures in three-dimensional cultures in a Shank3-dependent manner. Ret9 but not Ret51 has been previously shown to be required for kidney development. Shank3 protein mediates sustained Erk-MAPK and PI3K signaling, which is crucial for tubule formation, through recruitment of the adaptor protein Grb2. These results demonstrate that the Shank3 adaptor protein can mediate cellular signaling, and provide a molecular mechanism for the biological divergence between the Ret9 and Ret51 isoform.     

The calcium-independent receptor for alpha-latrotoxin from human and rodent brains interacts with members of the ProSAP/SSTRIP/Shank family of multidomain proteins

Subtypes of the calcium-independent receptors for alpha-latrotoxin (CIRL1-3) define a distinct subgroup within the large family of the seven-transmembrane region cell surface receptors. The physiological function of CIRLs is unknown because neither extracellular ligands nor intracellular coupling proteins (G-proteins) have been identified. Using yeast two-hybrid screening, we identified a novel interaction between the C termini of CIRL1 and -2 and the PSD-95/discs large/ZO-1 (PDZ) domain of a recently discovered multidomain protein family (ProSAP/SSTRIP/Shank) present in human and rat brain. In vitro, CIRL1 and CIRL2 interacted strongly with the PDZ domain of ProSAP1. The specificity of this interaction has been verified by in vivo experiments using solubilized rat brain membrane fractions and ProSAP1 antibodies; only CIRL1, but not CIRL2, was co-immunoprecipitated with ProSAP1. In situ hybridization revealed that ProSAP1 and CIRL1 are co-expressed in the cortex, hippocampus, and cerebellum. Colocalization was also observed at the subcellular level, as both CIRL1 and ProSAP1 are enriched in the postsynaptic density fraction from rat brain. Expression of all three CIRL isoforms is highly regulated during postnatal brain development, with CIRL3 exhibiting its highest expression levels immediately after birth, followed by CIRL2 and finally CIRL1 in aged rats.