Rozprawy doktorskie: „GPCR”

1

Blankenship, Elise. "Conserved solvent networks in GPCR activation". Case Western Reserve University School of Graduate Studies / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=case1458221506.

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Poudel, Sagar. "GPCR-Directed Libraries for High Throughput Screening". Thesis, University of Skövde, School of Humanities and Informatics, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-29.

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Guanine nucleotide binding protein (G-protein) coupled receptors (GPCRs), the largest receptor family, is enormously important for the pharmaceutical industry as they are the target of 50-60% of all existing medicines. Discovery of many new GPCR receptors by the “human genome project”, open up new opportunities for developing novel therapeutics. High throughput screening (HTS) of chemical libraries is a well established method for finding new lead compounds in drug discovery. Despite some success this approach has suffered from the near absence of more focused and specific targeted libraries. To improve the hit rates and to maximally exploit the full potential of current corporate screening collections, in this thesis work, identification and analysis of the critical drug-binding positions within the GPCRs were done, based on their overall sequence, their transmembrane regions and their drug binding fingerprints. A proper classification based on drug binding fingerprints on the basis for a successful pharmacophore modelling and virtual screening were done, which facilities in the development of more specific and focused targeted libraries for HTS.

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3

Majin, Wodu. "Mathematical modelling of GPCR-mediated calcium signalling". Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/12451/.

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Ca2+ is an important messenger which mediates several physiological functions, including muscle contraction, fertilisation, heart regulation and gene transcription. One major way its cytosolic level is raised is via a G-protein coupled receptor (GPCR)- mediated release from intracellular stores. GPCR’s are the target of approximately 50% of all drugs in clinical use. Hence, understanding the underlying mechanisms of signalling in this pathway could lead to improved therapy in disease conditions associated with abnornmal Ca2+ signalling, and to the identification of new drug targets. To gain such insight, this thesis builds and analyses a detailed mathematical model of key processes leading to Ca2+ mobilisation. Ca2+ signalling is considered in the particular context of the M3 muscarinic receptor system. Guided by available data, the Ca2+ mobilisation model is assembled, first by analysing a base G-protein activation model, and subsequently extending it with downstream details. Computationally efficient designs of a global parameter sensitivity analysis method are used to identify the key controlling parameters with respect to the main features of the Ca2+ data. The underlying mechanism behind the experimentally observed, rapid, amplified Ca2+ response is shown to be a rapid rate of inositol trisphosphate (IP3) formation from Phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. Using the same results, potential drug targets (apart fromthe GPCR) are identified, including the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and PIP2. Moreover, possible explanations for therapeutic failures were found when some parameters exerted a biphasic effect on the relative Ca2+ increase. The sensitivity analysis results are used to simplify the process of parameter estimation by a significant reduction of the parameter space of interest. An evolutionary algorithm is used to successfully fit the model to a significant portion of the Ca2+ data. Subsequent sensitivity analyses of the best-fitting parameter sets suggest that mechanistic modelling of kinase-mediated GPCR desensitisation, and SERCA dynamics may be required for a comprehensive representation of the data.

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4

Tang, Lisa Sarah. "GPCR expressions in Saccharomyces cerevisiae : engineering transductions". Thesis, University of Leeds, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.423190.

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5

Mishra, Satyakam. "Frequent Subgraph Mining Analysis of GPCR Activation". Case Western Reserve University School of Graduate Studies / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=case1613575702373053.

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6

Kiess, Alexandra. "Funktionelle Relevanz intrazellulärer Splicevarianten des Brain-specific Angiogenesis Inhibitor 2 (BAI2)". Doctoral thesis, Universitätsbibliothek Leipzig, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-156171.

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BAI2 gehört zu den Adhesion-G-Protein-gekoppelten Rezeptoren (aGPCR). Diese bisher wenig untersuchte Klasse von ca. 30 GPCR ist charakterisiert durch eine komplexe genomische Struktur, sehr große extrazelluläre Domänen und eine Vielzahl von Splicevarianten. Bisher ist bei den meisten aGPCR, wie auch bei BAI2, wenig über ihre Signaltransduktion und Funktion bekannt. Zum Verständnis der physiologischen Relevanz und zur Suche nach dem endogenen Agonist sind Kenntnisse über Proteinstruktur, Splicevarianten und Signaltransduktion essentiell. Ziel dieser Arbeit war es, mittels verschiedener in vitro-Methoden die Proteinstruktur des BAI2 in den transmembranären und intrazellulären Domänen näher zu untersuchen, sowie die natürlichen Splicevarianten in diesem Bereich, deren evolutionäre Konservierung, Gewebespezifität und Quantität zu erfassen. Für beide gefundenen Splicevarianten, eine im dritten intrazellulären Loop (ICL3) und eine im C-Terminus, konnte eine evolutionäre Konservierung auf Aminosäure- und genomischer Organisationsebene, sowie ihre Entstehung durch Exonskipping nachgewiesen werden. Nachfolgend wurden die Splicevarianten auf mögliche Interaktionen mit intrazellulären Komponenten untersucht. In dieser Arbeit konnte gezeigt werden, dass beide ICL3-Splicevarianten natürlicherweise in einem definierten Verhältnis auftreten. Außerdem konnte gezeigt werden, dass die lange ICL3-Variante des BAI2 nicht zu einer Änderung der Membrantopologie des Rezeptors, einer Homodimerisierung über die zusätzliche Aminosäuresequenz oder zu einer Interaktion mit dem C-Terminus führt. Die Splicevariante im humanen C-Terminus des BAI2 konnte als eine variable, durch Exonskipping entstandene Calcium-unabhängige Calmodulin-Bindungsstelle identifiziert werden. Diese Arbeit belegt die Existenz mehrerer BAI2-Isoformen in vivo. Die Struktur dieser Isoformen lässt unterschiedliche Funktionalitäten vermuten. Auch wenn erste Untersuchungen zwischen den beiden ICL3-Varianten keinen Unterschied ergaben, sind diese Erkenntnisse für die weitere Analyse der Signaltransduktion und Ligandensuche bedeutend. Es ist z.B. denkbar, dass sich die beiden ICL3-Varianten in der G-Protein-Kopplung oder bei der Rekrutierung von intrazellulären Interaktionspartnern unterscheiden oder dass die Splicevariante im C-Terminus zu einer Scaffold- Funktion des Calmodulins führt und/oder die Signaltransduktion durch eine permanente Bindung des Calmodulins an einer Isoform moduliert wird.

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7

Sladek, Barbara. "Structural studies of integral membrane GPCR accessory proteins". Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:09bf7ada-8e58-49f4-a979-bcd0cec95e8b.

