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Bursicon bioactivity is essential for tanning of the exoskeleton and for wing spreading behavior that occur in newly emerged adult insects. Previously, we demonstrated that in the fruit fly, Drosophila melanogaster, bursicon exists as a heterodimeric cystine knot protein that activates the leucine-rich repeats containing G protein-coupled receptor 2 (DLGR2). By performing similarity based in silico searches in genomic and complementary DNA databases, we identified bursicon homologous sequences in several protostomian as well as deuterostomian invertebrates. In the genome of the honeybee, Apis mellifera, the coding regions for bursicon cystine knot subunits are organized in a genomic locus of approximately 4 kilobase pairs. Reverse transcription PCR analysis indicates that this region likely codes for two distinct bursicon cystine knot subunits. Our results illustrate the remarkable conservation of bursicon in invertebrate species and provide an avenue for functional analyses of this hormone in a wide range of animal species. ispartof: General and Comparative Endocrinology vol:153 issue:1 pages:59-63 ispartof: location:ENGLAND, Univ Sheffield, Sheffield status: published

The animal olfactory system represents the gold standard of olfactory biosensors with its capability to identify and discriminate thousands of odorant compounds. In order to mimic the performances of natural olfactory sensors it is necessary to develop methods and techniques for the production, immobilization and electrical characterization of olfactory receptors. We review in this paper some of the advances we obtained in these fields. (c) 2006 Elsevier B.V. All rights reserved.

The animal olfactory system represents the gold standard of olfactory biosensors with its capability to identify and discriminate thousands of odorant compounds. In order to mimic the performances of natural olfactory sensors it is necessary to develop methods and techniques for the production, immobilization and electrical characterization of olfactory receptors. We review in this paper some of the advances we obtained in these fields. (c) 2006 Elsevier B.V. All rights reserved.

Smooth muscle cells exhibit phenotypic and mechanical plasticity. During maturation, signalling pathways controlling actin dynamics modulate contractile apparatus-associated gene transcription and contractile apparatus remodelling resulting from length change. Differentiated myocytes accumulate abundant caveolae that evolve from the structural association of lipid rafts with caveolin-1, a protein with domains that confer unique functional properties. Caveolae and caveolin-1 modulate and participate in receptor-mediated signalling, and thus contribute to functional diversity of phenotypically similar myocytes. In mature smooth muscle, caveolae are partitioned into discrete linear domains aligned with structural proteins that tether actin to the extracellular matrix. Caveolin-1 binds with β-dystroglycan, a subunit of the dystrophin glycoprotein complex (DGC), and with filamin, an actin binding protein that organizes cortical actin, to which integrins and focal adhesion complexes are anchored. The DGC is linked to the actin cytoskeleton by a dystrophin subunit and is a receptor for extracellular laminin. Thus, caveolae and caveolin-associated signalling proteins and receptors are linked via structural proteins to a dynamic filamentous actin network. Despite development of transgenic models to investigate caveolins and membrane-associated actin-linking proteins in skeletal and cardiac muscle function, only superficial understanding of this association in smooth muscle phenotype and function has emerged.Key words: caveolin, dystroglycan, filamin, mechanical plasticity, G-protein-coupled receptors.

Completion of the Caenorhabditis elegans genome sequencing project in 1998 has provided more insight into the complexity of nematode neuropeptide signaling. Several C. elegans neuropeptide precursor genes, coding for approximately 250 peptides, have been predicted from the genomic database. One can, however, not deduce whether all these peptides are actually expressed, nor is it possible to predict all post-translational modifications. Using two dimensional nanoscale liquid chromatography combined with tandem mass spectrometry and database mining, we analyzed a mixed stage C. elegans extract. This peptidomic setup yielded 21 peptides derived from formerly predicted neuropeptide-like protein (NLP) precursors and 28 predicted FMRFamide-related peptides. In addition, we were able to sequence 11 entirely novel peptides derived from nine peptide precursors that were not predicted or identified in any way previously. Some of the identified peptides display profound sequence similarities with neuropeptides from other invertebrates, indicating that these peptides have a long evolutionary history. ispartof: Biochemical And Biophysical Research Communications vol:335 issue:1 pages:76-86 ispartof: location:Louvain-La-Neuve, Belgium status: published

