The Shank/ProSAP N-Terminal (SPN) Domain of Shank3 Regulates Targeting to Postsynaptic Sites and Postsynaptic Signaling.

Members of the Shank family of postsynaptic scaffold proteins (Shank1-3) link neurotransmitter receptors to the actin cytoskeleton in dendritic spines through establishing numerous interactions within the postsynaptic density (PSD) of excitatory synapses. Large Shank isoforms carry at their N-termini a highly conserved domain termed the Shank/ProSAP N-terminal (SPN) domain, followed by a set of Ankyrin repeats. Both domains are involved in an intramolecular interaction which is believed to regulate accessibility for additional interaction partners, such as Ras family G-proteins, αCaMKII, and cytoskeletal proteins. Here, we analyze the functional relevance of the SPN-Ank module; we show that binding of active Ras or Rap1a to the SPN domain can differentially regulate the localization of Shank3 in dendrites. In Shank1 and Shank3, the linker between the SPN and Ank domains binds to inactive αCaMKII. Due to this interaction, both Shank1 and Shank3 exert a negative effect on αCaMKII activity at postsynaptic sites in mice in vivo. The relevance of the SPN-Ank intramolecular interaction was further analyzed in primary cultured neurons; here, we observed that in the context of full-length Shank3, a closed conformation of the SPN-Ank tandem is necessary for proper clustering of Shank3 on the head of dendritic spines. Shank3 variants carrying Ank repeats which are not associated with the SPN domain lead to the atypical formation of postsynaptic clusters on dendritic shafts, at the expense of clusters in spine-like protrusions. Our data show that the SPN-Ank tandem motif contributes to the regulation of postsynaptic signaling and is also necessary for proper targeting of Shank3 to postsynaptic sites. Our data also suggest how missense variants found in autistic patients which alter SPN and Ank domains affect the synaptic function of Shank3.

Structural deficits in key domains of Shank2 lead to alterations in postsynaptic nanoclusters and to a neurodevelopmental disorder in humans.

Postsynaptic scaffold proteins such as Shank, PSD-95, Homer and SAPAP/GKAP family members establish the postsynaptic density of glutamatergic synapses through a dense network of molecular interactions. Mutations in SHANK genes are associated with neurodevelopmental disorders including autism and intellectual disability. However, no SHANK missense mutations have been described which interfere with the key functions of Shank proteins believed to be central for synapse formation, such as GKAP binding via the PDZ domain, or Zn2+-dependent multimerization of the SAM domain. We identify two individuals with a neurodevelopmental disorder carrying de novo missense mutations in SHANK2. The p.G643R variant distorts the binding pocket for GKAP in the Shank2 PDZ domain and prevents interaction with Thr(-2) in the canonical PDZ ligand motif of GKAP. The p.L1800W variant severely delays the kinetics of Zn2+-dependent polymerization of the Shank2-SAM domain. Structural analysis shows that Trp1800 dislodges one histidine crucial for Zn2+ binding. The resulting conformational changes block the stacking of helical polymers of SAM domains into sheets through side-by-side contacts, which is a hallmark of Shank proteins, thereby disrupting the highly cooperative assembly process induced by Zn2+. Both variants reduce the postsynaptic targeting of Shank2 in primary cultured neurons and alter glutamatergic synaptic transmission. Super-resolution microscopy shows that both mutants interfere with the formation of postsynaptic nanoclusters. Our data indicate that both the PDZ- and the SAM-mediated interactions of Shank2 contribute to the compaction of postsynaptic protein complexes into nanoclusters, and that deficiencies in this process interfere with normal brain development in humans.

Regulation of Liprin-α phase separation by CASK is disrupted by a mutation in its CaM kinase domain.