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GPCR accessory proteins regulate the strength, efficiency and specificity of signal transfer upon receptor activation. Due to the inherent difficulties of studying membrane proteins in vitro and in vivo, little is known about the structure and topology of these small accessory proteins. Two examples of GPCR accessory proteins are the Melanocortin-2 receptor accessory protein (MRAP) and the Receptor-activity-modifying protein (RAMP) family. MRAP and RAMP1 are the main focus of this thesis in which they are thoroughly characterised by solution-state NMR and further biophysical techniques. The single-pass transmembrane domain protein MRAP regulates the class A GPCR melanocortin receptors. It is specifically required for trafficking the melanocortin-2-receptor from the endoplasmic reticulum to the cell surface and subsequent receptor activation. A remarkable characteristic of MRAP is its proposed native dual-topology, which leads to an antiparallel homodimeric conformation. Investigation of the biochemical and biophysical properties of MRAP revealed an α-helical transmembrane domain, and an α-helical N-terminal LD(Y/I)L-motif. Further efforts concentrated on establishing the homodimeric conformation of MRAP in vitro. RAMP1 facilitates receptor trafficking and alters the ligand specificity of the GPCR Class B receptors calcitonin receptors and calcitonin receptor-like receptors. Moreover, RAMP1 is required to act as a Calcitonin-gene-related peptide (CGRP) receptor (RAMP1). RAMP1 has been shown to form stable parallel homodimers in the absence of its cognate receptor. Its dimerisation and the possible dimerisation motif PxxxxP-motif were studied extensively. With the goal of understanding the mechanism of dimerisation and the role of GPCR accessory proteins I have used solution-state NMR in detergent micelles as my main technique. NMR provides unique possibilities for understanding the structure and dynamics of such small membrane proteins.

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8

Richardson, Kathryn. "Mechanisms of GPCR signal regulation in fission yeast". Thesis, University of Warwick, 2014. http://wrap.warwick.ac.uk/63554/.

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Cells communicate with each other and respond to environmental cues by sending and receiving signals. Many external signals (ligands) are detected through G protein-coupled receptors (GPCRs), a major class of transmembrane proteins. GPCRs transduce these external signals into appropriate intracellular responses, enabling the cell to adapt to its environment. Malfunctions in these signalling pathways can lead to a range of human diseases and hence GPCRs have become attractive candidates for pharmacological design. The activation of a single receptor has the ability to induce numerous intracellular responses. Coupling this with the great number of different GPCR-types expressed in human cells means that understanding the basic principles of signal transduction and termination in humans is complicated. This study utilises the more simplistic eukaryotic yeast Schizosaccharomyces pombe (S. pombe) to overcome this complexity, as it contains only two GPCR types and hence the cross-talk between pathways is greatly reduced, whilst the structure and signalling functions of GPCRs are often evolutionarily conserved between yeast and humans. Mathematical modelling was used to aid the understanding of GPCR signalling in S. pombe and to inform experimental design. Speci�cally, an ordinary differential equation model �rst developed by Croft et al. (2013) was extended to include all known downstream signal transduction, regulation and termination events. This model is the �rst of its kind to describe a whole GPCR signalling pathway within S. pombe. Although it accurately predicts the cellular response to GPCR signalling it could only reproduce the biological plateau in temporal response with the addition of a 'yet unknown mechanism' GPCR degradation term. This motivated the investigation of how GPCRs in S. pombe are internalised from the plasma membrane in response to ligand stimulation. The primary mechanism for signal termination is via internalisation of the GPCR. This study identi�ed three potential casein kinases (Cki1, Cki2 and Cki3) that promote internalisation of the S. pombe GPCR Mam2. Microscopy analyses in combination with quantitative transcriptional, cell growth and cell cycle position assays uncovered a novel role for these kinases: that Cki2 regulates cell size during vegetative growth, Cki1 and Cki3 regulate the GPCR-response pathway and that Cki3 is essential for completing cytokinesis in S. pombe that have already undergone formation of a conjugation tube in response to ligand. Confocal microscopy of uorescent labelled Mam2 indicated a role for Cki2 in the internalisation and hence termination of the GPCR-response pathway. These findings add to the growing body of evidence that casein kinases are implicated in GPCR desensitisation.

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9

Goddard, Alan David. "Functional analysis of GPCR signalling cascades in Schizosaccharomyces pombe". Thesis, University of Warwick, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.437696.

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10

Koyama, Hiroyuki. "Comprehensive Profiling of GPCR Expression in Ghrelin-producing Cells". Kyoto University, 2016. http://hdl.handle.net/2433/215953.

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11

Engemaier, Eva. "Die strukturelle und funktionelle Evolution des G-Protein-gekoppelten Rezeptors GPR34". Doctoral thesis, Universitätsbibliothek Leipzig, 2012. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-86270.

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Gegenstand der Arbeit ist eine umfassende Charakterisierung von Struktur und Funktion des G-Protein-gekoppelten Rezeptors GPR34 auf genomischer, mRNA und Proteinebene, die mögliche Rückschlüsse auf physiologische Funktionen oder den natürlichen Agonisten zulassen. Dazu wurde die genomische Organisation des GPR34 in Mensch, Maus und Ratte analysiert und festgestellt, dass neben der intronlosen kodierenden Sequenz auch der 5´-Bereich des GPR34 mit seiner nicht-kodierenden Intron-Exon-Struktur stark konserviert ist. Es wurden in der Ratte und der Maus mindestens zwei, beim Menschen ein putativer Transkriptionsstart identifiziert. Beim Menschen konnte ein kryptisches Intron innerhalb der kodierenden Sequenz des GPR34 gefunden werden, was zu einer Verkürzung des N-Terminus um 47 Aminosäuren führt. Auf der Transkriptionsebene wurde der GPR34 und der GPR34-like Rezeptor im Haushuhn (Gallus gallus) und die GPR34-Expression in der Ratte mittels quantitativer RT-PCR analysiert und die ubiquitäre Gewebeverteilung des Rezeptors bestätigt. Beim menschlichen GPR34 konnte festgestellt werden, dass die fünf putativen Translationsstarts innerhalb der kodierenden Sequenz auch als solche funktionstüchtig sind und der zweite Translationsstart bevorzugt genutzt wird. Die genetische Variabilität des GPR34 in der menschlichen Population ist sehr gering. Es konnte innerhalb einer weltweiten DNA-Probensammlung nur ein einziges Mal eine Mutation in der kodierenden Sequenz lokalisiert werden. Mithilfe des während dieser Arbeit entstandenen Mausmodelles ist eine weitere Charakterisierung der physiologischen Relevanz des GPR34 möglich.

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12

Thamm, Markus. "Charakterisierung der Serotonin-Rezeptoren der Honigbiene Apis mellifera : von den Genen zum Verhalten". Phd thesis, Universität Potsdam, 2009. http://opus.kobv.de/ubp/volltexte/2010/4073/.

