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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. Supplementary Information The online version contains supplementary material available at 10.1007/s12035-021-02504-9.

eLife 10, e66165 (1-31) (2021). doi:10.7554/eLife.66165, 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., Published by eLife Sciences Publications, Cambridge

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.

Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology and specific biological function of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators and small 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 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-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 the SHANK3-actin binding site, 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, dendritic spine morphology in neurons and autism-linked phenotypes in vivo.

ARGONAUTE-2 and associated miRNAs form the RNA-induced silencing complex (RISC), which targets mRNAs for translational silencing and degradation as part of the RNA interference pathway. Despite the essential nature of this process for cellular function, there is little information on the role of RISC components in human development and organ function. We identify 13 heterozygous mutations in AGO2 in 21 patients affected by disturbances in neurological development. Each of the identified single amino acid mutations result in impaired shRNA-mediated silencing. We observe either impaired RISC formation or increased binding of AGO2 to mRNA targets as mutation specific functional consequences. The latter is supported by decreased phosphorylation of a C-terminal serine cluster involved in mRNA target release, increased formation of dendritic P-bodies in neurons and global transcriptome alterations in patient-derived primary fibroblasts. Our data emphasize the importance of gene expression regulation through the dynamic AGO2-RNA association for human neuronal development., AGO2 binds to miRNAs to repress expression of cognate target mRNAs. Here the authors report that heterozygous AGO2 mutations result in defects in neurological development and impair RNA interference.

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.

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.

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. 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. 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.

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.

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 β-catenin, a component of the Wnt pathway. We show that full length as well as truncated variants of Shank3 interact with β-catenin via the PDZ domain of Shank3, and the armadillo repeats of β-catenin. As a result of this interaction, truncated forms of Shank3 and β-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 β-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.

Nitric oxide-sensitive guanylyl cyclase is a heterodimeric enzyme consisting of an α and a β subunit. Two different α subunits (α1 and α2) give rise to two heterodimeric enzymes α1/β1 and α2/β1. Both coexist in a wide range of tissues including blood vessels and the lung, but expression of the α2/β1 form is generally much lower and approaches levels similar to the α1/β1 form in the brain only. In the present paper, we show that the α2/β1 form interacts with Lin7a in mouse brain synaptosomes based on co-precipitation analysis. In HEK293 cells, we found that the overexpressed α2/β1 form, but not the α1/β1 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 β1 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/β1 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.

Somatostatin (SST) is a regulatory peptide hormone that acts through five different G protein-coupled receptors (SSTR1-5). Whereas expression of all five SSTR subtypes in epidermis has been shown, the biological relevance of the SST/SSTR system in the skin is completely unknown. We show here that SST is expressed in human skin and is present in a subset of Merkel cells and dendritic cells as well as in keratinocytes. We focused further on the somatostatin receptor subtype 3 (SSTR3) and its interacting protein MUPP1, as both were found to be localized at cellular junctions in epidermal keratinocytes. MUPP1 is a component of tight junctions (TJs); these cell-cell junctions contribute to barrier function of the paracellular pathway in cultured keratinocytes. We provide evidence that SSTR3 and MUPP1 interact in primary cultured human keratinocytes at high Ca(2+) conditions. Interestingly, SST, presumably via SSTR3/MUPP1, regulates TJ permeability in cultured keratinocytes. During long-term treatment of human keratinocytes, SST also affects the expression of distinct TJ proteins such as claudin-4. Our data are the first example of a peptide hormone regulating TJ functionality and composition in human keratinocytes, suggesting that control via peptide hormones provides the possibility to regulate the TJ barrier characteristics of the skin.

Jacob is a recently identified plasticity-related protein that couples N-methyl-d-aspartate receptor activity to nuclear gene expression. An expression analysis by Northern blot and in situ hybridization shows that Jacob is almost exclusively present in brain, in particular in the cortex and the limbic system. Alternative splicing gives rise to multiple mRNA variants, all of which exhibit a prominent dendritic localization in the hippocampus. Functional analysis in primary hippocampal neurons revealed that a predominant cis-acting dendritic targeting element in the 3'-untranslated region of Jacob mRNAs is responsible for dendritic mRNA localization. In the mouse brain, Jacob transcripts are associated with both the fragile X mental retardation protein, a well described trans-acting factor regulating dendritic mRNA targeting and translation, and the kinesin family member 5C motor complex, which is known to mediate dendritic mRNA transport. Jacob is susceptible to rapid protein degradation in a Ca(2+)- and Calpain-dependent manner, and Calpain-mediated clipping of the myristoylated N terminus of Jacob is required for its nuclear translocation after N-methyl-d-aspartate receptor activation. Our data suggest that local synthesis in dendrites may be necessary to replenish dendritic Jacob pools after truncation of the N-terminal membrane anchor and concomitant translocation of Jacob to the nucleus.

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.

Many G-protein-coupled receptors carry C-terminal ligand motifs for PSD-95/discs large/ZO-1 (PDZ) domains; via interaction with PDZ domain-containing scaffold proteins, this allows for integration of receptors into signaling complexes. However, the presence of PDZ domain proteins attached to intracellular membranes suggests that PDZ-type interactions may also contribute to subcellular sorting of receptors. The protein interacting specifically with Tc10 (PIST; also known as GOPC) is a trans-Golgi-associated protein that interacts through its single PDZ domain with a variety of cell surface receptors. Here we show that PIST controls trafficking of the interacting β1-adrenergic receptor both in the anterograde, biosynthetic pathway and during postendocytic recycling. Overexpression and knockdown experiments show that PIST leads to retention of the receptor in the trans-Golgi network (TGN), to the effect that overexpressed PIST reduces activation of the MAPK pathway by β1-adrenergic receptor (β1AR) agonists. Receptors can be released from retention in the TGN by coexpression of the plasma membrane-associated scaffold PSD-95, which allows for transport of receptors to the plasma membrane. Stimulation of β1 receptors and activation of the cAMP pathway lead to relocation of PIST from the TGN to an endosome-like compartment. Here PIST colocalizes with SNX1 and the internalized β1AR and protects endocytosed receptors from lysosomal degradation. In agreement, β1AR levels are decreased in hippocampi of PIST-deficient mice. Our data suggest that PIST contributes to the fine-tuning of β1AR sorting both during biosynthetic and postendocytic trafficking.