Background: neuropeptide S (NPS) and its receptor NPSR1 act along the hypothalamic-pituitary-adrenal axis to modulate anxiety, fear responses, nociception and inflammation. The importance of the NPS-NPSR1 signaling pathway is highlighted by the observation that, in humans, NPSR1 polymorphism associates with asthma, inflammatory bowel disease, rheumatoid arthritis, panic disorders, and intermediate phenotypes of functional gastrointestinal disorders. Because of the genetic complexity at the NPSR1 locus, however, true causative variations remain to be identified, together with their specific effects on receptor expression or function. To gain insight into the mechanisms leading to NPSR1 disease-predisposing effects, we performed a thorough functional characterization of all NPSR1 promoter and coding SNPs commonly occurring in Caucasians (minor allele frequency .0.02). Principal Findings: we identified one promoter SNP (rs2530547 [2103]) that significantly affects luciferase expression in gene reporter assays and NPSR1 mRNA levels in human leukocytes. We also detected quantitative differences in NPSinduced genome-wide transcriptional profiles and CRE-dependent luciferase activities associated with three NPSR1 nonsynonymous SNPs (rs324981 [Ile107Asn], rs34705969 [Cys197Phe], rs727162 [Arg241Ser]), with a coding variant exhibiting a loss-of-function phenotype (197Phe). Potential mechanistic explanations were sought with molecular modelling and bioinformatics, and a pilot study of 2230 IBD cases and controls provided initial support to the hypothesis that different ciscombinations of these functional SNPs variably affect disease risk. Significance: these findings represent a first step to decipher NPSR1 locus complexity and its impact on several human conditions NPS antagonists have been recently described, and our results are of potential pharmacogenetic relevance. Citation: Anedda F, Zucchelli M, Schepis D, Hellquist A, Corrado L, et al. (2011) Multiple Polymorphisms Affect Expression and Function of the Neuropeptide S

Deficits in sensorimotor gating measured by prepulse inhibition (PPI) of the startle have been known as characteristics of patients with schizophrenia and related neuropsychiatric disorders. PPI disruption is thought to rely on the activity of the mesocorticolimbic dopaminergic system and is inhibited by most antipsychotic drugs. These drugs however act also at the nigrostriatal dopaminergic pathway and exert adverse locomotor responses. Finding a way to inhibit the mesocorticolimbic- without affecting the nigrostriatal-dopaminergic pathway may thus be beneficial to antipsychotic therapies. The melanin-concentrating hormone (MCH) system has been shown to modulate dopamine-related responses. Its receptor (MCH1R) is expressed at high levels in the mesocorticolimbic and not in the nigrostriatal dopaminergic pathways. Interestingly a genomic linkage study revealed significant associations between schizophrenia and markers located in the MCH1R gene locus. We hypothesize that the MCH system can selectively modulate the behavior associated with the mesocorticolimbic dopamine pathway. Using mice, we found that central administration of MCH potentiates apomorphine-induced PPI deficits. Using congenic rat lines that differ in their responses to PPI, we found that the rats that are susceptible to apomorphine (APO-SUS rats) and exhibit PPI deficits display higher MCH mRNA expression in the lateral hypothalamic region and that blocking the MCH system reverses their PPI deficits. On the other hand, in mice and rats, activation or inactivation of the MCH system does not affect stereotyped behaviors, dopamine-related responses that depend on the activity of the nigrostriatal pathway. Furthermore MCH does not affect dizocilpine-induced PPI deficit, a glutamate related response. Thus, our data present the MCH system as a regulator of sensorimotor gating, and provide a new rationale to understand the etiologies of schizophrenia and related psychiatric disorders.