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Das serotonerge System besitzt sowohl bei Invertebraten als auch bei Vertebraten eine große Bedeutung für die Kontrolle und Modulation vieler physiologischer Prozesse und Verhaltensleistungen. Bei der Honigbiene Apis mellifera spielt Serotonin (5-Hydroxytryptamin, 5-HT) eine wichtige Rolle bei der Arbeitsteilung und dem Lernen. Die 5-HT-Rezeptoren, die überwiegend zur Familie der G-Protein gekoppelten Rezeptoren (GPCRs) gehören, besitzen eine Schlüsselstellung für das Verständnis der molekularen Mechanismen der serotonergen Signalweiterleitung. Ziel dieser Arbeit war es, 5-HT-Rezeptoren der Honigbiene zu charakterisieren. Dazu zählt die Identifizierung der molekularen Struktur, die Ermittlung der intrazellulären Signalwege, die Erstellung von pharmakologischen Profilen, die Ermittlung der Expressionsmuster und die Ermittlung der physiologischen Funktionen der Rezeptoren. Mit Hilfe der Informationen aus dem Honey Bee Genome Project, konnten drei RezeptorcDNAs kloniert werden. Vergleiche der abgeleiteten Aminosäuresequenzen mit den Aminosäuresequenzen bereits charakterisierter Rezeptoren legten nahe, dass es sich dabei um einen 5-HT1- (Am5-HT1) und zwei 5-HT2-Rezeptoren (Am5-HT2α und Am5-HT2β) handelt. Die strukturelle Analyse der abgeleiteten Aminosäuresequenz dieser Rezeptoren postuliert das Vorhandensein der charakteristischen heptahelikalen Architektur von GPCRs und zeigt starkkonservierte Motive, die bedeutend für die Ligandenbindung, die Rezeptoraktivierung und die Kopplung an G-Proteine sind. Für die beiden 5 HT2-Rezeptoren konnte zudem alternatives Spleißen nachgewiesen werden. Mit den cDNAs des Am5-HT1- und des Am5-HT2α-Rezeptors wurden HEK293-Zellen stabil transfiziert und anschließend die Rezeptoren funktionell und pharmakologisch analysiert. Am5-HT1 hemmt bei Aktivierung abhängig von der 5-HT-Konzentration die cAMPProduktion.Die Substanzen 5-Methoxytryptamin (5-MT) und 5-Carboxamidotryptamin konnten als Agonisten identifiziert werden. Methiothepin dagegen blockiert die 5-HTWirkung vollständig. Prazosin und WAY100635 stellen partielle Antagonisten des Am5-HT1-Rezeptors dar. Der Am5-HT2_-Rezeptor stimuliert bei Aktivierung die Synthese des sekundären Botenstoffs Inositoltrisphosphat, was wiederum zu einer messbaren Erhöhung der intrazellulären Ca2+-Konzentration führt. 5-MT und 8-OH-DPAT zeigen eine deutliche agonistische Wirkung auf Am5-HT2α. Dagegen besitzen Clozapin, Methiothepin, Mianserin und Cyproheptadin die Fähigkeit, die 5-HT-Wirkung um 51-64 % zu vermindern. Die bereits erwähnte alternative Spleißvariante von Am5-HT2α wurde ebenfalls in HEK293-Zellen exprimiert und analysiert, scheint jedoch eigenständig nicht funktionell zu sein. Gegen die dritte cytoplasmatische Schleife (CPL3) wurde ein polyklonales Antiserum generiert. Dieses erkennt in Western-Blot-Analysen ein Protein mit einer Masse von ca. 50 kDa. Durch immunhistochemische Analysen am Bienengehirn wurde die Verteilung des Rezeptors genauer untersucht. Dabei zeigten die optischen Neuropile, besonders die Lamina und die Ocellarnerven, stets eine starke Markierung. Außerdem wird der Rezeptor in den α- und β-Loben sowie der Lippe, dem Basalring und dem Pedunculus der Pilzkörper exprimiert. Doppelmarkierungen zeigen stets eine enge Nachbarschaft von serotonergen Fasern und dem Am5-HT1-Rezeptor. Weiterhin konnte gezeigt werden, dass der Am5-HT1-Rezeptor sehr wahrscheinlich an der Regulation des phototaktischen Verhalten der Honigbiene beteiligt ist. Verfütterung von 5-HT hat eine deutlich negative Wirkung auf das phototaktischen Verhalten. Diese kann durch den Am5-HT1-Rezeptor-Agonisten 5-CT imitiert werden. Schließlich konnte gezeigt werden, dass der Am5-HT1-Antagonist Prazosin die 5-HT-Wirkung deutlich vermindern kann.
The serotonergic system plays an important role in the control and modulation of many physiological and behavioral processes in both vertebrates and invertebrates. In the honeybee Apis mellifera, serotonin (5-hydroxytryptamine, 5-HT) has been implicated in the control and regulation of division of labor as well as learning and memory. A key role in understanding the serotonergic system plays the molecular and functional characterization of 5-HT receptor subtypes. In most cases, serotonin receptors represent G protein-coupled receptors (GPCRs). This work describes the characterization of honeybee serotonin receptors. This comprises the identification of their molecular structure, intracellular second messenger pathways, pharmacological properties, expression profiles and functions. By screening the honeybee genome, we found three candidate genes encoding for putative serotonin receptors. The cDNAs of these genes were cloned and the deduced amino acid sequences were analysed. The sequence information was used to isolate the cDNAs encoding for these three receptors. Comparison of the deduced amino acid sequences with sequences of other known receptors suggests that one receptor belongs to the 5-HT1 (Am5-HT1) and the other two receptors to the 5-HT2 receptor class (Am5-HT2α and Am5-HT2β). Major characteristics common to all GPCRs (e.g. the heptahelical architecture) were confirmed by structural analyses of the deduced amino acid sequences. Furthermore, truncated receptor transcripts representing alternative splice variants of both 5-HT2 receptors could be detected. HEK293 cells were stably transfected with the cDNAs of Am5-HT1 or Am5-HT2_ and functionally and pharmacologically analysed. The activation of Am5-HT1 by 5-HT results in the dose dependent attenuation of adenylyl cyclase activity. 5-methoxytryptamine (5-MT) and 5-carboxamidotryptamine are able to imitate the 5-HT effect. In contrast, methiothepin is able to block the entire 5-HT effect, whereas prazosine and WAY100635 block the 5-HT effect only partially. The Am5-HT2α receptor stimulates the synthesis of the second messenger inositol trisphosphate which in turn mediates an increase in the intracellular Ca2+. The substances 5-MT and 8-OH-DPAT were identified as agonists of the Am5-HT2α receptor. In contrast, clozapine, methiothepine, mianserine, and cyproheptadine show strong antagonistic actions. A truncated alternative splice variant of the Am5-HT2α-receptor was also analysed but didn’t show any functional coupling by itself. An antiserum was raised against the third cytoplasmic loop (CPL3) of the Am5-HT1 receptor. This antiserum detects a protein with a molecular mass of 50 kDa in western blot analyses. The expression of the Am5-HT1 receptor was studied in detail using immunohistochemistry. Strong Am5-HT1-like immunofluorescence was observed in the ocellar nerve, in the three optic ganglia and in the α- and β-lobes, the pedunculi, the lip and the basal ring of the mushroom bodies. Furthermore, co-labeling with an antibody against 5-HT showed that this receptor is expressed in close vicinity to serotonergic neurons. Finally, behavioral experiments suggest a possible role of the Am5-HT1 receptor in phototactic behavior. Feeding of 5-HT to worker honeybees results in a decrease of phototactic behavior. This 5-HT action could be mimiced by feeding of the Am5-HT1 agonist 5-CT. In contrast, the Am5-HT1 antagonist prazosine prevents the 5-HT-induced decrease in phototaxis.

