Competition and State Aid Policy in the European Community

This article discusses the effect of European Community state aid policy on competition. First, the article defines and discusses the types of state aid under the Treaty Establishing the European Community. Second, the article analyzes the distortion of competition and effect on trade that state aids have. Third, the article discusses whether state aid qualifies for an exemption because it fulfills some other goal of the Treaty

The Ca2+/Cl− dependent L-[3H]glutamate binding: a new receptor or a particular transport process?

AbstractCa2+/Cl− increases the L-[3H]glutamate binding to rat brain synaptic membranes. It was suggested that Ca2+/Cl− expresses a new class of glutamate receptors. We report several lines of evidence suggesting that Ca2+/Cl− in fact favours a glutamate transport into membrane vesicles. This finding may serve to reconcile most of the discrepancies found in the literature on the glutamate binding and its pharmacology

The ‘magic tail’ of G protein-coupled receptors: an anchorage for functional protein networks

AbstractAll cell types express a great variety of G protein-coupled receptors (GPCRs) that are coupled to only a limited set of G proteins. This disposition favors cross-talk between transduction pathways. However, GPCRs are organized into functional units. They promote specificity and thus avoid unsuitable cross-talk. New methodologies (mostly yeast two-hybrid screens and proteomics) have been used to discover more than 50 GPCR-associated proteins that are involved in building these units. In addition, these protein networks participate in the trafficking, targeting, signaling, fine-tuning and allosteric regulation of GPCRs. To date, proteins that interact with the GPCR C-terminus are the most abundant and are the focus of this review

Cloning, expression and pharmacology of the mouse 5-HT4L receptor

AbstractSince most of our knowledge on pharmacological properties of brain 5-HT4 receptors have been discussed for mouse colliculi neurons, we cloned the corresponding receptor using the RT-PCR approach. As expected, the homology with the already cloned rat 5-HT4L receptor was high, revealing only 16 differences at the amino-acid level. One of the differences, proline75 in mouse, alanine75 in the already published rat sequences was not confirmed. Therefore this proline is part of the consensus sequence present in all 5-HT receptor transmembrane domain II (LVMP). Comparing the affinities of 11 agonists and five antagonists for the cloned mouse receptor (5-HT4L) expressed in LLCPK1 and the corresponding receptor in mouse colliculi shows an excellent correlation. The transfected mouse 5-HT4L receptor stimulated cAMP production. When expressed at high density, it exhibited intrinsic activity. In contrast to the previously described distribution, we found that mRNA encoding for both the short (5-HT4S) and the long form (5-HT4L) of 5-HT4 receptors are expressed in all mouse and rat brain areas

Homer1a-Dependent Crosstalk Between NMDA and Metabotropic Glutamate Receptors in Mouse Neurons

A large number of evidences suggest that group-I metabotropic glutamate receptors (mGluR1a, 1b, 1c, 5a, 5b) can modulate NMDA receptor activity. Interestingly, a physical link exists between these receptors through a Homer-Shank multi-protein scaffold that can be disrupted by the immediate early gene, Homer1a. Whether such a versatile link supports functional crosstalk between the receptors is unknown.Here we used biochemical, electrophysiological and molecular biological approaches in cultured mouse cerebellar neurons to investigate this issue. We found that Homer1a or dominant negative Shank3 mutants that disrupt the physical link between the receptors allow inhibition of NMDA current by group-I mGluR agonist. This effect is antagonized by pertussis toxin, but not thapsigargin, suggesting the involvement of a G protein, but not intracellular calcium stores. Also, this effect is voltage-sensitive, being present at negative, but not positive membrane potentials. In the presence of DHPG, an apparent NMDA "tail current" was evoked by large pulse depolarization, only in neurons transfected with Homer1a. Co-immunoprecipitation experiments showed interaction between G-protein betagamma subunits and NMDA receptor in the presence of Homer1a and group-I mGluR agonist.Altogether these results suggest a direct inhibition of NMDA receptor-channel by Gbetagamma subunits, following disruption of the Homer-Shank3 complex by the immediate early gene Homer1a. This study provides a new molecular mechanism by which group-I mGluRs could dynamically regulate NMDA receptor function

