The human polyomavirus, JCV, uses serotonin receptors to infect cells

The human polyomavirus, JCV, causes the fatal demyelinating disease progressive multifocal leukoencephalopathy in immunocompromised patients. We found that the serotonergic receptor 5HT2AR could act as the cellular receptor for JCV on human glial cells. The 5HT2Areceptor antagonists inhibited JCV infection, and monoclonal antibodies directed at 5HT2Areceptors blocked infection of glial cells by JCV, but not by SV40. Transfection of 5HT2Areceptor–negative HeLa cells with a 5HT2A receptor rescued virus infection, and this infection was blocked by antibody to the 5HT2A receptor. A tagged 5HT2A receptor colocalized with labeled JCV in an endosomal compartment following internalization. Serotonin receptor antagonists may thus be useful in the treatment of progressive multifocal leukoencephalopathy

Physics Potential of the ICAL detector at the India-based Neutrino Observatory (INO)

The upcoming 50 kt magnetized iron calorimeter (ICAL) detector at the India-based Neutrino Observatory (INO) is designed to study the atmospheric neutrinos and antineutrinos separately over a wide range of energies and path lengths. The primary focus of this experiment is to explore the Earth matter effects by observing the energy and zenith angle dependence of the atmospheric neutrinos in the multi-GeV range. This study will be crucial to address some of the outstanding issues in neutrino oscillation physics, including the fundamental issue of neutrino mass hierarchy. In this document, we present the physics potential of the detector as obtained from realistic detector simulations. We describe the simulation framework, the neutrino interactions in the detector, and the expected response of the detector to particles traversing it. The ICAL detector can determine the energy and direction of the muons to a high precision, and in addition, its sensitivity to multi-GeV hadrons increases its physics reach substantially. Its charge identification capability, and hence its ability to distinguish neutrinos from antineutrinos, makes it an efficient detector for determining the neutrino mass hierarchy. In this report, we outline the analyses carried out for the determination of neutrino mass hierarchy and precision measurements of atmospheric neutrino mixing parameters at ICAL, and give the expected physics reach of the detector with 10 years of runtime. We also explore the potential of ICAL for probing new physics scenarios like CPT violation and the presence of magnetic monopoles.Comment: 139 pages, Physics White Paper of the ICAL (INO) Collaboration, Contents identical with the version published in Pramana - J. Physic

The interaction of a constitutively active arrestin with the arrestin-insensitive 5-HT(2A) receptor induces agonist-independent internalization.

5-HT(2A) serotonin receptors are unusual among G-protein coupled receptors in that they can be internalized and desensitized, in some cell types, in an arrestin-independent manner. The molecular basis of the arrestin-insensitivity of 5-HT(2A) receptors is unknown but is probably caused, in part, by the apparent lack of agonist-induced 5-HT(2A) receptor phosphorylation. Because the arrestin-insensitivity of 5-HT(2A) receptors is cell-type selective, we used a "constitutively active" arrestin mutant that can interact with agonist-activated but nonphosphorylated receptors. We show here that this "constitutively active" arrestin mutant (Arr2-R169E) can force 5-HT(2A) receptors to be regulated by arrestins. Cotransfection of 5-HT(2A) receptors with Arr2-R169E induced agonist-independent 5-HT(2A) receptor internalization, and a constitutive translocation of the Arr2-R169E mutant to the plasma membrane, whereas wild-type Arrestin-2 had no effect. Additionally, Arr2-R169E, unlike wild-type arrestin-2, induced a significant decrease in efficacy of agonist-induced phosphoinositide hydrolysis with an unexpected increase in agonist potency. Radioligand binding assays demonstrated that the fraction of receptors in the high-affinity agonist binding-state increased with expression of Arr2-R169E, indicating that Arr2-R169E stabilizes the agonist-high affinity state of the 5-HT(2A) receptor (R*). Intriguingly, the agonist-independent interaction of Arr2-R169E with 5-HT(2A) receptors was inhibited by inverse agonist treatment and is thus probably caused by the high level of 5-HT(2A) receptor constitutive activity. This is the first demonstration that a constitutively active arrestin mutant can both induce agonist-independent internalization and stabilize the agonist-high affinity state of an arrestin-insensitive G protein coupled receptor

Long Range Effect of Mutations on Specific Conformational Changes in the Extracellular Loop 2 of Angiotensin II Type 1 Receptor