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13

Zhang, Boyang. "Functional and Structural Insights into the First and Second Intracellular Domains for D1-Class Dopaminergic Receptors". Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/35932.

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Previous studies have shown that the subtype-specific pharmacological properties of D1-class receptors (D1R and D5R) can be attributed to their third intracellular domain and C-terminal tail. However, the importance of their first and second intracellular domains (IC1 and IC2) has yet to be explored. Using mutagenesis and bioinformatics, we examine the functional and structural roles of Ser/Thr spanning IC1 and IC2—most of which are conserved not only among D1-class receptors but also among other GPCRs. Mutant receptors of human D1-class receptors (hD1R and hD5R) were constructed whereby all Ser and Thr were mutated to the respective Ala and Val in the IC1 region (termed ST1 mutant receptors) and in the IC2 region (termed ST2 mutant receptors). We found that hD1-ST2 and hD5-ST2 exhibited contrasting properties of agonist affinity, constitutive activity, and dopamine potency. On the other hand, both ST2 mutants underwent internalization as wild-type but displayed weakened desensitization abilities. Homology models, which have been refined under membrane simulations, illustrate that the conserved Ser3.55 and Thr3.65 utilize their side chains to anchor the loop regions of IC2 to cytoplasmic helices. We also found multiple functional alterations in the hD1-ST1 and hD5-ST2, but in a subtype-similar manner. Mutating the conserved Thr2.39 recapitulated the ablated basal activity and drastic decrease in dopamine potency previously witnessed in the hD1-ST1. Based on the recurring theme observed in crystal structures, the side-chain of Thr2.39 may help to position IC2 to have proper contacts with the G protein. Mutating the conserved Ser2.45 was found to be solely responsible for the elevated Emax (maximal response) of the hD1-ST1. Using single point mutagenesis, we further found that breaking the potential molecular interactions of Ser2.45 in hD1R (i.e. with Asn3.42 and Trp4.50) mimicked its elevated Emax. This elevated Emax was not found to be caused by altered abilities to undergo agonist-induced desensitization or internalization relative to hD1R. Overall, our work highlights the important functional and structural roles of IC1 and IC2 that needs to be accounted for in our current canonical models of GPCR signalling. Given the conserved nature of these Ser/Thr, our work may also be pertinent towards understanding the roles of IC1 and IC2 for other GPCRs.

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Guerrero, Hernández Martina. "Targeting tumor microenvironment crosstalk through GPCR receptors and PI3K pathway". Doctoral thesis, Universitat de Barcelona, 2019. http://hdl.handle.net/10803/667975.

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The tumor microenvironment (TME) is gaining momentum due to its contribution to cancer progression and therapy resistance. This TME has a direct crosstalk with tumor cells that involves the activation of different pathways. Follicular lymphoma (FL) is the most common indolent non-Hodgkin lymphoma. Although FL is generally characterized by slow progression and high response rates to therapy, it is still considered incurable, because almost virtually all the patients relapse. FL is probably the NHL with the highest dependence on microenvironment. PI3K is a common denominator transducing the signaling from FL crosstalk with the TME and plays an important role in multiple cellular functions, and also contributes to cancer promoting aspects of the TME, such as angiogenesis and inflammatory cell recruitment. Idelalisib is a first-in-class δ isoform- specific PI3K inhibitor that receive regulatory approval for relapsed CLL, SLL and FL in 2014. Idelalisib blocks PI3K δ which is restricted to leukocytes. BCL2 deregulation is paramount in the pathogenesis of FL, as a consequence of the t(14;18), and therefore it is an attractive target for novel therapeutic approaches. Venetoclax (ABT-199, AbbVie) is a small BCL-2 inhibitors. Even though 85% of FL patients harbor the t(14;18), the results of the first clinical trial with venetoclax were not satisfactory (overall response 38%). From this first study we conclude that Idelalisib modulates key pathways in the germinal center and shapes the FL immune microenvironment by decreasing the recruitment of TFH and Treg to the tumor site leading to less immunesuppresive phenotype. Furthermore Idelalisib induces a moderate cytotoxic effect on FL cells in co-cultures. This co-culture decrease FL dependence on BCL-2 and consequently, venetoclax cytotoxicity, but Idelalisib sensitizes FL co-cultures to venetoclax. In summary, Idelalisib interferes with the crosstalk of FL and its immune microenvironment and potentiates the activity of venetoclax targeting the tumor cells, thus representing a promising combination therapy that may improve FL outcome. Colorectal cancer (CRC) is the third most common cancer in males and the second in females, and the fourth most common cause of cancer-related death worldwide. Patients with advanced and distal metastatic disease (stage IV), the survival rate drops to 10%, which accounts for approximately 18% of cases. The TME in CRC, is a complex structure composed by different type of cells, which are interacting each other’s and secreting a variety of growth factors and other molecules, such as cytokines and chemokines. Tumor development is based on the crosstalk between tumor cells and their surrounding microenvironment, and this crosstalk is mediated by the receptors and its ligand expression in both types of cells. G protein-coupled receptors (GPCRs) are an important family of membrane signaling receptors, which have an important role in cancer growth and development. Originally, GPCRs were considered as monomeric functional entities, nevertheless, in recent years has become evident that GPCRs form dimers and this dimers formation may modify the cellular response. In cancer, CXCR4 (has been studied extensively) plays an important role at different stages of cancer development, and is involved in the metastasis process of tumor cells. The up- regulation of CXCR4 in CRC correlates with a poor prognosis. Another GPCR, CB2 receptor modulates the downstream signaling and it is able to activate a wide range of signaling pathways, including extracellular signal-regulated kinases 1/2 (ERK1/2). In CRC, it has been described an up-regulation of CB2 receptor expression. GPCRs show differential expression in cancer cells and tissues, and they are highly druggable sites. From this second study we concluded that CXCR4 and CB2 expression is increased in primary colon tumor cells and in metastasis cells compared to normal epithelial cells from colon mucosa, and they formed heterodimers in colon tumoral cells and are associated with more aggressive phenotypes. Moreover, a bidirectional cross-antagonism crosstalk is established between these receptors. These heterodimers regulate in vitro CXCL12-induced migration, and in vivo, the simultaneous inhibition of both receptors shows superior anti-tumoral and anti-metastatic activities than the single agent inhibition. In summary, targeting the heterodimerization of CXCR4 and CB2 that are biologically relevant in cancer can be an effective way to reduce proliferation and dissemination in CRC.
El estudio del microambiente tumoral está ganando importancia en las últimas décadas debido a su contribución en la formación y desarrollo del cáncer, además de contribuir en la resistencia de las células tumorales a diferentes terapias. Este microambiente interactúa con las células tumorales y activa diferentes vías. El linfoma folicular (FL), es el linfoma no Hodgkin indolente más común y con mayor dependencia del microambiente tumoral, además es considerado incurable. PI3K desempeña un papel importante en la comunicación con el microambiente, y es importante en múltiples funciones celulares, además de contribuir en la angiogénesis, reclutamiento de células inflamatorias y promover el crecimiento tumoral. Idelalisib es un inhibidor de PI3K (específicamente de la isoforma δ), que se aprobó en 2014 por la FDA. Paralelamente la desregulación de BCL2 es primordial en la patogénesis de FL, como consecuencia de la t (14; 18), presente en un 85% de los pacientes, y por lo tanto es un objetivo atractivo para novedosos enfoques terapéuticos. Venetoclax (ABT-199, AbbVie) es un pequeño inhibidor de BCL2, que mostró unos resultados del primer ensayo clínico no satisfactorios (respuesta global del 38%). De este primer estudio concluimos que Idelalisib interfiere en la comunicación de FL y su microambiente inmune, además potencia la actividad de venetoclax atacando a las células tumorales, lo que representa una terapia de combinación prometedora que puede mejorar el resultado del tratamiento de FL.