Plant Insecticide L-Canavanine Repels Drosophila via the Insect Orphan GPCR DmX

An orphan G-protein-coupled gustatory receptor mediates detection of the plant poison L-canavanine in fruit flies

Quantitative Phosphoproteomics Unravels Biased Phosphorylation of Serotonin 2A Receptor at Ser 280 by Hallucinogenic versus Nonhallucinogenic Agonists

International audienceThe serotonin 5-HT 2A receptor is a primary target of psy-chedelic hallucinogens such as lysergic acid diethyl-amine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT 2A receptor ago-nists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hy-drophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT 2A receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT 2A ago-nist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser 280) located in the third intracellular loop of the 5-HT 2A receptor, a region important for its desensitiza-tion. The specific phosphorylation of Ser 280 by hallucino-gens was further validated by quantitative mass spec-trometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific anti-body. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT 2A receptors at Ser 280 in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser 280 to as-partic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucino-gens to desensitize the receptor. This study reveals a biased phosphorylation of the 5-HT 2A receptor in response to hallucinogenic versus nonhallucinogenic ago-nists, which underlies their distinct capacity to desensi-tize the receptor. Molecular & Cellular Proteomics 13: 10.1074/mcp.M113.036558, 1273-1285, 2014. Among the G Protein-Coupled Receptors (GPCRs) 1 activated by serotonin (5-hydroxytryptamine, 5-HT), the 5-HT 2A receptor continues to attract particular attention in view of its broad physiological role and implication in the actions of numerous psychotropic agents (1, 2). It is a primary target of widely used atypical antipsychotics such as clozapine, risperi-done, and olanzapine, which act as antagonists or inverse agonists (1, 3). The activation of 5-HT 2A receptors expressed in the prefrontal cortex has also been implicated in the psy-cho-mimetic effects of psychedelic hallucinogens, such as lysergic acid diethylamide (LSD), mescaline, and psilocybin, which are often used to model positive symptoms of schizo-phrenia (4-8). However, these psychoactive effects are not reproduced by structurally-related agonists, such as ergota-mine and the anti-Parkinson agent lisuride, despite the fact that they exhibit comparable affinities and efficacies at 5-HT 2A receptors (7, 9). This paradox was partially resolved by the demonstration that hallucinogens induce a specific transcrip-From the ‡CNRS

Computational Modeling for the Activation Cycle of G-proteins by G-protein-coupled Receptors

In this paper, we survey five different computational modeling methods. For comparison, we use the activation cycle of G-proteins that regulate cellular signaling events downstream of G-protein-coupled receptors (GPCRs) as a driving example. Starting from an existing Ordinary Differential Equations (ODEs) model, we implement the G-protein cycle in the stochastic Pi-calculus using SPiM, as Petri-nets using Cell Illustrator, in the Kappa Language using Cellucidate, and in Bio-PEPA using the Bio-PEPA eclipse plug in. We also provide a high-level notation to abstract away from communication primitives that may be unfamiliar to the average biologist, and we show how to translate high-level programs into stochastic Pi-calculus processes and chemical reactions.Comment: In Proceedings MeCBIC 2010, arXiv:1011.005

Bright, highly water soluble triazacyclononane europium complexes to detect ligand binding with time-resolved FRET microscopy

Luminescent europium complexes are used in a broad range of applications as a result of their particular emissive properties. The synthesis and application of bright, highly water-soluble, and negatively charged sulfonic- or carboxylic acid derivatives of para-substituted aryl–alkynyl triazacyclononane complexes are described. Introduction of the charged solubilizing moieties suppresses cellular uptake or adsorption to living cells making them applicable for labeling and performing assays on membrane receptors. These europium complexes are applied to monitor fluorescent ligand binding on cell-surface proteins with time-resolved Förster resonance energy transfer (TR-FRET) assays in plate-based format and using TR-FRET microscopy