The topology of the second extracellular loop (ECL2) and its interaction with ligands is unique in each G protein-coupled receptor. When the orthosteric ligand pocket located in the transmembrane (TM) domain is occupied, ligand-specific conformational changes occur in the ECL2. In more than 90% of G protein-coupled receptors, ECL2 is tethered to the third TM helix via a disulfide bond. Therefore, understanding the extent to which the TM domain and ECL2 conformations are coupled is useful. To investigate this, we examined conformational changes in ECL2 of the angiotensin II type 1 receptor (AT1R) by introducing mutations in distant sites that alter the activation state equilibrium of the AT1R. Differential accessibility of reporter cysteines introduced at four conformation-sensitive sites in ECL2 of these mutants was measured. Binding of the agonist angiotensin II (AngII) and inverse agonist losartan in wild-type AT1R changed the accessibility of reporter cysteines, and the pattern was consistent with ligand-specific "lid" conformations of ECL2. Without agonist stimulation, the ECL2 in the gain of function mutant N111G assumed a lid conformation similar to AngII-bound wild-type AT1R. In the presence of inverse agonists, the conformation of ECL2 in the N111G mutant was similar to the inactive state of wild-type AT1R. In contrast, AngII did not induce a lid conformation in ECL2 in the loss of function D281A mutant, which is consistent with the reduced AngII binding affinity in this mutant. However, a lid conformation was induced by [Sar(1), Gln(2), Ile(8)] AngII, a specific analog that binds to the D281A mutant with better affinity than AngII. These results provide evidence for the emerging paradigm of domain coupling facilitated by long range interactions at distant sites on the same receptor

Angiotensin II-regulated microRNA 483-3p directly targets multiple components of the renin-angiotensin system

Improper regulation of signaling in vascular smooth muscle cells (VSMCs) by angiotensin II (AngII) can lead to hypertension, vascular hypertrophy and atherosclerosis. The extent to which the homeostatic levels of the components of signaling networks are regulated through microRNAs (miRNA) modulated by AngII type 1 receptor (AT(1)R) in VSMCs is not fully understood. Whether AT(1)R blockers used to treat vascular disorders modulate expression of miRNAs is also not known. To report differential miRNA expression following AT(1)R activation by AngII, we performed microarray analysis in 23 biological and technical replicates derived from humans, rats and mice. Profiling data revealed a robust regulation of miRNA expression by AngII through AT(1)R, but not the AngII type 2 receptor (AT(2)R). The AT(1)R-specific blockers, losartan and candesartan antagonized >90% of AT(1)R-regulated miRNAs and AngII-activated AT(2)R did not modulate their expression. We discovered VSMC-specific modulation of 22 miRNAs by AngII, and validated AT(1)R-mediated regulation of 17 of those miRNAs by real-time polymerase chain reaction analysis. We selected miR-483-3p as a novel representative candidate for further study because mRNAs of multiple components of the renin-angiotensin system (RAS) were predicted to contain the target sequence for this miRNA. MiR-483-3p inhibited the expression of luciferase reporters bearing 3'-UTRs of four different RAS genes and the inhibition was reversed by antagomir-483-3p. The AT(1)R-regulated expression levels of angiotensinogen and angiotensin converting enzyme 1 (ACE-1) proteins in VSMCs are modulated specifically by miR-483-3p. Our study demonstrates that the AT(1)R-regulated miRNA expression fingerprint is conserved in VSMCs of humans and rodents. Furthermore, we identify the AT(1)R-regulated miR-483-3p as a potential negative regulator of steady-state levels of RAS components in VSMCs. Thus, miRNA-regulation by AngII to affect cellular signaling is a novel aspect of RAS biology, which may lead to discovery of potential candidate prognostic markers and therapeutic targets. (C) 2014 Elsevier Ltd. All rights reserved

Caveolin-1 and Lipid Microdomains Regulate Gs Trafficking and Attenuate Gs/Adenylyl Cyclase Signaling