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Gardner, Jacob Andrew. "GPCR Signaling in the Genesis and Progression of Pancreatic Cancer". Diss., Temple University Libraries, 2009. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/35453.

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Molecular Biology and Genetics
Ph.D.
Ductal adenocarcinomas of the pancreas are the 4th most common cause of cancer death. The 1 and 5 year survival rates for all stages combined are currently 26% and 5% respectively. Median survival is less than 6 months. Despite remarkable progress in the fields of genetics, cancer biology, and advances in surgical techniques as well as chemotherapeutics, our ability to recognize and treat patients with pancreatic cancer remains poor. GPCR signaling modules have been increasingly implicated in the genesis and progression of pancreatic cancers. Aberrant agonist production, receptor expression and dysfunctional signaling resulting from genomic instability in a background of a heterotopic tumor-stromal microenvironment, contribute to the initiation, progression, and eventual metastasis of the disease. Numerous GPCR agonists, including lysophosphatidic acid (LPA), along with their cognate receptors have been implicated in this oncogenic process. LPA, one of the simplest bioactive lipids, has been shown to be a potent stimulant of metastatic behavior in in vitro models. It also acts as a mitogen by inducing proliferation and cell survival pathways in various normal and transformed cell lines. In patients with pancreatic cancer both the receptors and ligand have been found to be overexpressed. It has been noted that pancreatic cancer cell lines expressing higher levels of the LPA receptors present with greater motility. This has led to the hypothesis that LPA contributes to the progression of pancreatic cancer through the promotion of a metastatic phenotype. However, the underlying mechanisms have not been well described. LPA receptors have been shown to couple to the Gi, Gq, or G12 family of heterotrimeric G proteins. Consequently, signals transduced through these receptors have been shown to stimulate Gαi, Gαq, and Gα12/13 dependent pathways. While earlier studies have linked Gαi to LPA induced migration, there is recent evidence to suggest that Gα13 may provide a major signaling mechanism for LPA receptors stimulating migration in diverse cell types including cancer cell lines. Given the ominous nature of pancreatic cancers it is of critical importance to understand the mechanisms that promote more malignant phenotypes and to assess the role of Gα13 in this process. The goal of this thesis therefore is to define the role of Gα13 in LPA-mediated migration of pancreatic cancer cells. To assess the oncogenic potential of LPA and the role of Gα13 in stimulating the migration of pancreatic cancer cells, a panel of pancreatic cancer cell lines was assembled and characterized with regard to their expression of the LPA receptors as well as the Gα subunits of the heterotrimeric G proteins. These cell lines were further studied through a series of proliferation, wound healing, and transwell migration assays to assess the role of LPA in the induction of proliferation and migration in pancreatic cancer cells. The results demonstrated that LPA functions as a mitogen in certain pancreatic cancer cell lines, but is a potent stimulant of cell motility and invasive migration. Interestingly, these studies indicated that this response proceeds through routes that may not involve Gαi, as a potent migratory response was observed in MDAPanc28 cells which lack expression of the Gαi subunit. This was verified through the transwell assays conducted in the presence of PTX demonstrating that migration occurs independently of PTX sensitive mechanism and thus independently of Gαi.. Using a dominant negative mutant strategy, the studies presented in this thesis establishes the role of Gα13 in mediating LPA-LPAR stimulated migration of pancreatic cancer cells. Using pancreatic cancer cell lines that stably express the competitively inhibitory dominant negative mutant of Gα13, the ability of these mutants to inhibit a LPA mediated migratory response was monitored by wound-healing as well as transwell migration assays The results of these studies indicated a substantial attenuation of the migratory response and demonstrated for the first time the critical role of Gα13in LPA induced migration in a pancreatic cancer cell line.
Temple University--Theses

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Senarath, Kanishka D. "Interrogation of GPCR-G Protein Signaling using Novel Optogenetic Tools". University of Toledo / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=toledo155681039815937.

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Jha, Ankita. "Quantitative control of GPCR organization and signaling by endocytosis in epithelial morphogenesis". Thesis, Aix-Marseille, 2017. http://www.theses.fr/2017AIXM0393/document.