CASK is a unique membrane-associated guanylate kinase (MAGUK) because of its Ca2+/calmodulin-dependent kinase (CaMK) domain. We describe four male patients with a severe neurodevelopmental disorder with microcephaly carrying missense variants affecting the CaMK domain. One boy who carried the p.E115K variant and died at an early age showed pontocerebellar hypoplasia (PCH) in addition to microcephaly, thus exhibiting the classical MICPCH phenotype observed in individuals with CASK loss-of-function variants. All four variants selectively weaken the interaction of CASK with Liprin-α2, a component of the presynaptic active zone. Liprin-α proteins form spherical phase-separated condensates, which we observe here in Liprin-α2 overexpressing HEK293T cells. Large Liprin-α2 clusters were also observed in transfected primary-cultured neurons. Cluster formation of Liprin-α2 is reversed in the presence of CASK; this is associated with altered phosphorylation of Liprin-α2. The p.E115K variant fails to interfere with condensate formation. As the individual carrying this variant had the severe MICPCH disorder, we suggest that regulation of Liprin-α2-mediated phase condensate formation is a new functional feature of CASK which must be maintained to prevent PCH.

Mutations affecting the N-terminal domains of SHANK3 point to different pathomechanisms in neurodevelopmental disorders.

Shank proteins are major scaffolds of the postsynaptic density of excitatory synapses. Mutations in SHANK genes are associated with autism and intellectual disability. The effects of missense mutations on Shank3 function, and therefore the pathomechanisms are unclear. Several missense mutations in SHANK3 affect the N-terminal region, consisting of the Shank/ProSAP N-terminal (SPN) domain and a set of Ankyrin (Ank) repeats. Here we identify a novel SHANK3 missense mutation (p.L270M) in the Ankyrin repeats in patients with an ADHD-like phenotype. We functionally analysed this and a series of other mutations, using biochemical and biophysical techniques. We observe two major effects: (1) a loss of binding to δ-catenin (e.g. in the p.L270M variant), and (2) interference with the intramolecular interaction between N-terminal SPN domain and the Ank repeats. This also interferes with binding to the α-subunit of the calcium-/calmodulin dependent kinase II (αCaMKII), and appears to be associated with a more severe neurodevelopmental pathology.

Variant-specific effects define the phenotypic spectrum of HNRNPH2-associated neurodevelopmental disorders in males.

Bain type of X-linked syndromic intellectual developmental disorder, caused by pathogenic missense variants in HRNRPH2, was initially described in six female individuals affected by moderate-to-severe neurodevelopmental delay. Although it was initially postulated that the condition would not be compatible with life in males, several affected male individuals harboring pathogenic variants in HNRNPH2 have since been documented. However, functional in-vitro analyses of identified variants have not been performed and, therefore, possible genotype-phenotype correlations remain elusive. Here, we present eight male individuals, including a pair of monozygotic twins, harboring pathogenic or likely pathogenic HNRNPH2 variants. Notably, we present the first individuals harboring nonsense or frameshift variants who, similarly to an individual harboring a de novo p.(Arg29Cys) variant within the first quasi-RNA-recognition motif (qRRM), displayed mild developmental delay, and developed mostly autistic features and/or psychiatric co-morbidities. Additionally, we present two individuals harboring a recurrent de novo p.(Arg114Trp), within the second qRRM, who had a severe neurodevelopmental delay with seizures. Functional characterization of the three most common HNRNPH2 missense variants revealed dysfunctional nucleocytoplasmic shuttling of proteins harboring the p.(Arg206Gln) and p.(Pro209Leu) variants, located within the nuclear localization signal, whereas proteins with p.(Arg114Trp) showed reduced interaction with members of the large assembly of splicing regulators (LASR). Moreover, RNA-sequencing of primary fibroblasts of the individual harboring the p.(Arg114Trp) revealed substantial alterations in the regulation of alternative splicing along with global transcriptome changes. Thus, we further expand the clinical and variant spectrum in HNRNPH2-associated disease in males and provide novel molecular insights suggesting the disorder to be a spliceopathy on the molecular level.

SHANK3 conformation regulates direct actin binding and crosstalk with Rap1 signaling.

Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology, and specific biological functions of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators, and small guanosine triphosphatases (GTPases). SHANK3 is a multifunctional scaffold protein, interacting with several actin-binding proteins and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its Shank/ProSAP N-terminal (SPN) domain. Here, we demonstrate that, in addition to scaffolding actin regulators and actin-binding proteins, SHANK3 interacts directly with actin through its SPN domain. Molecular simulations and targeted mutagenesis of the SPN-ankyrin repeat region (ARR) interface reveal that actin binding is inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3, and mutation of SHANK3, resulting in reduced actin binding, augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for cell morphology and integrin activity in cancer cells. In addition, SHANK3-actin interaction regulates dendritic spine morphology in neurons and autism-linked phenotypes in vivo.

The Golgi-Associated PDZ Domain Protein Gopc/PIST Is Required for Synaptic Targeting of mGluR5.

In neuronal cells, many membrane receptors interact via their intracellular, C-terminal tails with PSD-95/discs large/ZO-1 (PDZ) domain proteins. Some PDZ proteins act as scaffold proteins. In addition, there are a few PDZ proteins such as Gopc which bind to receptors during intracellular transport. Gopc is localized at the trans-Golgi network (TGN) and binds to a variety of receptors, many of which are eventually targeted to postsynaptic sites. We have analyzed the role of Gopc by knockdown in primary cultured neurons and by generating a conditional Gopc knockout (KO) mouse line. In neurons, targeting of neuroligin 1 (Nlgn1) and metabotropic glutamate receptor 5 (mGlu5) to the plasma membrane was impaired upon depletion of Gopc, whereas NMDA receptors were not affected. In the hippocampus and cortex of Gopc KO animals, expression levels of Gopc-associated receptors were not altered, while their subcellular localization was disturbed. The targeting of mGlu5 to the postsynaptic density was reduced, coinciding with alterations in mGluR-dependent synaptic plasticity and deficiencies in a contextual fear conditioning paradigm. Our data imply Gopc in the correct subcellular sorting of its associated mGlu5 receptor in vivo.

Functional analysis of CASK transcript variants expressed in human brain.

The calcium-/calmodulin dependent serine protein kinase (CASK) belongs to the membrane-associated guanylate kinases (MAGUK) family of proteins. It fulfils several different cellular functions, ranging from acting as a scaffold protein to transcription control, as well as regulation of receptor sorting. CASK functions depend on the interaction with a variety of partners, for example neurexin, liprin-α, Tbr1 and SAP97. So far, it is uncertain how these seemingly unrelated interactions and resulting functions of CASK are regulated. Here, we show that alternative splicing of CASK can guide the binding affinity of CASK isoforms to distinct interaction partners. We report seven different variants of CASK expressed in the fetal human brain. Four out of these variants are not present in the NCBI GenBank database as known human variants. Functional analyses showed that alternative splicing affected the affinities of CASK variants for several of the tested interaction partners. Thus, we observed a clear correlation of the presence of one splice insert with poor binding of CASK to SAP97, supported by molecular modelling. The alternative splicing and distinct properties of CASK variants in terms of protein-protein interaction should be taken into consideration for future studies.

Autism-associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses.

Members of the SH3- and ankyrin repeat (SHANK) protein family are considered as master scaffolds of the postsynaptic density of glutamatergic synapses. Several missense mutations within the canonical SHANK3 isoform have been proposed as causative for the development of autism spectrum disorders (ASDs). However, there is a surprising paucity of data linking missense mutation-induced changes in protein structure and dynamics to the occurrence of ASD-related synaptic phenotypes. In this proof-of-principle study, we focus on two ASD-associated point mutations, both located within the same domain of SHANK3 and demonstrate that both mutant proteins indeed show distinct changes in secondary and tertiary structure as well as higher conformational fluctuations. Local and distal structural disturbances result in altered synaptic targeting and changes of protein turnover at synaptic sites in rat primary hippocampal neurons.

