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Available from: Fuad Bahram, Feb 13, 2014
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    • "To confirm and extend this finding, we performed in situ proximity ligation assays (PLA) with transfected HeLa cells. These assays yield a fluorescent signal only when the distance between two proteins is no more than $30 nm (Soderberg et al., 2006; Figure 1 S100B interacts with a consensus motif in the third intracellular loop of the human 5-HT 7 receptor. (A) Schematic illustration of the 5-HT 7 receptor and its third intracellular loop (IC3). "
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    ABSTRACT: The serotonin 5-HT7 receptor (5-HT7) is an emerging target for psychiatric pharmacotherapy. Recent observations in rodent models and humans suggest that its blockade mediates antidepressant efficacy. In the present study, we identify the Ca(2+)-binding protein S100B as an interacting partner of 5-HT7 and show that S100B negatively regulates inducible cyclic AMP (cAMP) accumulation in transfected HeLa cells and mouse cortical astrocytes. Overexpression of S100B causes brain region-specific dysregulation of the cAMP pathway in vivo, such that concentrations of cAMP in the frontal cortex are higher in S100B transgenic female mice compared to wild-types. Finally, S100B transgenic female mice show depressive-like behavior in the forced swim test (FST) and pharmacological blockade of 5-HT7 with SB269970 normalizes FST behavior. Taken together, our results show that S100B affects behavioral despair in female mice through functional interaction with the 5-HT7 receptor. Furthermore, we identify S100B as a cAMP-regulatory protein in cultured astrocytes and the murine frontal cortex. Future experiments will clarify whether there is a direct link between the 5-HT7-associated and cAMP-regulatory actions of S100B.
    Full-text · Article · Oct 2015 · European neuropsychopharmacology: the journal of the European College of Neuropsychopharmacology
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    • "To determine whether Mop and Hrs exist in a complex, we used a modified in situ proximity ligation assay (PLA; Söderberg et al., 2006; Jarvius et al., 2007) in the Drosophila wing disk. This assay can detect whether proteins are in close proximity to one another in vivo. "
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    ABSTRACT: Endosomal trafficking of signaling proteins plays an essential role in cellular homeostasis. The seven-pass transmembrane protein, Frizzled (Fz) is a critical component of the Wnt signaling. Although Wnt signaling is proposed to be regulated by endosomal trafficking of Fz, the molecular events that enable this regulation are not completely understood. Here, we show that the endosomal protein Myopic (Mop) regulates Fz trafficking in the Drosophila wing disk by inhibiting the ubiquitination and degradation of Hrs. Deletion of Mop or Hrs results in endosomal accumulation of Fz and therefore reduced Wnt signaling. The in situ Proximity Ligation Assay revealed a strong association between Mop and Hrs in the Drosophila wing disk. Overexpression of Hrs rescues the trafficking defect caused by mop knockdown. Mop aids in the maintenance of Ubpy which deubiquitinates (and thus stabilizes) Hrs. In the absence of the ubiquitin ligase Cbl, Mop is dispensable. These findings support a previously unknown role for Mop in endosomal trafficking of Fz in Wnt-receiving cells. © 2015 by The American Society for Cell Biology.
    Full-text · Article · Jul 2015 · Molecular biology of the cell
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    • "In this model, high RGS4 activity can limit diffusion of GTP-bound Ga i to <20 nm. This suggestion is supported by our proximity ligation assay data, which suggest preferential postsynaptic coupling of a2Rs/AMPARs and GABA B Rs/ NMDARs within 20 nm (Sö derberg et al., 2006). There is mounting anatomical evidence that synaptic proteins, including glutamate receptors, are organized into 70-to 80-nm clusters within the postsynaptic density (MacGillavry et al., 2013; Nair et al., 2013). "
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    ABSTRACT: A diverse array of neuromodulators governs cellular function in the prefrontal cortex (PFC) via the activation of G-protein-coupled receptors (GPCRs). However, these functionally diverse signals are carried and amplified by a relatively small assortment of intracellular second messengers. Here, we examine whether two distinct Gαi-coupled neuromodulators (norepinephrine and GABA) act as redundant regulators of glutamatergic synaptic transmission. Our results reveal that, within single dendritic spines of layer 5 pyramidal neurons, alpha-2 adrenergic receptors (α2Rs) selectively inhibit excitatory transmission mediated by AMPA-type glutamate receptors, while type B GABA receptors (GABABRs) inhibit NMDA-type receptors. We show that both modulators act via the downregulation of cAMP and PKA. However, by restricting the lifetime of active Gαi, RGS4 promotes the independent control of these two distinct target proteins. Our findings highlight a mechanism by which neuromodulatory microdomains can be established in subcellular compartments such as dendritic spines. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Jul 2015 · Cell Reports
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