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Au cours de la gastrulation de l’embryon de Drosophile, l’activation apicale du cytosquelette d’acto-myosine orchestre la constriction apicale dans le mésoderme en invagination ainsi que l’intercalation cellulaire dans l’ectoderme en extension. Un contrôle quantitatif de l’activité des GPCRs et, par conséquent, de l’activation de Rho1 est à l’origine des différences de déformation des cellules du mésoderme et de l’ectoderme mais ces mécanismes demeurent incompris. L’activité du GPCR Smog se concentre respectivement en deux compartiments distincts à la surface de la membrane plasmique (PM) et dans ses invaginations (PMI). Au moyen de la FCS, nous avons étudié la surface de la PM et pu montrer que Smog oligomérise en homo-clusters en réponse à son activation par le ligand Fog. L’endocytose de Smog est facilitée par la kinase Gprk2 et sa protéine adaptatrice la β-Arrestine-2 qui retire Smog actif de la PM. Lorsque que la concentration de Fog est élevée ou que l’endocytose est réduite, Smog s’organise en homo-clusters et s’accumule au niveau des PMI qui agissent comme des centres d’activation de Rho1. Une concentration plus importante d’homo-clusters de Smog et un nombre plus important PMI dans le mésoderme par comparaison avec l’ectoderme. Répartition dynamique de Smog actif à la surface de la PM ou dans ses invaginations impacte directement sur la signalisation Rho1. Les PMI accumulent de hauts niveaux de Rho1-GTP suggérant qu’elles forment des centres de signalisation. La concentration de Fog et l’endocytose de Smog sont des processus régulateurs couplés qui contrôlent la différence quantitative d’activation de Rho1 dans le mésoderme et l’ectoderme de la Drosophile
During Drosophila gastrulation, apical activation of the actomyosin networks drives apical constriction in the invaginating mesoderm and cell-cell intercalation in the extending ectoderm. Here, we show that cell-surface G-protein coupled receptor, Smog activates G-proteins, Rho1 and Rho-kinase that is required for apical constriction and cell-cell intercalation. Quantitative control over GPCR activity and thereby Rho1 activation underlies differences in deformation of the mesoderm and ectoderm cells but the mechanisms remain elusive. We show that GPCR-Smog activity is concentrated on two different apical plasma membrane compartments i.e. the surface and the plasma membrane invaginations. Using FCS, we probe the surface of the plasma membrane (PM) and show that Smog homo-clusters in response to its activating ligand Fog. Endocytosis of Smog is facilitated by the kinase Gprk2 and the adaptor protein β-Arrestin-2 that clears active Smog from the surface of PM. When Fog concentration is high or endocytosis is low, Smog arranges in homo-clusters and accumulates in plasma membrane invaginations (PMI), that are hubs for Rho1 activation. Lastly, we find high Smog homo-cluster concentrations and numerous apical PMIs in the mesoderm compared to the ectoderm. We identify that dynamic partitioning of active Smog on the surface of the PM or PMI directly impact on Rho1 signaling. PMIs accumulate high Rho1-GTP suggesting they form signaling centers. Fog concentration and Smog endocytosis form coupled regulatory processes that regulate quantitative differential Rho1/MyoII activation in the Drosophila mesoderm and ectoderm

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Cutolo, Pasquale. "Etude de l'interaction structurelle et fonctionnelle entre la chimiokine CXCL12 et ses récepteurs : CXCR4 et ACKR3/CXCR7". Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS550/document.

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L'axe formé par la chimiokine CXCL12 et son récepteur CXCR4 est conservé chez les vertébrés où il joue un rôle important dans l'embryogenèse et la vie adulte, régule de nombreux processus des réponses immunitaires grâce à ses fonctions dans la migration cellulaire, la survie et la prolifération.En outre, cet axe est impliqué dans les processus pathologiques tels que les cancers (croissance et métastase) et immunodéficiences ainsi que des dysfonctionnements (par exemple l'expression dérégulée, polymorphismes ou mutations) et est également détourné par certains agents pathogènes (par exemple le virus de l'immunodéficience humaine, virus du papillome humain).Un grand groupe de travail est consacré à cette paire comme cible thérapeutique, mais seulement un composé (à savoir Plérixafor) a atteint l'approbation pour une utilisation clinique faisant le potentiel de cet axe comme cible de médicament encore inexploré.Bien que cet axe est l'objet d'un grand intérêt, des questions demeurent quant aux déterminants structurels impliqués dans l'interaction CXCL12/CXCR4.Cependant, la structure récemment résolue par diffraction de CXCR4 a donné quelque indice au sujet de ces questions, et au delà, la possible stoichiométrie entre CXCL12 et CXCR4.Plusieurs éléments de preuve appuient le concept que les formes CXCR4 homo- et hétéro- oligomères (qui peut contribuer à la diversité des fonctions de récepteur), telles que la structure de diffraction, le gain de fonction d'un récepteur CXCR4 mutant responsable du syndrome WHIM et la modulation allostérique des fonctions de CXCR4 par CXCR7 (ACKR3), le second récepteur de CXCL12. La possibilité de former des oligomères ouvre de nombreuses questions en matière de CXCL12 et ses interactions avec CXCR4 et CXCR7/ACKR3. La stoichiométrie de cette interaction reste une question ouverte, comme le récepteur est capable de former des oligomères avec le même récepteur ou autre récepteurs, en particulier CXCR7/ACKR3. Ce récepteur, connu comme scavenger, n'a pas de structure résolue et son mécanisme d'interaction avec CXCL12 reste inconnu.Afin d'étudier les interactions CXCL12/CXCR4/CXCR7, nous avons appliqué plusieurs techniques de modélisation moléculaire tels que peptid-peptide docking et simulations de dynamique moléculaire.Objets du projet ont étés : la résolution des possibles formes stoichiométriques de l'interaction CXCR4/CXCL12 (modélisation moléculaire, docking et dynamique); la modélisation de la structure du récepteur CXCR7/ACKR3 et son interaction avec CXCL12 (homology modeling), avec caractérisation des domaines et des résidus clef de l'activation des pathways de signalisation en aval du récepteur (mutants CXCR7/ACKR3); l'étude et la caractérisation de nouveaux outils innovants pour la détection de l'oligomerisation de ces récepteurs en conditions endogènes. (Nanobodies, HTRF)Les résultats du premier objectif ont été publiés en janvier 2016 : PMID 26813575.La modélisation de CXCR7/ACKR3 nous a permit de générer plusieurs mutants du récepteur pour tester nos hypothèses sur l’activation.Les nanobodies caractérisés pour CXCR4 seront utilisé dans une deuxième étude pour l’identification des formes oligomériques du récepteur sur tissus et cellules
The axis formed by the chemokine CXCL12 and its receptor CXCR4 is conserved in vertebrates where it plays an important role in embryogenesis and adult life, regulates many processes of immune responses through its functions in cell migration, survival and proliferation.In addition, this axis is involved in pathological processes such as cancers (growth and metastasis) and immune deficiencies and malfunctions (eg deregulated expression, mutations or polymorphisms) and is also hijacked by certain pathogens (eg HIV, human papilloma virus).A large working group is dedicated to this pair as a therapeutic target, but only a compound (ie Plerixafor) achieved approval for clinical use by the potential of this area as a drug target unexplored.Although this axis is the subject of great interest, questions remain about the structural determinants involved in CXCL12 / CXCR4 interaction.However, the recently resolved diffraction structure of CXCR4 gave some clue about these questions, and beyond possible stoichiometry between CXCL12 and CXCR4.Several lines of evidence support the concept that forms CXCR4 homo- and hetero-oligomers (which can contribute to the diversity of the receptor functions), as shown in the diffraction structure, the gain function of a mutant CXCR4 receptor responsible for the syndrome WHIM and allosteric modulation of CXCR4 functions by CXCR7 (ACKR3), the second receptor of the chemokine CXCL12. The ability to form oligomers opens many issues of CXCL12 and its interaction with CXCR4 and CXCR7 / ACKR3.The stoichiometry of this interaction still remains an open question, as the receptor is capable to form oligomers with the same receptor or other receptors, particularly CXCR7 / ACKR3. This receptor, known as scavenger, has not solved structure and the mechanism of interaction with CXCL12 is unknown.To study the interactions CXCL12 / CXCR4 / CXCR7, we applied several molecular modeling techniques such as peptide-peptide docking and molecular dynamics simulations.Objectives of this project were: the resolution of the different stoichiometric forms for the interaction of CXCR4 and CXCL12 (molecular modeling, docking and dynamic); modeling the CXCR7 / ACKR3 receptor structure and its interaction with CXCL12 (homology modeling), with the characterization of domains and residues key in the activation of downstream signaling pathways of the receptor (CXCR7 / ACKR3 mutants); the study and characterization of new innovative tools for the detection of oligomerization of these receptors in endogenous conditions. (Nanobodies, HTRF)The results of the first objective were published in January 2016: PMID 26813575.Modeling of CXCR7 / ACKR3 allowed us to generate several mutants of the receptor to test our hypothesis about the activation pathways.Nanobodies were fully characterized for CXCR4 to be used in a second study to identify oligomeric forms of the receptor in tissues and cells

