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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
21

Analysis of two D1-like dopamine receptors from the honey bee Apis mellifera reveals agonist-independent activity

Blenau, Wolfgang, Mustard, Julie A., Hamilton, Ingrid S., Ward, Vernon K., Ebert, Paul R., Mercer, Alison R. January 2003 (has links)
Dopamine is found in many invertebrate organisms, including insects, however, the mechanisms through which this amine operates remain unclear. We have expressed two dopamine receptors cloned from honey bee (AmDOP1 and AmDOP2) in insect cells (Spodoptera frugiperda), and compared their pharmacology directly using production of cAMP as a functional assay. In each assay, AmDOP1 receptors required lower concentrations of dopamine and 6,7-ADTN for maximal activation than AmDOP2 receptors. Conversely, butaclamol and cis(Z)-flupentixol were more potent at blocking the cAMP response mediated through AmDOP2 than AmDOP1 receptors. Expression of AmDOP1, but not AmDOP2, receptors significantly increased levels of cAMP even in the absence of ligand. This constitutive activity was blocked by cis(Z)-flupentixol. This work provides the first evidence of a constitutively activated dopamine receptor in invertebrates and suggests that although AmDOP1 and AmDOP2 share much less homology than their vertebrate counterparts, they display a number of functional parallels with the mammalian D1-like dopamine receptors.
22

Molecular and functional characterization of an octopamine receptor from honeybee (Apis mellifera) brain

Blenau, Wolfgang, Grohmann, Lore, Erber, Joachim, Ebert, Paul R., Strünker, Timo, Baumann, Arnd January 2003 (has links)
Biogenic amines and their receptors regulate and modulate many physiological and behavioural processes in animals. In vertebrates, octopamine is only found in trace amounts and its function as a true neurotransmitter is unclear. In protostomes, however, octopamine can act as neurotransmitter, neuromodulator and neurohormone. In the honeybee, octopamine acts as a neuromodulator and is involved in learning and memory formation. The identification of potential octopamine receptors is decisive for an understanding of the cellular pathways involved in mediating the effects of octopamine. Here we report the cloning and functional characterization of the first octopamine receptor from the honeybee, Apis mellifera . The gene was isolated from a brain-specific cDNA library. It encodes a protein most closely related to octopamine receptors from Drosophila melanogaster and Lymnea stagnalis . Signalling properties of the cloned receptor were studied in transiently transfected human embryonic kidney (HEK) 293 cells. Nanomolar to micromolar concentrations of octopamine induced oscillatory increases in the intracellular Ca2+ concentration. In contrast to octopamine, tyramine only elicited Ca2+ responses at micromolar concentrations. The gene is abundantly expressed in many somata of the honeybee brain, suggesting that this octopamine receptor is involved in the processing of sensory inputs, antennal motor outputs and higher-order brain functions.
23

Pharmacology of serotonin-induced salivary secretion in Periplaneta americana

Blenau, Wolfgang, Troppmann, Britta, Walz, Bernd January 2007 (has links)
The acinar salivary gland of the cockroach, Periplaneta americana, is innervated by dopaminergic and serotonergic nerve fibers. Stimulation of the glands by serotonin (5-hydroxytryptamine, 5-HT) results in the production of a protein-rich saliva, whereas stimulation by dopamine results in saliva that is protein-free. Thus, dopamine acts selectively on ion-transporting peripheral cells within the acini, and 5-HT acts on protein-producing central cells. We have investigated the pharmacology of the 5-HT-induced secretory activity of isolated salivary glands of P. americana by testing several 5-HT receptor agonists and antagonists. The effects of 5-HT can be mimicked by the non-selective 5-HT receptor agonist 5-methoxytryptamine. All tested agonists that display at least some receptor subtype specificity in mammals, i.e., 5-carboxamidotryptamine, (+/-)-8-OH-DPAT, (+/-)-DOI, and AS 19, were ineffective in stimulating salivary secretion. 5-HT-induced secretion can be blocked by the vertebrate 5-HT receptor antagonists methiothepin, cyproheptadine, and mianserin. Our pharmacological data indicate that the pharmacology of arthropod 5-HT receptors is remarkably different from that of their vertebrate counterparts. (C) 2007 Elsevier Ltd. All rights reserved.
24

Molecular characterization and localization of the first tyramine receptor of the American cockroach (Periplaneta americana)