Leucine-rich repeat and PDZ [postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1] domain proteins such as scribble and Densin-180 have been implicated in the establishment of cell-cell contacts. Here, we show that Densin-180, which has been identified as a constituent of the postsynaptic density in excitatory synapses interacts with the postsynaptic scaffold protein shank (shank1-3). The interaction involves a two-point attachment of the C-terminal region of Densin-180 with the Src homology 3 domain and the N-terminal part of the proline-rich region of shank proteins. The N-terminal leucine-rich repeat region, which is not involved in binding shank, targets Densin-180 to the plasma membrane in transfected cells and to the basolateral membrane of epithelial cells. Nevertheless, coexpression of shank leads to a redirection of Densin-180 into intracellular clusters. In cultured hippocampal neurons, Densin-180 overexpression induces excessive branching of neuronal dendrites, which occurs at the expense of clusters for the postsynaptic marker PSD-95. Coexpression of shank3 abrogates branch formation and targets Densin-180 into postsynaptic clusters instead. Shank blocks binding of δ-catenin but not αCaM kinase II to Densin-180; because δ-catenin has been shown to induce branching and neurite formation, our data suggest a mechanism where shank could block the activation of a Densin-180-dependent signaling pathway by δ-catenin.

In Drosophila oocytes and neuroblasts, the double-stranded RNA binding protein Staufen assembles into ribonucleoprotein particles, which mediate cytoplasmic mRNA trafficking and translation. Two different mammalian orthologues also appear to reside in distinct RNA-containing particles. To date, relatively little is known about the molecular composition of Staufen-containing ribonucleoprotein complexes. Here, we have used a novel one-step affinity purification protocol to identify components of Staufen 1-containing particles. Whereas the nucleocytoplasmic RNA-binding protein nucleolin is linked to Staufen in an RNA-dependent manner, the association of protein phosphatase 1, the microtubule-dependent motor protein kinesin and several components of the large and small ribosomal subunits with Staufen ribonucleoprotein complexes is RNA-independent. Notably, all these components do not co-purify with a second RNA-binding protein, hnRNPK (heterogeneous ribonucleoprotein K), demonstrating the high specificity of the purification protocol. Furthermore, pull-down and immunoprecipitation experiments suggest a direct interaction between Staufen 1 and the ribosomal protein P0 in vitro as well as in cells. In cell fractionation and sucrose gradient assays, Staufen co-fractionates with intact ribosomes and polysomes, but not with the isolated 40 S ribosomal subunit. Taken together, these findings imply that, in the cytoplasm of mammalian cells, an association with the ribosomal P-stalk protein P0 recruits Staufen 1 into ribosome-containing ribonucleoprotein particles, which also contain kinesin, protein phosphatase 1 and nucleolin.

The insulin receptor substrate of 53 kDa (IRSp53) is a target of the small GTPase cdc42 which is strongly enriched in the postsynaptic density of excitatory synapses. IRSp53 interacts with the postsynaptic shank1 scaffolding molecule in a cdc42 regulated manner. The functional significance of the cdc42/IRSp53 pathway in postsynaptic sites is however, unclear. Here we identify PSD-95 as a second synaptic interaction partner of IRSp53. Interaction is mediated by a C-terminal PDZ binding motif in IRSp53 and the second PDZ domain of PSD-95. In HEK cells, overexpressed IRSp53 induces filopodia and targets PSD-95 into these processes. Immunoprecipitation and immunocytochemistry experiments demonstrate that the interaction occurs at postsynaptic sites in the brain. By virtue of its PDZ-binding and SH3 domains, IRSp53 is capable of inducing the formation of a triple complex (shank1/IRSp53/PSD-95).

Shank proteins are scaffolding proteins in the postsynaptic density of excitatory synapses in the mammalian brain. In situ hybridization revealed that Shank1/SSTRIP and Shank2/ProSAP1 mRNAs are widely expressed early in postnatal brain development whereas Shank3/ProSAP2 expression increases during postnatal development especially in the cerebellum and thalamus. Shank1 and Shank3 (but not Shank2) mRNAs are present in the molecular layers of the hippocampus, consistent with a dendritic transcript localization. Shank1 and Shank2 transcripts are detectable in the dendritic fields of Purkinje cells, whereas Shank3 mRNA is restricted to cerebellar granule cells. The appearance of dendritic Shank mRNAs in cerebellar Purkinje cells coincides with the onset of dendrite formation. Expression of reporter transcripts in hippocampal neurons identifies a 200-nucleotide dendritic targeting element (DTE) in the Shank1 mRNA. The widespread presence of Shank mRNAs in dendrites suggests a role for local synthesis of Shanks in response to stimuli that induce alterations in synaptic morphology.

By combining a Drosophila genome data base search and reverse transcriptase-PCR-based cDNA isolation, two G-protein-coupled receptors were cloned, which are the closest known invertebrate homologs of the mammalian opioid/somatostatin receptors. However, when functionally expressed in Xenopus oocytes by injection of Drosophila orphan receptor RNAs together with a coexpressed potassium channel, neither receptor was activated by known mammalian agonists. By applying a reverse pharmacological approach, the physiological ligands were isolated from peptide extracts from adult flies and larvae. Edman sequencing and mass spectrometry of the purified ligands revealed two decapentapeptides, which differ only by an N-terminal pyroglutamate/glutamine. The peptides align to a hormone precursor sequence of the Drosophila genome data base and are almost identical to allatostatin C from Manduca sexta. Both receptors were activated by the synthetic peptides irrespective of the N-terminal modification. Site-directed mutagenesis of a residue in transmembrane region 3 and the loop between transmembrane regions 6 and 7 affect ligand binding, as previously described for somatostatin receptors. The two receptor genes each containing three exons and transcribed in opposite directions are separated by 80 kb with no other genes predicted between. Localization of receptor transcripts identifies a role of the new transmitter system in visual information processing as well as endocrine regulation.