Missense mutations in CASK, coding for the calcium-/calmodulin-dependent serine protein kinase, interfere with neurexin binding and neurexin-induced oligomerization.

Mutations in the X-linked gene coding for the calcium-/calmodulin-dependent serine protein kinase (CASK) are associated with severe neurological disorders ranging from intellectual disability (in males) to mental retardation and microcephaly with pontine and cerebellar hypoplasia. CASK is involved in transcription control, in the regulation of trafficking of the post-synaptic NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and acts as a presynaptic scaffolding protein. For CASK missense mutations, it is mostly unclear which of CASK's molecular interactions and cellular functions are altered and contribute to patient phenotypes. We identified five CASK missense mutations in male patients affected by neurodevelopmental disorders. These and five previously reported mutations were systematically analysed with respect to interaction with CASK interaction partners by co-expression and co-immunoprecipitation. We show that one mutation in the L27 domain interferes with binding to synapse-associated protein of 97 kDa. Two mutations in the guanylate kinase (GK) domain affect binding of CASK to the nuclear factors CASK-interacting nucleosome assembly protein (CINAP) and T-box, brain, 1 (Tbr1). A total of five mutations in GK as well as PSD-95/discs large/ZO-1 (PDZ) domains affect binding of CASK to the pre-synaptic cell adhesion molecule Neurexin. Upon expression in neurons, we observe that binding to Neurexin is not required for pre-synaptic localization of CASK. We show by bimolecular fluorescence complementation assay that Neurexin induces oligomerization of CASK, and that mutations in GK and PDZ domains interfere with the Neurexin-induced oligomerization of CASK. Our data are supported by molecular modelling, where we observe that the cooperative activity of PDZ, SH3 and GK domains is required for Neurexin binding and oligomerization of CASK.

Targeting of δ-catenin to postsynaptic sites through interaction with the Shank3 N-terminus.

BACKGROUND: Neurodevelopmental disorders such as autism spectrum disorder (ASD) may be caused by alterations in genes encoding proteins that are involved in synapse formation and function. This includes scaffold proteins such as Shank3, and synaptic adhesion proteins such as Neurexins or Neuroligins. An important question is whether the products of individual risk genes cooperate functionally (exemplified in the interaction of Neurexin with Neuroligin isoforms). This might suggest a common pathway in pathogenesis. For the SHANK3 gene, heterozygous loss of function, as well as missense mutations have been observed in ASD cases. Several missense mutations affect the N-terminal part of Shank3 which contains the highly conserved Shank/ProSAP N-terminal (SPN) and Ankyrin repeat (Ank) domains. The role of these domains and the relevance of these mutations for synaptic function of Shank3 are widely unknown. METHODS: We used purification from a synaptic protein fraction, as well as a variety of biochemical and cell biological approaches to identify proteins which associate with the Shank3 N-terminus at postsynaptic sites. RESULTS: We report here that δ-catenin, which is encoded by CTNND2, an autism candidate gene, directly interacts with the Ank domain of Shank3 at postsynaptic sites through its Armadillo-repeat domain. The interaction is not affected by well-known posttranslational modifications of δ-catenin, i.e. by phosphorylation or palmitoylation. However, an ASD-associated mutation in the SPN domain of Shank3, L68P, significantly increases the interaction of Shank3 with δ-catenin. By analysis of postsynaptic fractions from mice, we show that the lack of SPN-Ank containing, large isoforms of Shank3 results in the loss of postsynaptic δ-catenin. Further, expression of Shank3 variants containing the N-terminal domains in primary cultured neurons significantly increased the presence of coexpressed δ-catenin at postsynaptic sites. LIMITATIONS: Work in model organisms such as mice, and in primary cultured neurons may not reproduce faithfully the situation in human brain neurons. Work in primary cultured neurons was also hampered by lack of a specific antibody for endogenous δ-catenin. CONCLUSIONS: Our data show that the interaction between Shank3 N-terminus and δ-catenin is required for the postsynaptic targeting of δ-catenin. Failure of proper targeting of δ-catenin to postsynaptic sites may contribute to the pathogenesis of autism spectrum disorder.