Blenau, Wolfgang, Rotte, Cathleen, Krach, Christian, Balfanz, Sabine, Baumann, Arnd, Walz, Bernd January 2009 (has links)
The phenolamines octopamine and tyramine control, regulate, and modulate many physiological and behavioral processes in invertebrates. Vertebrates possess only small amounts of both substances, and thus, octopamine and tyramine, together with other biogenic amines, are referred to as “trace amines.” Biogenic amines evoke cellular responses by activating G-protein-coupled receptors. We have isolated a complementary DNA (cDNA) that encodes a biogenic amine receptor from the American cockroach Periplaneta americana, viz., Peatyr1, which shares high sequence similarity to members of the invertebrate tyramine-receptor family. The PeaTYR1 receptor was stably expressed in human embryonic kidney (HEK) 293 cells, and its ligand response has been examined. Receptor activation with tyramine reduces adenylyl cyclase activity in a dose-dependent manner (EC50 350 nM). The inhibitory effect of tyramine is abolished by co-incubation with either yohimbine or chlorpromazine. Receptor expression has been investigated by reverse transcription polymerase chain reaction and immunocytochemistry. The mRNA is present in various tissues including brain, salivary glands, midgut, Malpighian tubules, and leg muscles. The effect of tyramine on salivary gland acinar cells has been investigated by intracellular recordings, which have revealed excitatory presynaptic actions of tyramine. This study marks the first comprehensive molecular, pharmacological, and functional characterization of a tyramine receptor in the cockroach.
25

Inverse agonist and neutral antagonist actions of synthetic compounds at an insect 5-HT1 receptor

Troppmann, Britta, Balfanz, Sabine, Baumann, Arnd, Blenau, Wolfgang January 2010 (has links)
Background and purpose: 5-Hydroxytryptamine (5-HT) has been shown to control and modulate many physiological and behavioural functions in insects. In this study, we report the cloning and pharmacological properties of a 5-HT1 receptor of an insect model for neurobiology, physiology and pharmacology. Experimental approach: A cDNA encoding for the Periplaneta americana 5-HT1 receptor was amplified from brain cDNA. The receptor was stably expressed in HEK 293 cells, and the functional and pharmacological properties were determined in cAMP assays. Receptor distribution was investigated by RT-PCR and by immunocytochemistry using an affinity-purified polyclonal antiserum. Key results: The P. americana 5-HT1 receptor (Pea5-HT1) shares pronounced sequence and functional similarity with mammalian 5-HT1 receptors. Activation with 5-HT reduced adenylyl cyclase activity in a dose-dependent manner. Pea5-HT1 was expressed as a constitutively active receptor with methiothepin acting as a neutral antagonist, and WAY 100635 as an inverse agonist. Receptor mRNA was present in various tissues including brain, salivary glands and midgut. Receptor-specific antibodies showed that the native protein was expressed in a glycosylated form in membrane samples of brain and salivary glands. Conclusions and implications: This study marks the first pharmacological identification of an inverse agonist and a neutral antagonist at an insect 5-HT1 receptor. The results presented here should facilitate further analyses of 5-HT1 receptors in mediating central and peripheral effects of 5-HT in insects.
26

OX1 Orexin Receptor Signalling to Phospholipases

Ekholm, Marie January 2010 (has links)
The neuropeptides orexin-A and orexin-B were discovered in 1998 and were first described as regulators of feeding behaviour. Later research has shown that they have an important role in the regulation of sleep. Two G protein-coupled receptors, OX1 and OX2 orexin receptors, mediate the cellular responses to orexins. The overall aim of this thesis was to investigate the OX1 orexin receptors signalling to phospholipases. Previous investigations have determined that orexin receptors induce Ca2+ elevations through both receptor-operated Ca2+ channels (ROCs) and store-operated Ca2+ channels (SOCs). In this thesis we investigated the importance of these influxpathways on orexin-mediated phospholipase (PLC) activation. The results demonstrate that ROC influx is enough to fully support orexin-stimulated PLC activation but that SOC influx has a further amplifying role. We also investigated the metabolites generated after PLC activation, inositolphosphates and diacylglycerol (DAG). The results indicate involvement of two different PLC activities with different substrate specificities one of them leading to DAG production without co-occurring IP3 production at low orexin receptor stimulation. The results also suggest that at even lower orexin receptor stimulation DAG is produced via the activation of phospholipase D. In this thesis we also investigated if the ubiquitous phospholipase A2 (PLA2) signalling system is involved in orexin receptor signalling. The results demonstrate that stimulation of the OX1 orexin receptors leads to arachidonic acid (AA) release. This release is fully dependent on Ca2+ influx, probably through ROC, and at the same time the studies demonstrate that ROC influx is partly dependent on PLA2 activation. At low orexin receptor activation the AA release seemed to in part rely on extracellular signal-regulated kinase. We also devised two methods to aid in these investigations. The first method enabled studies of the receptor-operated Ca2+ influx without interference of the co-occurring store-operated Ca2+ influx. This was done by the expression of IP3-metabolising enzymes IP3-3-kinase-A and IP3-5-phosphatase-I. The second method enables quantification of DAG and IP3 signalling in fixed cells using GFP-fused indicators, leading to a semi-quantitative but easily applicable pharmacological assay.
27