Signalling by receptors coupled to heterotrimeric G proteins primarily involves intermediates (G-protein subunits and second messengers) that can reach their targets by diffusion either through the cytosol or within the plane of the membrane. However, in many cases additional proteins have been identified which, by providing a scaffold, link several components of a signal transduction pathway in order to control speed and specificity of signalling. Scaffolding proteins such as PSD-95 or shank/ProSAP proteins organise rather large signalling complexes. Recent proteomic approaches have now been used to study the molecular composition of receptor complexes in detail, for example glutamate receptors and 5HT 2C receptors, providing a much more complicated picture of G-protein-coupled receptor signalling than previously anticipated.

Using the yeast-two-hybrid system a novel protein was identified from human brain that interacts with the C-terminus of melanin-concentrating hormone receptor 1 (MCH-R1). This protein, characterized by a Myeloid translocation protein 8, Nervy, DEAF1 proteins (MYND) zinc-finger domain, is termed MCH-R1-interacting zinc-finger protein, MIZIP. It is fully conserved in man, rat, mouse and highly conserved in Xenopus and zebrafish, but not detectable in invertebrates. MIZIP gene organization in human (six exons on chromosome 9q34.3) and mouse is highly conserved, yet in rodents an additional exon is generated giving rise to alternatively spliced mRNAs. MIZIP is expressed in brain, testis and stomach, where expression of MCH and MCH-R1 was previously reported. MIZIP interaction with MCH-R1 was verified by overlay and pull-down assays as well as by co-transfection experiments in human embryonic kidney-293 cells. MIZIP is cytoplasmically localized but gets recruted to the plasma membrane when cells are co-transfected with MCH-R1 supporting the notion that MIZIP is involved in the function of MCH-R1.

PSD-95/discs large/ZO-1 (PDZ) domain proteins integrate many G-protein coupled receptors (GPCRs) into membrane associated signalling complexes. Additional PDZ proteins are involved in intracellular receptor trafficking. We show that three PDZ proteins (SNX27, PIST and NHERF1/3) regulate the mouse somatostatin receptor subtype 5 (SSTR5). Whereas the PDZ ligand motif of SSTR5 is not necessary for plasma membrane targeting or internalization, it protects the SSTR5 from postendocytic degradation. Under conditions of lysosomal inhibition, recycling of the SSTR5 to the plasma membrane does not depend on the PDZ ligand. However, recycling of the wild type receptor carrying the PDZ binding motif depends on SNX27 which interacts and colocalizes with the receptor in endosomal compartments. PIST, implicated in lysosomal targeting of some membrane proteins, does not lead to degradation of the SSTR5. Instead, overexpressed PIST retains the SSTR5 at the Golgi. NHERF family members release SSTR5 from retention by PIST, allowing for plasma membrane insertion. Our data suggest that PDZ proteins act sequentially on the GPCR at different stages of its subcellular trafficking.

The postsynaptic density is the ultrastructural entity containing the neurotransmitter reception apparatus of excitatory synapses in the brain. A recently identified family of multidomain proteins termed Src homology 3 domain and ankyrin repeat-containing (Shank), also known as proline-rich synapse-associated protein/somatostatin receptor-interacting protein, plays a central role in organizing the subsynaptic scaffold by interacting with several synaptic proteins including the glutamate receptors. We used the N-terminal ankyrin repeats of Shank1 and -3 to search for interacting proteins by yeast two-hybrid screening and by affinity chromatography. By cDNA sequencing and mass spectrometry the cytoskeletal protein α-fodrin was identified as an interacting molecule. The interaction was verified by pull-down assays and by coimmunoprecipitation experiments from transfected cells and brain extracts. Mapping of the interacting domains of α-fodrin revealed that the highly conserved spectrin repeat 21 is sufficient to bind to the ankyrin repeats. Both interacting partners are coexpressed widely in the rat brain and are colocalized in synapses of hippocampal cultures. Our data indicate that the Shank1 and -3 family members provide multiple independent connections between synaptic glutamate receptor complexes and the cytoskeleton.

Several recent studies suggest that G protein-coupled receptors can assemble as heterodimers or hetero-oligomers with enhanced functional activity. However, inactivation of a fully functional receptor by heterodimerization has not been documented. Here we show that the somatostatin receptor (sst) subtypes sst2A and sst3 exist as homodimers at the plasma membrane when expressed in human embryonic kidney 293 cells. Moreover, in coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst2A and sst3. The sst2A-sst3 heterodimer exhibited high affinity binding to somatostatin-14 and the sst2-selective ligand L-779,976 but not to the sst3-selective ligand L-796,778. Like the sst2A homodimer, the sst2A-sst3 heterodimer stimulated guanosine 5′-3-O-(thio)triphosphate (GTPγS) binding, inhibition of adenylyl cyclase, and activation of extracellular signal-regulated kinases after exposure to the sst2-selective ligand L-779,976. However, unlike the sst3 homodimer, the sst2A-sst3 heterodimer did not promote GTPγS binding, adenylyl cyclase inhibition, or extracellular signal-regulated kinase activation in the presence of the sst3-selective ligand L-796,778. Interestingly, during prolonged somatostatin-14 exposure, the sst2A-sst3 heterodimer desensitized at a slower rate than the sst2A and sst3 homodimers. Both sst2A and sst3 homodimers underwent agonist-induced endocytosis in the presence of somatostatin-14. In contrast, the sst2A-sst3 heterodimer separated at the plasma membrane, and only sst2A but not sst3 underwent agonist-induced endocytosis after exposure to somatostatin-14. Together, heterodimerization of sst2A and sst3results in a new receptor with a pharmacological and functional profile resembling that of the sst2A receptor, however with a greater resistance to agonist-induced desensitization. Thus, inactivation of sst3 receptor function by heterodimerization with sst2A or possibly other G protein-coupled receptors may explain some of the difficulties in detecting sst3-specific binding and signaling in mammalian tissues.