We have screened an odorant compound library and discovered molecules acting as chemical signals that specifically activate both G-protein-coupled olfactory receptors (ORs) on the cell surface of olfactory sensory neurons and the human nuclear estrogen receptor alpha (ER) involved in transcriptional regulation of cellular differentiation and proliferation in a wide variety of tissues. Hence, these apparent dual active odorants induce distinct signal transduction pathways at different subcellular localizations, which affect both neuronal signaling, resulting in odor perception, and the ER-dependent transcriptional control of specific genes. We demonstrate these effects using fluorescence-based in vitro and cellular assays. Among these odorants, we have identified synthetic sandalwood compounds, an important class of molecules used in the fragrance industry. For one estrogenic odorant we have also identified the cognate OR. This prompted us to compare basic molecular recognition principles of odorants on the two structurally and apparent functionally non-related receptors using computational modeling in combination with functional assays. Faced with the increasing evidence that ORs may perform chemosensory functions in a number of tissues outside of the nasal olfactory epithelium, the unraveling of these molecular ligand-receptor interaction principles is of critical importance. In addition the evidence that certain olfactory sensory neurons naturally co-express ORs and ERs may provide a direct functional link between the olfactory and hormonal systems in humans. Our results are therefore useful for defining the structural and functional characteristics of ER-specific odorants and the role of odorant molecules in cellular processes other than olfaction.

Photoactivation ('uncaging’) is a powerful approach for releasing bioactive small-molecules in living cells. Current uncaging methods are limited by the random distribution of caged molecules within cells. We have developed a mitochondria-specific photoactivation method, which permitted us to release free sphingosine inside mitochondria and thereafter monitor local sphingosine metabolism by lipidomics. Our results indicate that sphingosine was quickly phosphorylated into sphingosine 1-phosphate (S1P) driven by sphingosine kinases. In time-course studies, the mitochondria-specific uncaged sphingosine demonstrated distinct metabolic patterns compared to globally-released sphingosine, and did not induce calcium spikes. Our data provide direct evidence that sphingolipid metabolism and signaling are highly dependent on the subcellular location and opens up new possibilities to study the effects of lipid localization on signaling and metabolic fate., eLife digest Fatty or oily molecules called lipids are essential components of the membranes of cells and important signaling molecules too. They are made in specific compartments of the cell, but most are found in all membranes, albeit in varying amounts. Their widespread distribution suggests that there are extensive networks for transporting lipids within cells. Yet scientists know little about lipid transport inside living cells because it is difficult to detect their movements. Mitochondria are cellular compartments that are often referred to as the “powerhouses of the cell”. Many lipids are found in mitochondria including one called sphingosine, which is a common component of many other cell membranes too. Sphingosine can increase the concentration of calcium ions inside the cells, and when converted to a molecule called sphingosine 1 phosphate it forms a signaling molecule that regulates fundamental processes like cell survival and migration. However, it was not known if sphingosine localized in the mitochondria was processed differently to the same molecule elsewhere in the cell, or if its signaling activity was affected by its location. In the laboratory, Feng et al. synthesized an inactive sphingosine-like molecule that would only localize to mitochondria and which could be activated with a flash of light. By adding this molecule to human cells, they showed that sphingosine could be converted to sphingosine 1 phosphate within the mitochondria, before being exported rapidly to another compartment in the cell. The experiments allowed Feng et al. to observe the process in enough detail to to conclude that, despite its rapid transport, when localized only inside mitochondria, sphingosine could not trigger its normal signaling response. This new light-activated lipid molecule will be a useful tool for many researchers studying both metabolism and signaling. In principle, a similar tool could be developed for many compounds and it should also be possible to localize the compound to different locations within the cell. This new generation of compounds would give scientists a better understanding of mitochondria biology. They could be applied to the study of diseases where the mitochondria do not function as they should, for example Barth syndrome, where a mitochondria specific lipid called cardiolipin is not properly synthesized.