Identification, Characterization and Evolution of Membrane-bound Proteins

Höglund, Pär J. January 2008 (has links)
Membrane proteins constitute approximately 30% of all genes in the human genome and two large families of membrane proteins are G protein-coupled receptors (GPCRs) and Solute Carriers (SLCs) with about 800 and 380 human genes, respectively. In Papers I, II and IV, we report 16 novel human Adhesion GPCRs found by searches in NCBI and Celera databases. In Paper I, we report eight novel human GPCRs, and six in Paper II. We identified two new human Adhesion GPCRs and 17 mouse orthologs in Paper IV. Phylogenetic analysis demonstrates that the 16 novel human genes are additional members of the Adhesion GPCR family and can be divided into eight phylogenetic groups. EST expression charts for the entire repertoire of Adhesions in human and mouse were established, showing widespread distribution in both central and peripheral tissues. Different domains were found in their N-terminus, some, such as pentraxin in GPR112, indicates that they take part in immunological processes. In Paper III, we discovered seven new human Rhodopsin GPCRs. In Paper V, we present the identification of two new human genes, termed SLC6A17 and SLC6A18 from the Solute Carriers family 6 (SLC6). We also identified the corresponding orthologs and additional genes from the mouse and rat genomes. We analysed, in total, 430 unique SLC6 proteins from 10 animal, one plant, two fungi and 196 bacterial genomes. In Paper VI, we provide the first systematic analysis of the evolutionary history of the different SLC families in Eukaryotes. In all, we analysed 2403 sequences in eight species and we delineate the evolutionary history of each of the 46 SLC families.
28

Classification, Evolution, Pharmacology and Structure of G protein-coupled Receptors

Lagerström, Malin C January 2006 (has links)
G protein-coupled receptors (GPCR) are integral membrane proteins with seven α-helices that translate a remarkable diversity of signals into cellular responses. The superfamily of GPCRs is among the largest and most diverse protein families in vertebrates. We have searched the human genome for GPCRs and show that the family includes approximately 800 proteins, which can divided into five main families; Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2 and Secretin. This study represents one of the first overall road maps of the GPCR family in a mammalian genome. Moreover, we identified eight novel members of the human Adhesion family which are characterized by long N-termini with various domains. We also investigated the GPCR repertoire of the chicken genome, where we manually verified a total of 557 chicken GPCRs. We detected several specific expansions and deletions that may reflect some of the functional differences between human and chicken. Substantial effort has been made over the years to find compounds that can bind and activate the melanocortin 4 receptor (MC4R). This receptor is involved in food intake and is thus an important target for antiobesity drugs. We used site-directed mutagenesis to insert micromolar affinity binding sites for zinc between transmembrane (TM) regions 2 and 3. We generated a molecular model of the human MC4R which suggests that a rotation of TM3 is important for activation of the MC4R. Furthermore, we present seven new vertebrate prolactin releasing hormone receptors (PRLHRs) and show that they form two separate subtypes, PRLHR1 and PRLHR2. We performed a pharmacological characterization of the human PRLHR which showed that the receptor can bind neuropeptide Y (NPY) related ligands. We propose that an ancestral PRLH peptide has coevolved with a redundant NPY binding receptor, which then became PRLHR. This suggests how gene duplication events can lead to novel peptide ligand/receptor interactions and hence spur the evolution of new physiological functions.
29

Nature and Function of the Signaling Complex Formed by the M2 Muscarinic Cholinergic Receptor