Interaction between the C terminus of a G-protein-coupled receptor and intracellular constituents may represent a crucial step in regulating signal transduction. To identify potential interacting candidates the C terminus of the somatostatin receptor subtype 1 was used as bait in a yeast two hybrid screen of a human brain cDNA library. We identified the human Skb1 sequence (Skb1Hs) as interacting protein, which is homologous to the yeast protein known Skb1 to down-regulate mitosis in Schizosaccharomyces pombe via binding to the Shk1 protein kinase; the latter is a homolog to the mammalian p21(cdc42/Rac)-activated protein kinases. Interaction required almost the entire C terminus of the somatostatin receptor subtype 1 including the conserved NPXXY motif of transmembrane region seven; in the case of the Skb1Hs most of the N terminus and an S-adenosylmethionine binding domain were mandatory, whereas the C terminus was not essential. Interaction was verified by coexpression experiments in human embryonic kidney cells. As revealed by immunocytochemical analysis Skb1Hs expressed alone aggregates in large cytosolic clusters. When coexpressed, receptor subtype 1 and Skb1Hs were colocalized at the cell surface; these cells showed a strong increase in somatostatin binding compared with cells expressing the receptor only. This may suggest that Skb1Hs acts like a chaperone by correctly targeting the receptor to the cell surface.

Internalization of G protein-coupled receptors is crucial for resensitization of phosphorylation-desensitized receptors, but also for their long term desensitization through sequestration. To elucidate the mechanisms regulating cell surface availability of the somatostatin (SRIF) receptor subtype sst5, we characterized its internalization properties in transfected COS-7 cells using biochemical, confocal microscopic, and electron microscopic techniques. Our results demonstrated rapid and efficient sequestration of specifically bound[ 125I]Tyr0-d-Trp8-SRIF (up to 45% of bound radioactivity). Combined immunocytochemical detection of sst5 and visualization of a fluorescent SRIF analog by confocal microscopy revealed that whereas the internalized ligand progressively clustered toward the cell center with time, immunoreactive receptors remained predominantly associated with the plasma membrane. The preservation of cell surface receptors was confirmed by binding experiments on whole cells revealing a lack of sat...

By using the yeast two-hybrid system we identified a novel protein from the human brain interacting with the C terminus of somatostatin receptor subtype 2. This protein termed somatostatin receptor interacting protein is characterized by a novel domain structure, consisting of six N-terminal ankyrin repeats followed by SH3 and PDZ domains, several proline-rich regions, and a C-terminal sterile alpha motif. It consists of 2185 amino acid residues encoded by a 9-kilobase pair mRNA; several splice variants have been detected in human and rat cDNA libraries. Sequence comparison suggests that the novel multidomain protein, together with cortactin-binding protein, forms a family of cytoskeletal anchoring proteins. Fractionation of rat brain membranes indicated that somatostatin receptor interacting protein is enriched in the postsynaptic density fraction. The interaction of somatostatin receptor subtype 2 with its interacting protein was verified by overlay assays and coimmunoprecipitation experiments from transfected human embryonic kidney cells. Somatostatin receptor subtype 2 and the interacting protein display a striking overlap of their expression patterns in the rat brain. Interestingly, in the hippocampus the mRNA for somatostatin receptor interacting protein was not confined to the cell bodies but was also observed in the molecular layer, suggesting a dendritic localization of this mRNA.

We investigated the 5′-flanking promoter region of the rat somatostatin receptor subtype 3 (rSSTR3). Using a cDNA probe, genomic clones containing the 5′-flanking promoter region of the rSSTR3 gene were isolated. A sequence of 5.4 kb directly upstream from the start codon was analyzed and two introns were found in the 5′ untranslated region (UTR) of the cDNA sequence. The transcriptional initiation site was determined by 5′ rapid amplification of cDNA ends (RACE), primer extension and RNase protection analysis with cerebellar RNA. Two major transcriptional initiation sites were found at position −1040 (tsp1) and −856 (tsp2) relative to the translational initiation site. Like a number of other promoters of G-protein-coupled receptors, the rSSTR3 gene lacks TATA and CAAT motifs and includes G+C-rich regions. Functional analysis of the promoter region by transfecting rSSTR3 luciferase-reporter gene constructs into rat pituitary GH3 cells and HEK 293 cells indicated that a 107-bp region upstream of tsp2 was sufficient to drive transcription. Furthermore a 562-bp region at position −1304 to −1865 upstream of the ATG start codon exerted a negative regulatory effect on transcriptional activity.

A sequence motif of 20 amino acid residues within the C-terminal portion of the rat somatostatin receptor subtype 4 (SSTR4) has been shown to prevent rapid agonist-dependent receptor internalization in transfected human embryonic kidney (HEK) cells. Molecular dissection of this motif by biochemical ligand-binding assays revealed that the block was released by mutating a single residue (threonine 331) to an alanine. These data are in line with confocal microscopic analysis of cultured primary neurons microinjected with cDNA constructs encoding either SSTR4 or the mutant T331A. Immunocytochemical analysis showed that the mutant receptor, but not SSTR4, was internalized. However, internalized T331A was not recycled to the cell surface, suggesting that it lacks sequence elements that determine intracellular sorting after endocytosis. Neither wildtype SSTR nor the mutant T331A exhibited functional desensitization when assayed for their ability to inhibit adenylate cyclase. In agreement with this, the wt receptor and its mutant were not phosphorylated in response to agonist treatment. Lack of desensitization of SSTR4 has been electrophysiologically verified by coexpressing the receptor with a G-protein-gated, inwardly rectifying potassium channel in Xenopus oocytes. A strong somatostatin 14 (SST14)-activated inward potassium current was observed that was long-lasting and which decayed only slowly after washout of the agonist. This is in contrast to another somatostatin receptor subtype, SSTR3, which mediates rapidly desensitizing currents. Binding experiments on HEK cells transfected with either SSTR3 or 4 indicated that this difference is not attributable to slow dissociation of the agonist from the receptor, suggesting that SSTR4 mediates long-lasting signalling, a property which may be relevant for clinical therapy.