Characterization of agonist-dependent somatostatin receptor subtype 2 trafficking in neuroendocrine cells.

BACKGROUND: Somatostatin (SOM) receptor subtype 2 (SSTR2) is the major receptor subtype mediating SOM effects throughout the neuraxis. We previously demonstrated that the non-selective agonist [D-Trp8]-SOM induces intracellular sequestration of SSTR2, whereas this receptor is maintained at the cell surface after treatment with the SSTR2-selective agonist L-779,976 in cells co-expressing SSTR2 and SSTR5. METHODS AND RESULTS: In this study, we knocked-out SSTR5 in AtT20 cells endogenously expressing both SSTR2 and SSTR5 and used immuno-labeling and confocal microscopy to investigate the effect of SSTR5 on regulation of SSTR2 trafficking. Our results indicate that unlike [D-Trp8]-SOM-induced intracellular sequestration, L-779,976 stimulation results in the maintenance of SSTR2 at the cell surface regardless of whether SSTR5 is present or not. We then examined the trafficking pathways of SSTR2 upon stimulation by either agonist. We found that both [D-Trp8]-SOM and L-779,976 induce SSTR2 internalization via transferrin-positive vesicles. However, SSTR2 internalized upon L-779,976 treatment undergoes rapid recycling to the plasma membrane, whereas receptors internalized by [D-Trp8]-SOM recycle slowly after washout of the agonist. Furthermore, [D-Trp8]-SOM stimulation induces degradation of a fraction of internalized SSTR2 whereas L-779,976-dependent, rapid SSTR2 recycling appears to protect internalized SSTR2 from degradation. In addition, Octreotide which has preferential SSTR2 affinity, induced differential effects on both SSTR2 trafficking and degradation. CONCLUSION: Our results indicate that the biased agonistic property of L-779,976 protects against SSTR2 surface depletion by rapidly initiating SSTR2 recycling while SSTR5 does not regulate L-779-976-dependent SSTR2 trafficking.

Truncating mutations in SHANK3 associated with global developmental delay interfere with nuclear ß-catenin signaling.

Mutations in SHANK3, coding for a large scaffold protein of excitatory synapses in the CNS, are associated with neurodevelopmental disorders including autism spectrum disorders and intellectual disability (ID). Several cases have been identified in which the mutation leads to truncation of the protein, eliminating C-terminal sequences required for post-synaptic targeting of the protein. We identify here a patient with a truncating mutation in SHANK3, affected by severe global developmental delay and intellectual disability. By analyzing the subcellular distribution of this truncated form of Shank3, we identified a nuclear localization signal (NLS) in the N-terminal part of the protein which is responsible for targeting Shank3 fragments to the nucleus. To determine the relevance of Shank3 for nuclear signaling, we analyze how it affects signaling by ß-catenin, a component of the Wnt pathway. We show that full length as well as truncated variants of Shank3 interact with ß-catenin via the PDZ domain of Shank3, and the armadillo repeats of ß-catenin. As a result of this interaction, truncated forms of Shank3 and ß-catenin strictly co-localize in small intra-nuclear bodies both in 293T cells and in rat hippocampal neurons. On a functional level, the sequestration of both proteins in these nuclear bodies is associated with a strongly repressed transcriptional activation by ß-catenin owing to interaction with the truncated Shank3 fragment found in patients. Our data suggest that truncating mutations in SHANK3 may not only lead to a reduction in Shank3 protein available at postsynaptic sites but also negatively affect the Wnt signaling pathway.

Cognitive impairment and autistic-like behaviour in SAPAP4-deficient mice.