Ma, Amy Wing-Shan 05 December 2012 (has links)
G protein-coupled receptors (GPCRs) are known to exist as oligomers, but there is much uncertainty over the oligomeric size, the number of interacting G proteins and the stability of that interaction. The present approach to these questions has been threefold. Monomers of the M2 muscarinic receptor were purified from Spodoptera frugiperda (Sf9) cells and reconstituted in phospholipid vesicles, where they spontaneously formed tetramers. The size of the reconstituted complex was determined from its electrophoretic mobility after cross-linking and inferred from a quantitative, model-based assessment of cooperative effects in the binding of two muscarinic antagonists: N-methylscopolamine and quinuclidinylbenzilate. Binding of the agonist oxotremorine-M to receptor reconstituted with purified G proteins revealed at least three classes of sites that interconverted from higher to lower affinity upon the addition of guanylylimidotriphosphate (GMP-PNP). The binding properties resemble those of muscarinic receptors in myocardial preparations, thereby implying the existence of tetramers in native tissues. G proteins that copurify with the M2 receptor from cardiac membranes also were found to exist as oligomers, some of which contain both alpha(o) and alpha(i2), and the purified complexes contained receptor and G protein in near-equal amounts. A tetrameric receptor implies a tetramer of G proteins, a conclusion that is supported by the distribution of sites between different states identified in the binding of [35S]GTPgammaS to the purified complex. Covalent adducts of a GPCR fused to a Galpha-subunit provide a model system in which the relationship between receptor and G protein complex is defined with respect to stability and composition. Such a fusion of the M2 receptor and Galpha(i1) underwent a cleavage near the amino terminus of the alpha-subunit, however, flagging the likelihood of similar effects in other such adducts. Truncation of the amino terminus prior to fusion generated a stable product that revealed GMP-PNP-sensitive, biphasic binding of oxotremorine-M and noncompetitive interactions between N-methylscopolamine and quinuclidinylbenzilate. A covalent RG complex therefore exhibits the functional properties of M2 receptors in native systems. These observations are consistent with the notion that signaling through the M2 receptor occurs via cooperative interactions within a stable complex that comprises four receptors and four G proteins.
30

Molecular dynamics simulations of seven-transmembrane receptors

Cordomí Montoya, Arnau 11 March 2008 (has links)
Seven transmembrane (7-TM) G protein coupled receptors (GPCR) constitute the largest family of integral membrane proteins in eukaryotes with more than 1000 members and encoding more than 2% of the human genome. These proteins play a key role in the transmission and transduction of cellular signals responding to hormones, neurotransmitters, light and other agonists, regulating basic biological processes. Their natural abundance together with their localization in the cell membrane makes them suitable targets for therapeutic intervention. Consequently, GPCR are proteins with enormous pharmacologic interest, representing the targets of about 50% of the currently marketed drugs. The current limitations in the experimental techniques necessary for microscopic studies of the membrane as well as membrane proteins emerged the use of computational methods and specifically molecular dynamics simulations. The lead motif of this thesis is the study of GPCR by means of this technique, with the ultimate goal of developing a methodology that can be generalized to the study of most 7-TM as well as other membrane proteins. Since the bovine rhodopsin was the only protein of the GPCR family with a known threedimensional structure at an atomic level until very recently, most of the effort is centered in the study of this receptor as a model of GPCR.The scope of this thesis is twofold. On the one hand it addresses the study of the simulation conditions, including the procedure as well as the sampling box to get optimal results, and on the other, the biological implications of the structural and dynamical behavior observed in the simulations. Specifically, regarding the methodological aspects of the work, the bovine rhodopsin has been studied using different treatments of long-range electrostatic interactions and sampling conditions, as well as the effect of sampling the protein embedded in different one-component lipid bilayers. The binding of ions to lipid bilayers in the absence of the protein has also been investigated. Regarding the biological consequences of the analysis of the MD trajectories, it has been carefully addressed the binding site of retinal and its implications in the process of isomerization after photon uptake, the alteration a group of residues constituting the so-called electrostatic lock between helices TM3 and TM6 in rhodopsin putatively used as common activation mechanism of GPCR, and the structural effects caused by the dimerization based on a recent semi-empirical model. Finally, the specific binding of ions to bacteriorhodopsin has also been studied. The main conclusion of this thesis is provide support to molecular dynamics as technique capable to provide structural and dynamical informational about membranes and membrane proteins, not currently accessible from experimental methods). Moreover, the use of an explicit lipidic environment is crucial for the study the membrane protein dynamics as well as for the protein-protein and lipidprotein interactions.

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