Agonist-dependent internalization of the rat somatostatin receptor subtype 3 (SSTR3) requires four hydroxyl amino acids (Ser341, Ser346, Ser351, and Thr357) in the receptor C terminus (Roth, A., Kreienkamp, H.-J., Nehring, R., Roostermann, D., Meyerhof, W. and Richter, D. (1997) DNA Cell Biol. 16, 111-119). Here we report on the molecular mechanism responsible for the endocytotic process by analyzing the agonist-dependent phosphorylation of wild-type and mutant receptors expressed in human embryonic kidney cells. Wild-type SSTR3 is phosphorylated in response to agonist treatment. Phosphorylation is markedly reduced in a S341A/S346A/S351A triple mutant and is also reduced, but to a lesser extent, in the T357A point mutant. Internalization of the wild-type receptor is preceded by a functional desensitization of the receptor; in contrast, the triple serine mutant does not desensitize after treatment with agonists as assayed by its ability to inhibit forskolin-stimulated adenylate cyclase activity. After internalization via a clathrin-coated vesicle mediated endocytotic pathway, SSTR3 efficiently recycles to the cell surface, suggesting that agonist mediated endocytosis is necessary for the functional resensitization of a phosphorylated and desensitized receptor.

The molecular evolution of the opioid receptor family has been studied by isolating cDNAs that encode six distinct opioid receptor-like proteins from a lower vertebrate, the teleost fish Catostomus commersoni . One of these, which has been obtained in full-length form, encodes a 383-amino acid protein that exhibits greatest sequence similarity to mammalian μ-opioid receptors; the corresponding gene is expressed predominantly in brain and pituitary. Transfection of the teleost cDNA into HEK 293 cells resulted in the appearance of a receptor having high affinity for the μ-selective agonist [ d -Ala 2 , MePhe 4 -Gly-ol 5 ]enkephalin (DAMGO) ( K d = 0.63 ± 0.15 nM) and for the nonselective antagonist naloxone ( K d = 3.1 ± 1.3 nM). The receptor had negligible affinity for U50488 and [ d -Pen 2 , d -Pen 5 ]enkephalin (DPDPE), which are κ- and δ-opioid receptor selective agonists, respectively. Stimulation of transfected cells with 1 μM DAMGO lowered forskolin-induced cAMP levels, an effect that could be reversed by naloxone. Experiments in Xenopus oocytes have demonstrated that the fish opioid receptor can, in an agonist-dependent fashion, activate a coexpressed mouse G-protein-gated inward-rectifying potassium channel (GIRK1). The identification of six distinct fish opioid receptor-like proteins suggests that additional mammalian opioid receptors remain to be identified at the molecular level. Furthermore, our data indicate that the μ-opioid receptor arose very early in evolution, perhaps before the appearance of vertebrates, and that the pharmacological and functional properties of this receptor have been conserved over a period of ≈400 million years implying that it fulfills an important physiological role.

Shank/ProSAP proteins are major scaffold proteins of the postsynaptic density; mutations in the human SHANK3 gene are associated with intellectual disability or autism spectrum disorders. We have analyzed the functional relevance of several SHANK3 missense mutations affecting the N-terminal portion of the protein by expression of wild-type and mutant Shank3 in cultured neurons and by binding assays in heterologous cells. Postsynaptic targeting of recombinant Shank3 was unaltered. In electrophysiological experiments, both wild-type and L68P mutant forms of Shank3 were equally effective in restoring synaptic function after knockdown of endogenous Shank3. We observed that several mutations affected binding to interaction partners of the Shank3 ankyrin repeat region. One of these mutations, L68P, improved binding to both ligands. Leu-68 is located N-terminal to the ankyrin repeats, in a highly conserved region that we identify here as a novel domain termed the Shank/ProSAP N-terminal (SPN) domain. We show that the SPN domain interacts with the ankyrin repeats in an intramolecular manner, thereby restricting access of either Sharpin or α-fodrin. The L68P mutation disrupts this blockade, thus exposing the Shank3 ankyrin repeat region to its ligands. Our data identify a new type of regulation of Shank proteins and suggest that mutations in the SHANK3 gene do not necessarily induce a loss of function, but may represent a gain of function with respect to specific interaction partners. Background: Missense mutations in the SHANK3 gene have been detected in autism patients. Results: A mutation in the conserved SPN region of Shank3 improves ligand binding to the ankyrin repeats. Conclusion: The SPN domain regulates accessibility of the ankyrin repeats through an intramolecular interaction. Significance: Autism-associated mutations of Shank3 result in gain-of-function with respect to specific interaction partners.

Dendritic targeting of mRNAs encoding the microtubule-associated protein 2 (MAP2) in neurons involves a cis-acting dendritic targeting element. Two rat brain proteins, MAP2-RNA trans-acting protein (MARTA)1 and MARTA2, bind to the cis-element with both high affinity and specificity. In this study, affinity-purified MARTA2 was identified as orthologue of human far-upstream element binding protein 3. In neurons, it resides in somatodendritic granules and dendritic spines and associates with MAP2 mRNAs. Expression of a dominant-negative variant of MARTA2 disrupts dendritic targeting of endogenous MAP2 mRNAs, while not noticeably altering the level and subcellular distribution of polyadenylated mRNAs as a whole. Finally, MAP2 transcripts associate with the microtubule-based motor KIF5 and inhibition of KIF5, but not cytoplasmic dynein function disrupts extrasomatic trafficking of MAP2 mRNA granules. Thus, in neurons MARTA2 appears to represent a key trans-acting factor involved in KIF5-mediated dendritic targeting of MAP2 mRNAs.

Sequences of the alpha-subunits of the nicotinic acetylcholine receptor from the snake and mongoose contain several differences in the region between amino acids 183 and 200. Receptors from both of these species reveal resistance to the snake alpha-toxins presumably arising as a protective evolutionary mechanism. Sequence differences include the added glycosylation signals at residue 187 in the mongoose and at residues 189 and 111 in snake. Although previous observations with peptides and fusion proteins either synthesized chemically or in a bacterial expression system indicate that certain amino acid residues may contribute to the resistance, our findings with the intact receptor in an eukaryotic expression system indicate the major role for glycosylation. In this study, we show that addition of glycosylation signals gives rise to virtually complete glycosylation at the added sites, although heterogeneity of oligosaccharide processing is evident. By analysis of combinations of mutants, we document that glycosylation exerts the predominant influence on alpha-toxin binding. Substitutions at other residues are largely without influence as single mutations but appear to decrease affinity further in multiple mutants, particularly where the receptor is glycosylated at the 187 and 189 positions. Glycosylation exerts a major influence on the dissociation as well as the association rates of the alpha-toxin-receptor complex, suggesting that the decrease for alpha-toxin affinity is not simply a consequence of restricted diffusional access, rather glycosylation affects the conformation and stability of the bound complex.