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Adamson, Roslin Jane. "Probing GPCR-Gα interactions : a functional study by EM and SPR". Thesis, University of Oxford, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.669745.

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Mason, Vicki Louise. "Effects of different assay configurations on pharmacological profiling of GPCR targets". Thesis, University of Reading, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.494820.

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The effect of assay type and assay configuration on the pharmacological profile of a range of ligands acting at the D2 dopamine receptor was investigated in this thesis. The ligands explored have previously been shown to display a spectrum of efficacy at the D2 dopamine receptor. In competition ligand binding studies versus [³H]spiperone in HEK-293CREβlacD₂ˡ cell membranes, receptor-G protein coupling was increased in the absence of Na⁺, however the pK/ values for some agonists were higher in the presence of Na⁺, which suggested that different ligands are able to induce different receptor conformations.

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Santos, Geisa Aparecida dos. "Análise comparativa de perfis de sinalização do receptor AT1 ativado por agonistas seletivos para a via de -arrestinas". Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/17/17131/tde-31102013-150124/.

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Os receptores acoplados à proteína G (GPCRs), também chamados de receptores 7TM, são conhecidos por regular virtualmente todos os processos fisiológicos em mamíferos e cerca de 40% de todas as drogas comerciais agem através destes receptores. A sinalização mediada por eles é classicamente atribuída à proteína G, que é ativada pela troca de GDP por GTP, promovendo a separação das subunidades G e G, e leva à produção de mensageiros secundários como cAMP, Ca2+ e DAG. Após a resposta os GPCRs são fosforilados pelas quinases de GPCRs (GRKs), sinalizando para recrutamento das -arrestinas citoplasmáticas, que por sua vez desencadeiam a formação de endossomos internalizando e dessensibilizando o receptor. Entretanto, estudos mostram que este endossomo, contendo o complexo ligante-receptor--arrestina, pode interagir com proteínas sinalizadoras no citoplasma desencadeando vias de sinalização independentes de proteína G. Recentemente foram descritos para diferentes receptores, ligantes capazes de ativar seletivamente uma das duas vias, proteína G ou -arrestina, chamados agonistas seletivos. O receptor AT1 é um GPCR particularmente interessante no estudo do agonismo seletivo, tanto por sua vasta expressão em tecidos quanto pelo conhecimento de agonistas seletivos já estabelecidos, tais como os ligantes SII e TRV120027. O objetivo deste trabalho foi analisar comparativamente os perfis de sinalização decorrente da ativação de AT1 por SII ou TRV120027 através do uso de arranjos de quinases e da modulação de genes relacionados a sinalização de GPCRs. Ang II que é ligante natural e total (ativa via dependente de proteína G e de -arrestina) neste receptor foi usada como controle para fins de comparação. Nossos dados mostraram que o perfil da sinalização mediada pelo receptor AT1 varia não só entre AngII e os agonistas seletivos, mas também entre os dois ligantes seletivos SII e TRV120027, mostrando que a interação receptor-ligante pode influenciar a sinalização em um grau mais refinado, além da ativação dependente de -arrestina ou proteína G. Estes dados mostram que existem perspectivas para o desenvolvimento futuro de ligantes com ainda maior grau de seletividade.
G protein coupled receptors (GPCRs), also known as 7TM receptors, are known to regulate virtually all physiological processes in mammals and approximately 40% of all current clinical drugs act by modulating such receptors. The signaling mediated by them is classically by coupling to G protein, which is activated by exchanging bound GDP for GTP, dissociation of G and G subunits, then leading to production of second messengers such as cAMP, Ca2+, and DAG. After the signal transduction, GPCR are phosphorylated by GPCR kinases (GRKs), followed by recruitment of cytoplasmic -arrestins, which initiate the endosome formation with consequent internalization and desensitization of the receptor. However, is has been demonstrated that the endosome assembling the ligand-receptor--arrestin complex can interact with cytoplasmic signaling proteins, therefore activating signaling pathways independently of G protein coupling. Recently, for different receptors, it has been described ligands capable of selectively activating one of these signaling pathways, G protein or -arrestin, called biased agonists. The AT1 receptor is a particularly interesting GPCR for the study of biased agonism, either due to its wide tissue expression as well as also due the existence of known and established biased ligands, such as SII and TRV120027. The aim of our study was to comparatively analyze the AT1 receptor signaling pathways profiles after activation by SII or TRV120027, using kinases arrays, and expression modulation of genes related to GPCRs signaling. AngII is the natural and full agonist of this receptor (activates both G protein and -arrestin signaling pathways) was used for comparison. Our data show that the signaling profile mediated by AT1 receptor can be distinct not only when comparing the profiles from AngII and the biased agonists, but also when comparing the profiles from the two biased ligands SII and TRv120027; revealing that the complex ligand-receptor can influence the downstream signaling pathways in a fine-tune way, further to the activation of -arrestin or G-protein. This data show that there are perspectives for the future development of ligands with even higher degree of selectivity.