In humans, genetic variants of DLGAP1-4 have been linked with neuropsychiatric conditions, including autism spectrum disorder (ASD). While these findings implicate the encoded postsynaptic proteins, SAPAP1-4, in the etiology of neuropsychiatric conditions, underlying neurobiological mechanisms are unknown. To assess the contribution of SAPAP4 to these disorders, we characterized SAPAP4-deficient mice. Our study reveals that the loss of SAPAP4 triggers profound behavioural abnormalities, including cognitive deficits combined with impaired vocal communication and social interaction, phenotypes reminiscent of ASD in humans. These behavioural alterations of SAPAP4-deficient mice are associated with dramatic changes in synapse morphology, function and plasticity, indicating that SAPAP4 is critical for the development of functional neuronal networks and that mutations in the corresponding human gene, DLGAP4, may cause deficits in social and cognitive functioning relevant to ASD-like neurodevelopmental disorders.

International Union of Basic and Clinical Pharmacology. CV. Somatostatin Receptors: Structure, Function, Ligands, and New Nomenclature.

Somatostatin, also known as somatotropin-release inhibitory factor, is a cyclopeptide that exerts potent inhibitory actions on hormone secretion and neuronal excitability. Its physiologic functions are mediated by five G protein-coupled receptors (GPCRs) called somatostatin receptor (SST)1-5. These five receptors share common structural features and signaling mechanisms but differ in their cellular and subcellular localization and mode of regulation. SST2 and SST5 receptors have evolved as primary targets for pharmacological treatment of pituitary adenomas and neuroendocrine tumors. In addition, SST2 is a prototypical GPCR for the development of peptide-based radiopharmaceuticals for diagnostic and therapeutic interventions. This review article summarizes findings published in the last 25 years on the physiology, pharmacology, and clinical applications related to SSTs. We also discuss potential future developments and propose a new nomenclature.

Functional Relevance of Missense Mutations Affecting the N-Terminal Part of Shank3 Found in Autistic Patients.

Genetic defects in SHANK genes are associated with autism. Deletions and truncating mutations suggest haploinsufficiency for Shank3 as a major cause of disease which may be analyzed in appropriate Shank deficient mouse models. Here we will focus on the functional analysis of missense mutations found in SHANK genes. The relevance of most of these mutations for Shank function, and their role in autism pathogenesis is unclear. This is partly due to the fact that mutations spare the most well studied functional domains of Shank3, such as the PDZ and SAM domains, or the short proline-rich motifs which are required for interactions with postsynaptic partners Homer, Cortactin, dynamin, IRSp53 and Abi-1. One set of mutations affects the N-terminal part, including the highly conserved SPN domain and ankyrin repeats. Functional analysis from several groups has indicated that these mutations (e.g., R12C; L68P; R300C, and Q321R) interfere with the critical role of Shank3 for synapse formation. More recently the structural analysis of the SPN-ARR module has begun to shed light on the molecular consequences of mutations in the SPN of Shank3. The SPN was identified as a Ras association domain, with high affinities for GTP-bound, active forms of Ras and Rap. The two autism related mutations in this part of the protein, R12C and L68P, both abolish Ras binding. Further work is directed at identifying the consequences of Ras binding to Shank proteins at postsynaptic sites.

De Novo Missense Mutations in DHX30 Impair Global Translation and Cause a Neurodevelopmental Disorder.

DHX30 is a member of the family of DExH-box helicases, which use ATP hydrolysis to unwind RNA secondary structures. Here we identified six different de novo missense mutations in DHX30 in twelve unrelated individuals affected by global developmental delay (GDD), intellectual disability (ID), severe speech impairment and gait abnormalities. While four mutations are recurrent, two are unique with one affecting the codon of one recurrent mutation. All amino acid changes are located within highly conserved helicase motifs and were found to either impair ATPase activity or RNA recognition in different in vitro assays. Moreover, protein variants exhibit an increased propensity to trigger stress granule (SG) formation resulting in global translation inhibition. Thus, our findings highlight the prominent role of translation control in development and function of the central nervous system and also provide molecular insight into how DHX30 dysfunction might cause a neurodevelopmental disorder.