Selective targeting of specific mRNAs into neuronal dendrites and their locally regulated translation at particular cell contact sites contribute to input-specific synaptic plasticity. Thus, individual synapses become decision-making units, which control gene expression in a spatially restricted and nucleus-independent manner. Dendritic targeting of mRNAs is achieved by active, microtubule-dependent transport. For this purpose, mRNAs are packaged into large ribonucleoprotein (RNP) particles containing an array of trans-acting RNA-binding proteins. These are attached to molecular motors, which move their RNP cargo into dendrites. A variety of proteins may be synthesized in dendrites, including signalling and scaffold proteins of the synapse and neurotransmitter receptors. In some cases, such as the alpha subunit of the calcium/calmodulin-dependent protein kinase II (αCaMKII) and the activity-regulated gene of 3.1 kb (Arg3.1, also referred to as activity-regulated cDNA, Arc), their local synthesis at synapses can modulate long-term changes in synaptic efficiency. Local dendritic translation is regulated by several signalling cascades including Akt/mTOR and Erk/MAP kinase pathways, which are triggered by synaptic activity. More recent findings show that miRNAs also play an important role in protein synthesis at synapses. Disruption of local translation control at synapses, as observed in the fragile X syndrome (FXS) and its mouse models and possibly also in autism spectrum disorders, interferes with cognitive abilities in mice and men.

Studies with site-directed labeling reagents have identified residues near the ligand binding pocket of the nicotinic acetylcholine receptor. Among these residues are three conserved tyrosines, Tyr-93, Tyr-190, and Tyr-198 of the alpha subunit. Previous studies combined mutagenesis, expression in Xenopus oocytes, and dose-response analysis to examine contributions of these tyrosines to agonist affinity. In this study, we prepared a series of mutants at each position, expressed them in 293 HEK cells, and studied binding of agonists and antagonists to mutant receptors on intact cells. We show that all three tyrosines contribute to binding of agonists, and that each tyrosine contributes roughly equally to the binding energy. Although the contributions are roughly equivalent, the nature of the contribution is not equivalent at each position. For Tyr-93 and Tyr-190 the aromatic hydroxyl is essential, whereas for Tyr-198 aromaticity of the side chain is essential. Nearly identical results were obtained for the elementary quaternary ligand tetramethylammonium, indicating that these tyrosines contribute to stabilization of the quaternary ammonium portion of agonist. Tyr-190 and Tyr-198 also contribute to binding of the competitive antagonist dimethyl-d-tubocurarine; the side chain specificity for binding supports tyrosine interactions with one of two quaternary ammonium groups in dimethyl-d-tubocurarine. Y190F, in addition to altering binding affinity, also affects the equilibrium between activatable and desensitized receptor states.

Sequence analysis reveals unique features in the alpha-subunit of nicotinic acetylcholine receptors from the alpha-toxin-resistant cobra and mongoose. Included are N-linked glycosylation signals just amino-terminal to the Tyr190, Cys192-Cys193 region of the ligand binding domain, substitution of Trp187 and Phe189 by non-aromatic residues and alteration of the proline sequence Pro194-X-X-Pro197. Glycosylation signals were inserted into the toxin-sensitive mouse alpha-subunit by the mutations F189N and W187N/F189T. The F189N alpha-subunit, when transfected with beta, gamma and delta, showed a 140-fold loss of alpha-bungarotoxin affinity, whereas the W187N/F189T double mutation exhibited a divergence in alpha-toxin affinities at the two sites, one class showing a 600-fold and the other showing an 11-fold reduction. The W187N mutant and the double mutant F189N/S191A lacking the requisite glycosylation signals exhibited little alteration in affinity, as did the P194L and P197H mutations. The glycosylation sites had little or no influence on binding of toxins of intermediate (alpha-conotoxin, 1500 Da) or small mass (lophotoxin, 500 Da) and of the agonist, carbamylcholine. The two sites for the binding of alpha-conotoxin M1 have widely divergent dissociation constants of 2.1 and 14,800 nM. Expression of alpha/gamma- and alpha/delta-subunit pairs indicated that the high and low affinity sites are formed by the alpha/delta and alpha/gamma contacts, respectively.

Functional absence of fragile X mental retardation protein (FMRP) causes the fragile X syndrome, a hereditary form of mental retardation characterized by a change in dendritic spine morphology. The RNA-binding protein FMRP has been implicated in regulating postsynaptic protein synthesis. Here we have analyzed whether the abundance of scaffold proteins and neurotransmitter receptor subunits in postsynaptic densities (PSDs) is altered in the neocortex and hippocampus of FMRP-deficient mice. Whereas the levels of several PSD components are unchanged, concentrations of Shank1 and SAPAP scaffold proteins and various glutamate receptor subunits are altered in both adult and juvenile knock-out mice. With the exception of slightly increased hippocampal SAPAP2 mRNA levels in adult animals, altered postsynaptic protein concentrations do not correlate with similar changes in total and synaptic levels of corresponding mRNAs. Thus, loss of FMRP in neurons appears to mainly affect the translation and not the abundance of particular brain transcripts. Semi-quantitative analysis of RNA levels in FMRP immunoprecipitates showed that in the mouse brain mRNAs encoding PSD components, such as Shank1, SAPAP1–3, PSD-95, and the glutamate receptor subunits NR1 and NR2B, are associated with FMRP. Luciferase reporter assays performed in primary cortical neurons from knock-out and wild-type mice indicate that FMRP silences translation of Shank1 mRNAs via their 3′-untranslated region. Activation of metabotropic glutamate receptors relieves translational suppression. As Shank1 controls dendritic spine morphology, our data suggest that dysregulation of Shank1 synthesis may significantly contribute to the abnormal spine development and function observed in brains of fragile X syndrome patients.