Lipid rafts and caveolae are specialized membrane microdomains implicated in regulating G protein-coupled receptor signaling cascades. Previous studies have suggested that rafts/caveolae may regulate β-adrenergic receptor/Gαs signaling, but underlying molecular mechanisms are largely undefined. Using a simplified model system in C6 glioma cells, this study disrupts rafts/caveolae using both pharmacological and genetic approaches to test whether caveolin-1 and lipid microdomains regulate Gs trafficking and signaling. Lipid rafts/caveolae were disrupted in C6 cells by either short-term cholesterol chelation using methyl-β-cyclodextrin or by stable knockdown of caveolin-1 and -2 by RNA interference. In imaging studies examining Gαs-GFP during signaling, stimulation with the βAR agonist isoproterenol resulted in internalization of Gαs-GFP; however, this trafficking was blocked by methyl-β-cyclodextrin or by caveolin knockdown. Caveolin knockdown significantly decreased Gαs localization in detergent insoluble lipid raft/caveolae membrane fractions, suggesting that caveolin localizes a portion of Gαs to these membrane microdomains. Methyl-β-cyclodextrin or caveolin knockdown significantly increased isoproterenol or thyrotropin-stimulated cAMP accumulation. Furthermore, forskolin- and aluminum tetrafluoride-stimulated adenylyl cyclase activity was significantly increased by caveolin knockdown in cells or in brain membranes obtained from caveolin-1 knockout mice, indicating that caveolin attenuates signaling at the level of Gαs/adenylyl cyclase and distal to GPCRs. Taken together, these results demonstrate that caveolin-1 and lipid microdomains exert a major effect on Gαs trafficking and signaling. It is suggested that lipid rafts/caveolae are sites that remove Gαs from membrane signaling cascades and caveolins might dampen globally Gαs/adenylyl cyclase/cAMP signaling

Interaction of G-Protein beta gamma Complex with Chromatin Modulates GPCR-Dependent Gene Regulation

Heterotrimeric G-protein signal transduction initiated by G-protein-coupled receptors (GPCRs) in the plasma membrane is thought to propagate through protein-protein interactions of subunits, G alpha and G beta gamma in the cytosol. In this study, we show novel nuclear functions of G beta gamma through demonstrating interaction of G beta(2) with integral components of chromatin and effects of G beta(2) depletion on global gene expression. Agonist activation of several GPCRs including the angiotensin II type 1 receptor specifically augmented G beta(2) levels in the nucleus and G beta(2) interacted with specific nucleosome core histones and transcriptional modulators. Depletion of G beta(2) repressed the basal and angiotensin II-dependent transcriptional activities of myocyte enhancer factor 2. G beta(2) interacted with a sequence motif that was present in several transcription factors, whose genome-wide binding accounted for the G beta(2)-dependent regulation of approximately 2% genes. These findings suggest a wide-ranging mechanism by which direct interaction of G beta gamma with specific chromatin bound transcription factors regulates functional gene networks in response to GPCR activation in cells

Correction: Interaction of G-Protein βγ Complex with Chromatin Modulates GPCR-Dependent Gene Regulation

<p>Correction: Interaction of G-Protein βγ Complex with Chromatin Modulates GPCR-Dependent Gene Regulation</p

3-D model of the proposed Gβ₂ interaction with MEF2A and histone H4.

<p>(a) Multiple sequence alignment using the CLUSTAL W program revealed that the phosphopeptide motif (–LLpTPPG–) was conserved in the TFs that associated with Gβ₂ (MEF2A, STAT1, STAT3 and NFAT), but not in NFκB and GATA4. (b) Surface model of Gβ₂γ₁₂ based on Gβ₁γ₂ crystal coordinates. Gβ₂γ₁₂ is shown in gray, and the common site of interaction with cytoplasmic effectors (Gα and PLCβ) is shown in teal. The –LLpTPPG– peptide (shown as red ball and stick) anchors to the central core of the β-propeller structure and makes contact with the amino acid residues shown in green and purple. The purple side chains contacting the peptide are conserved charge interactions. The histone H4 tail peptide, shown in brown, may interact on the surface of the WD7 repeat. (c) Co-immunoprecipitation of Gβ₂ with the AngII-responsive TFs, NFAT, STAT1, and STAT3, but not with GATA4 and p65 NFκB. The nuclear fractions (100 µg) prepared from HEK-AT₁R cells treated with AngII (1 µM for 30 min) were subjected to pull-down with only ProtG (−) or with a Gβ₂ antibody and ProtG (+). The immunoblot on the right shows the abundance of the respective proteins in the immunoprecipitates (− and +) and input lysates for the − and + samples. (d) Gβ₂ interaction with selective AngII-responsive TFs, suggesting a role for Gβ₂ in genome wide transcription that eventually leads to changes in cellular functions.</p

G-protein β₂ and γ₁₂ subunits are components of the nuclear proteome.

<p>(a) Schematic for the isolation of intact nuclei from the cytosol and characterization of the nuclear proteome by mass spectrometry analysis. (b) Composition of the nuclear proteome of HEK-AT₁R cells 30 min after AngII activation of AT₁R (list of proteins is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052689#pone.0052689.s004" target="_blank">Fig. S3</a>). (c) CID spectrum of the Gβ₂-specific tryptic peptide; peptide coverage is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052689#pone.0052689.s012" target="_blank">Table S1</a>. (d) CID spectrum of the Gγ₁₂-specific tryptic peptide.</p