Heterodimerization with the ß1 subunit directs the α2 subunit of nitric oxide-sensitive guanylyl cyclase to calcium-insensitive cell-cell contacts in HEK293 cells: Interaction with Lin7a.

Nitric oxide-sensitive guanylyl cyclase is a heterodimeric enzyme consisting of an α and a ß subunit. Two different α subunits (α1 and α2) give rise to two heterodimeric enzymes α1/ß1 and α2/ß1. Both coexist in a wide range of tissues including blood vessels and the lung, but expression of the α2/ß1 form is generally much lower and approaches levels similar to the α1/ß1 form in the brain only. In the present paper, we show that the α2/ß1 form interacts with Lin7a in mouse brain synaptosomes based on co-precipitation analysis. In HEK293 cells, we found that the overexpressed α2/ß1 form, but not the α1/ß1 form is directed to calcium-insensitive cell-cell contacts. The isolated PDZ binding motif of an amino-terminally truncated α2 subunit was sufficient for cell-cell contact localization. For the full length α2 subunit with the PDZ binding motif this was only the case in the heterodimer configuration with the ß1 subunit, but not as isolated α2 subunit. We conclude that the PDZ binding motif of the α2 subunit is only accessible in the heterodimer conformation of the mature nitric oxide-sensitive enzyme. Interaction with Lin7a, a small scaffold protein important for synaptic function and cell polarity, can direct this complex to nectin based cell-cell contacts via MPP3 in HEK293 cells. We conclude that heterodimerization is a prerequisite for further protein-protein interactions that direct the α2/ß1 form to strategic sites of the cell membrane with adjacent neighbouring cells. Drugs increasing the nitric oxide-sensitivity of this specific form may be particularly effective.

Severe learning deficits of IRSp53 mutant mice are caused by altered NMDA receptor-dependent signal transduction.

Learning and memory is dependent on postsynaptic architecture and signaling processes in forebrain regions. The insulin receptor substrate protein of 53 kDa (IRSp53, also known as Baiap2) is a signaling and adapter protein in forebrain excitatory synapses. Mice deficient in IRSp53 display enhanced levels of postsynaptic N-methyl-D-aspartate receptors (NMDARs) and long-term potentiation (LTP) associated with severe learning deficits. In humans, reduced IRSp53/Baiap2 expression is associated with a variety of neurological disorders including autism, schizophrenia, and Alzheimer's disease. Here, we analyzed mice lacking one copy of the gene coding for IRSp53 using behavioral tests including contextual fear conditioning and the puzzle box. We show that a 50% reduction in IRSp53 levels strongly affects the performance in fear-evoking learning paradigms. This correlates with increased targeting of NMDARs to the postsynaptic density (PSD) in hippocampi of both heterozygous and knock out (ko) mice at the expense of extrasynaptic NMDARs. As hippocampal NMDAR-dependent LTP is enhanced in IRSp53-deficient mice, we investigated signaling cascades important for the formation of fear-evoked memories. Here, we observed a dramatic increase in cAMP response element-binding protein-dependent signaling in heterozygous and IRSp53-deficient mice, necessary for the transcriptional dependent phase of LTP. In contrast, activation of the MAPK and Akt kinase pathways required for translation-dependent phase of LTP are reduced. Our data suggest that loss or even the reduction in IRSp53 increases NMDAR-dependent cAMP responsive element-binding protein activation in the hippocampus, and interferes with the ability of mice to learn upon anxiety-related stimuli. We show here that a moderate reduction in the postsynaptic protein IRSp53 in mice leads to an increase in postsynaptic NMDA receptors. Both in heterozygous and IRSp53 deficient mice, this is associated with altered postsynaptic signal transduction, and poor performance of mice in fear-associated learning paradigms, indicating that precise control of postsynaptic NMDA receptor density is essential for memory formation.