Dendritic mRNA transport coupled with local regulation of translation enables neurons to selectively alter the protein composition of individual postsynaptic sites. We have analyzed dendritic localization of shank1 mRNAs; shank proteins (shank1-3) are scaffolding molecules of the postsynaptic density (PSD) of excitatory synapses, which are crucial for PSD assembly and the formation of dendritic spines. Live cell imaging demonstrates saltatory movements of shank1 mRNA containing granules along microtubules in both anterograde and retrograde directions. A population of brain messenger ribonucleoprotein particles (mRNPs) containing shank1 mRNAs associates with the cargo-binding domain of the motor protein KIF5C. Through expression of dominant negative proteins, we show that dendritic targeting of shank1 mRNA granules involves KIF5C and the KIF5-associated RNA-binding protein staufen1. While transport of shank1 mRNAs follows principles previously outlined for other dendritic transcripts, shank1 mRNAs are distinguished by their translational regulation. Translation is strongly inhibited by a GC-rich 5(')untranslated region; in addition, internal ribosomal entry sites previously detected in other dendritic transcripts are absent in the shank1 mRNA. A concept emerges from our data in which dendritic transport of different mRNAs occurs collectively via a staufen1- and KIF5-dependent pathway, whereas their local translation is controlled individually by unique cis-acting elements.

IRSp53 is an essential intermediate between the activation of Rac and Cdc42 GTPases and the formation of cellular protrusions; it affects cell shape by coupling membrane-deforming activity with the actin cytoskeleton. IRSp53 is highly expressed in neurons where it is also an abundant component of the postsynaptic density (PSD). Here we analyze the physiological function of this protein in the mouse brain by generating IRSp53-deficient mice. Neurons in the hippocampus of young and adult knock-out (KO) mice do not exhibit morphological abnormalities in vivo. Conversely, primary cultured neurons derived from IRSp53 KO mice display retarded dendritic development in vitro. On a molecular level, Eps8 cooperates with IRSp53 to enhance actin bundling and interacts with IRSp53 in developing neurons. However, postsynaptic Shank proteins which are expressed at high levels in mature neurons compete with Eps8 to block actin bundling. In electrophysiological experiments the removal of IRSp53 increases synaptic plasticity as measured by augmented long term potentiation and paired-pulse facilitation. A primarily postsynaptic role of IRSp53 is underscored by the decreased size of the PSDs, which display increased levels of N-methyl-d-aspartate receptor subunits in IRSp53 KO animals. Our data suggest that the incorporation of IRSp53 into the PSD enables the protein to limit the number of postsynaptic glutamate receptors and thereby affect synaptic plasticity rather than dendritic morphology. Consistent with altered synaptic plasticity, IRSp53-deficient mice exhibit cognitive deficits in the contextual fear-conditioning paradigm.

The regulation of filopodia plays a crucial role during neuronal development and synaptogenesis. Axonal filopodia, which are known to originate presynaptic specializations, are regulated in response to neurotrophic factors. The structural components of filopodia are actin filaments, whose dynamics and organization are controlled by ensembles of actin-binding proteins. How neurotrophic factors regulate these latter proteins remains, however, poorly defined. Here, using a combination of mouse genetic, biochemical, and cell biological assays, we show that genetic removal of Eps8, an actin-binding and regulatory protein enriched in the growth cones and developing processes of neurons, significantly augments the number and density of vasodilator-stimulated phosphoprotein (VASP)-dependent axonal filopodia. The reintroduction of Eps8 wild type (WT), but not an Eps8 capping-defective mutant, into primary hippocampal neurons restored axonal filopodia to WT levels. We further show that the actin barbed-end capping activity of Eps8 is inhibited by brain-derived neurotrophic factor (BDNF) treatment through MAPK-dependent phosphorylation of Eps8 residues S624 and T628. Additionally, an Eps8 mutant, impaired in the MAPK target sites (S624A/T628A), displays increased association to actin-rich structures, is resistant to BDNF-mediated release from microfilaments, and inhibits BDNF-induced filopodia. The opposite is observed for a phosphomimetic Eps8 (S624E/T628E) mutant. Thus, collectively, our data identify Eps8 as a critical capping protein in the regulation of axonal filopodia and delineate a molecular pathway by which BDNF, through MAPK-dependent phosphorylation of Eps8, stimulates axonal filopodia formation, a process with crucial impacts on neuronal development and synapse formation.

Somatostatin (SST) inhibits the secretion of many peptide hormones including growth hormone (GH). The various functions of SST are mediated through at least five different receptor subtypes (SSTR1–5), their precise physiological roles have not been solved yet. Here we report on studies concerning the functional role of SSTR1 in the modulation of GH release from somatotrophs. Primary cell cultures from pituitaries of wild-type SSTR1 mice exposed to the SSTR1 selective somatostatin analog CH-275 show reduction of basal levels of GH secretion whereas somatotrophs isolated from SSTR1 null mutant mice did not respond to the agonist-mediated effect. This suggests that SSTR1 is involved in modulating basal GH levels in primary pituitary cell cultures and, together with SSTR2, may control the secretion of GH in the body.

Congenital stationary night blindness (CSNB) is a non-progressive Mendelian condition resulting from a functional defect in rod photoreceptors. A small number of unique missense mutations in the genes encoding various members of the rod phototransduction cascade, e.g. rhodopsin (RHO), cGMP phosphodiesterase beta-subunit (PDE6B), and transducin alpha-subunit (GNAT1) have been reported to cause autosomal dominant (ad) CSNB. While the RHO and PDE6B mutations result in constitutively active proteins, the only known adCSNB-associated GNAT1 change (p.Gly38Asp) produces an alpha-transducin that is unable to activate its downstream effector molecule in vitro. In a multigeneration Danish family with adCSNB, we identified a novel heterozygous C to G transversion (c.598C>G) in exon 6 of GNAT1 that should result in a p.Gln200Glu substitution in the evolutionarily highly conserved Switch 2 region of alpha-transducin, a domain that has an important role in binding and hydrolyzing GTP. Computer modeling based on the known crystal structure of transducin suggests that the p.Gln200Glu mutant exhibits impaired GTPase activity, and thereby leads to constitutive activation of phototransduction. This assumption is in line with our results of trypsin protection assays as well as previously published biochemical data on mutants of this glutamine in the GTPase active site of alpha-transducin following in vitro expression, and observations that inappropriately activating mutants of various members of the rod phototransduction cascade represent one of the major molecular causes of adCSNB.