The mechanism of signal transduction in G-protein-coupled receptors (GPCRs) is a crucial step in cell signaling. However, the molecular details of this process are still largely undetermined. Carrying out submicrosecond molecular dynamics simulations of beta-adrenergic receptors, we found that cooperation between a number of highly conserved residues is crucial to alter the equilibrium between the active state and the inactive state of diffusible ligand GPCRs. In particular, "ionic-lock" formation in beta-adrenergic receptors is directly correlated with the protonation state of a highly conserved aspartic acid residue [Asp(2.50)] even though the two sites are located more than 20 angstrom away from each other. Internal polar residues, acting as local microswitches, cooperate to propagate the signal from Asp(2.50) to the G-protein interaction site at the helix III-helix VI interface. Evolutionarily conserved differences between opsin and non-opsin GPCRs in the surrounding of Asp(2.50) influence the acidity of this residue and can thus help in rationalizing the differences in constitutive activity of class A GPCRs. (C) 2010 Elsevier Ltd. All rights reserved.

Understanding the binding mode of agonists to adrenergic receptors is crucial to enabling improved rational design of new therapeutic agents. However, so far the high conformational flexibility of G protein-coupled receptors has been an obstacle to obtaining structural information on agonist binding at atomic resolution. In this study, we report microsecond classical molecular dynamics simulations of β1 and β2 adrenergic receptors bound to the full agonist isoprenaline and in their unliganded form. These simulations show a novel agonist binding mode that differs from the one found for antagonists in the crystal structures and from the docking poses reported by in silico docking studies performed on rigid receptors. Internal water molecules contribute to the stabilization of novel interactions between ligand and receptor, both at the interface of helices V and VI with the catechol group of isoprenaline as well as at the interface of helices III and VII with the ethanolamine moiety of the ligand. Despite the fact that the characteristic N-C-C-OH motif is identical in the co-crystallized ligands and in the full agonist isoprenaline, the interaction network between this group and the anchor site formed by Asp(3.32) and Asn(7.39) is substantially different between agonists and inverse agonists/antagonists due to two water molecules that enter the cavity and contribute to the stabilization of a novel network of interactions. These new binding poses, together with observed conformational changes in the extracellular loops, suggest possible determinants of receptor specificity., Author Summary G-protein coupled receptors are the largest family of membrane proteins in the human genome and they constitute the largest class of drug targets. Amongst them, beta adrenergic receptors are involved in the regulation of muscular and vascular tone and are thus molecular targets for the treatment of various diseases including hypertension, heart failure and asthma. The function of these receptors is regulated via the binding of endogenous or exogenous ligands that can either lead to activation (agonists) or inactivation (inverse agonists/antagonists). However, structure determination of these receptors has been very elusive, and the few atomic resolution structures that are available so far have only been obtained in the presence of inverse agonists or antagonists. In order to study the binding mode of agonists inside the binding pocket, we employ all-atom molecular dynamics. This facilitates the study of the details of the interaction between agonist and receptor in full atomistic detail. We find that agonists binding to beta adrenergic receptors require the formation of a highly structured hydrogen bond network that is further stabilized by the presence of internal water molecules. The observed local rearrangements also help provide insights into the molecular origin of the differences between agonist and inverse agonist binding.

Olfactory receptors (ORs) constitute the largest family of sensory membrane proteins in mammals. They play a key role within the olfactory system in recognizing and discriminating a nearly unlimited number of structurally diverse odorous molecules. The molecular basis of OR-mediated signal detection and transduction is poorly understood. This is due to difficulties in functional expression of ORs in high yields, preventing structural and biophysical studies at the level of the receptor protein. Here we report on recombinant expression of mouse receptor mOR256-17 yielding 10(6) ORs per cell in transiently transfected mammalian cells. For quantification and optimization of OR expression, we employed different fluorescent probes. Green fluorescent protein fused to the C-terminus of mOR256-17 allowed quantification of total cellular OR biosynthesis, and post-translational fluorescence labeling of a 12-amino acid polypeptide sequence at the N-terminus permitted the selective visualization and quantification of ORs at the plasma membrane using cell flow cytometry. Our dual-color labeling approach is generally applicable to quantification of membrane proteins for mammalian cell-based expression. By screening a large odorant compound library, we discovered a selective spectrum of potent mOR256-17-specific agonists essential for probing the receptor function for future scaled-up productions.