Formation of postsynaptic specializations requires the positioning of a huge macromolecular complex, the postsynaptic density, at the tip of the dendritic spine. Assembly of this protein complex is enabled by tight control of intra- and intermolecular interactions between PSD scaffold proteins. In parallel, rearrangements of the actin cytoskeleton are necessary to generate a spine and bring it into its mature form. Actin treadmilling in spines is controlled by small GTPases of the rho family via a range of downstream effectors, including PAK, WAVE, and IRSp53 proteins. Aberrant spine formation is a hallmark of the pathology of inherited forms of mental retardation (MR), and proteins of the rho G-protein cycle are mutated in specific forms of MR. In addition, the loss of the RNA-binding protein FMRP in patients suffering from fragile X MR syndrome suggests that translational control at or near synapses may also contribute to the correct assembly of the postsynaptic apparatus.

The five different rat somatostatin receptor subtypes (SSTR1–SSTR5) were coexpressed with a subunit of G-protein gated inwardly rectifying potassium channel (GIRK1) in Xenopus oocytes. SSTR2–SSTR5, but not SSTR1 coupled efficiently to the activation of GIRK currents when stimulated by SST14 or SST28. A comparison of the dose-response curves and of the maximum currents obtained indicates that SSTR2 couples most efficiently to this effector, supporting the notion that SSTR2 is involved in activation of potassium conductances by SST in vivo.

The multi-domain protein PIST (protein interacting specifically with Tc10) interacts with the SSTR5 (somatostatin receptor 5) and is responsible for its intracellular localization. Here, we show that PIST is expressed in pancreatic β-cells and interacts with SSTR5 in these cells. PIST expression in MIN6 insulinoma cells is reduced by somatostatin (SST). After stimulation with SST, SSTR5 undergoes internalization together with PIST. MIN6 cells over-expressing PIST display enhanced glucose-stimulated insulin secretion and a decreased sensitivity to SST-induced inhibition of insulin secretion. These data suggest that PIST plays an important role in insulin secretion by regulating SSTR5 availability at the plasma membrane.

The physiological responses of somatostatin are mediated by five different G protein-coupled receptors. Although agonist-induced endocytosis of the various somatostatin receptor subtypes (sst(1)-sst(5)) has been studied in detail, little is known about their postendocytic trafficking. Here we show that somatostatin receptors profoundly differ in patterns of beta-arrestin mobilization and endosomal sorting. The beta-arrestin-dependent trafficking of the sst(2A) somatostatin receptor resembled that of a class B receptor in that upon receptor activation, beta-arrestin and the receptor formed stable complexes and internalized together into the same endocytic vesicles. This pattern was dependent on GRK2 (G protein-coupled receptor kinase 2)-mediated phosphorylation of a cluster of phosphate acceptor sites within the cytoplasmic tail of the sst(2A) receptor. Unlike other class B receptors, however, the sst(2A) receptor was rapidly resensitized and recycled to the plasma membrane. The beta-arrestin mobilization of the sst(3) and the sst(5) somatostatin receptors resembled that of a class A receptor in that upon receptor activation, beta-arrestin and the receptor formed relatively unstable complexes that dissociated at or near the plasma membrane. Consequently, beta-arrestin was excluded from sst(3)-containing vesicles. Unlike other class A receptors, a large proportion of sst(3) receptors was subject to ubiquitin-dependent lysosomal degradation and did not rapidly recycle to the plasma membrane. The sst(4) somatostatin receptor is unique in that it did not exhibit agonist-dependent receptor phosphorylation and beta-arrestin recruitment. Together, these findings may provide important clues about the regulation of receptor responsiveness during long-term administration of somatostatin analogs.

Amphiphysins are SH3 domain-containing proteins thought to function in clathrin-mediated endocytosis. To investigate the potential role of amphiphysin II in cellular trafficking of G protein-coupled somatostatin (SRIF) receptors, we generated an AtT-20 cell line stably overexpressing amphiphysin IIb, a splice variant that does not bind clathrin. Endocytosis of (125)I-[d-Trp(8)]SRIF was not affected by amphiphysin IIb overexpression. However, the maximal binding capacity (B(max)) of the ligand on intact cells was significantly lower in amphiphysin IIb overexpressing than in non-transfected cells. This difference was no longer apparent when the experiments were performed on crude cell homogenates, suggesting that amphiphysin IIb overexpression interferes with SRIF receptor targeting to the cell surface and not with receptor synthesis. Accordingly, immunofluorescence experiments demonstrated that, in amphiphysin overexpressing cells, sst(2A) and sst(5) receptors were segregated in a juxtanuclear compartment identified as the trans-Golgi network. Amphiphysin IIb overexpression had no effect on corticotrophin-releasing factor 41-stimulated adrenocorticotropic hormone secretion, suggesting that it is not involved in the regulated secretory pathway. Taken together, these results suggest that amphiphysin II is not necessary for SRIF receptor endocytosis but is critical for its constitutive targeting to the plasma membrane. Therefore, amphiphysin IIb may be an important component of the constitutive secretory pathway.

We identified and cloned the mouse orthologue of human GPR6 as a new member of the lysophospholipid-receptor family. Sphingosine-1-phosphate (S1P) activated GPR6, transiently expressed in frog oocytes or in Chinese hamster ovary (CHO) cells, with high specificity and nanomolar affinity. The GPR6 gene was found to be located on chromosome 10B1 and a single exon coded for the entire open-reading frame. Signal transduction of S1P was inhibited by pertussis toxin, suggesting a coupling of GPR6 to an inhibitory G protein. In CHO cells transfected with GPR6, the sphingosine-kinase pathway mediated Ca2+ mobilization from internal stores. Apoptotic cell death was induced by serum deprivation or H2O2 treatment and was prevented by S1P in GPR6-, but not in vector-transfected CHO cells. The antiapoptotic effect of S1P required activation of sphingosine kinase and was accompanied by an increase in MAP-kinase phosphorylation.