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Jaén, Cristina. "Differential coupling of RGS3s and RGS4 to GPCR-GIRK channel signaling complexes". [Tampa, Fla] : University of South Florida, 2006. http://purl.fcla.edu/usf/dc/et/SFE0001533.

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Altosaar, Katrin. "Dimer-dependent allosteric modulation within GPCR signalling complexes can influence signalling diversity". Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=114353.

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G protein-coupled receptors (GPCRs) comprise the largest group of cell surface receptors, translating environmental signals into cellular responses via cognate G protein partners. Contrary to our initial understanding, most GPCRs do not function in living cells as monomers, but most likely dimers, or even larger arrays of receptors. Standard drug design approaches rely on the notion that drugs binding the two receptors in a given dimer likely function independently of one another. However, this view has been challenged by recent work showing that ligand binding at both receptors can modulate dimeric receptors via allosteric communication. While one receptor may actually be needed to drive signalling, the other acts to control or modulate these signals, without a direct signalling outcome itself. Based on the notion of allosteric modulation within homo- and heterodimers, I tested and compared changes in signalling downstream as well as at the level of the receptor-G protein-effector (RGE) complex in response to different combinations of ligands at each protomer. Using a combination of calcium, cyclic adenosine monophosphate, and mitogen-activated protein kinase signalling assays, I have demonstrated functional interactions for a putative D2 dopamine receptor, oxytocin receptor heterodimer (D2R/OTR), in HEK 293 cells. Immunoprecipitation, bioluminescence resonance energy transfer (BRET) and confocal microscopy experiments reveal D2R and OTR do in fact form a heterodimer in vitro, which may explain the nature of these potential allosteric functional interactions. Using BRET, I assessed the RGE complex conformational dynamics in HEK 293 cells for two other heterodimers, β2-adrenergic receptor with cannabinoid CB1 receptor (β2AR/CB1R) and β2AR/OTR, in order to determine how they manifest in parallel to signalling events themselves. These studies reveal functional interactions can occur in terms of signalling complex conformation. Thus GPCR signalling can be modulated by its partner receptor at the level of downstream effector signalling or at the level of the signalling complex itself. With that said, putative heterodimers need to be reanalyzed in vivo for their allosteric properties, which may explain some of the side effects of so many drugs, and may have implications in drug design.
Les récepteurs couplés aux protéines G (RCPG) constituent le plus grand groupe de récepteurs de la surface cellulaire, qui traduisent les signaux environnementaux en réponses cellulaires via leurs protéines G associées. Contrairement à notre compréhension initiale, la majorité des RCPG ne fonctionnent pas en tant que monomères, mais possiblement en tant que dimères ou même oligomères. Les approches actuelles de conception de médicament estiment que lors de la liaison d'un médicament aux deux récepteurs d'un dimère quelconque, ces derniers fonctionnent potentiellement indépendamment l'un de l'autre. Cependant, cette notion a été reconsidérée par une étude récente montrant que la liaison d'un ligand aux deux récepteurs peut les altérer par voie de communication allostérique. Alors qu'un premier récepteur peut être requis pour initialiser la signalisation, un second peut contrôler ou modifier ces signaux, n'ayant pas nécessairement une signalisation directe comme résultante. Dans l'étude suivante, basée sur la notion de modulation allostérique au sein d'homodimère et d'hétérodimère, les changements de signalisation en aval ainsi qu'au niveau du complexe récepteur/protéine G/effecteur (RGE) ont été étudiés et comparés en réponse à différentes combinaisons de ligands pour chaque protomère. En utilisant une combinaison d'essais de signalisation de calcium, d'adénosine monophosphate cyclique (cAMP) et de protéine kinase activée par des agents mitogènes (MAPK), une interaction fonctionnelle entre le récepteur dopaminergique D2 et le récepteur de l'ocytocine (D2R/OTR) a été démontrée dans les cellules HEK 293. Des expériences d'immunoprécipitation, de transfert d'énergie de résonance par bioluminescence (BRET) et de microscopie confocale ont révélé la présence d'hétérodimère entre le D2R et l'OTR in vitro, ce qui pourrait expliquer la nature des interactions fonctionnelles allostériques. En utilisant la technique de BRET, la dynamique fonctionnelle du complexe RGE dans les cellules HEK 293 a été examinée chez deux autres hétérodimères, soit celui composé du récepteur adrénergique β2 et du récepteur cannabinoïde CB1 (β2AR/CB1R) et l'hétérodimère β2AR/OTR, afin de déterminer comment ils traduisent les évènements de signalisation. Ces études démontrent donc qu'une interaction fonctionnelle peut survenir sur le plan de la conformation du complexe de signalisation. Par conséquent, la signalisation d'un RCPG peut être modulée par son récepteur partenaire au niveau des effecteurs ou au niveau du complexe de signalisation lui-même. Pour cette raison, il serait impératif de réanalyser in vivo les propriétés allostériques d'hétérodimères putatifs, ce qui pourrait expliquer certains effets secondaires d'une multitude de médicaments et ce qui pourrait impliquer des changements majeurs dans la façon de concevoir de nouveaux médicaments.

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Hall, Caroline Jane. "The effect of GPCR crosstalk on intracellular Ca2+ responses and downstream signalling". Thesis, University of Leicester, 2011. http://hdl.handle.net/2381/9528.

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Jaén, Cristina. "Differential coupling of RGS3s and RGS4 to GPCR-GIRK channel signaling complexes". Scholar Commons, 2006. http://scholarcommons.usf.edu/etd/2571.

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'Regulators of G protein signaling' (RGS proteins) modulate the G proteincycle by enhancing the GTPase activity of Ga subunits. These changesaccelerate the kinetics of ion channel modulation by Gai/o-coupled receptors(GPCRs) such as the G protein-gated inward rectifier K+ (GIRK/Kir3) channel. Myexperiments indicate that a single cerebellar granule (CG) neuron, a cell type thatendogenously expresses GIRK channels is able to express a wide variety ofRGS proteins. I selected two of them, which are widely expressed andtranscriptionally regulated during pathophysiologic conditions, to compare theirfunctional properties. I originally described the differential modulatory effects oftwo RGS proteins, the RGS3 short isoform (RGS3s) and RGS4, on muscarinicm2 and serotonin 1A receptor-coupled Kir3.1/Kir3.2a channels expressed inChinese hamster ovary (CHO-K1) cells. Both RGS3s and RGS4 acceleratedGIRK activation and deactivation current kinetics in a similar way. However, onlyRGS3s si gnificantly decreased the maximal GIRK current (Imax) elicited by ACh(~45% inhibition) and significantly increased the EC50 for both GPCRs. Thehypothesis that emerged from this initial study was that the distinct RGS4 Nterminaldomain mediated a direct coupling of RGS4 to GPCR-GIRK channelsignaling complexes that was not shared by RGS3s. To test this hypothesis, Iepitope-tagged several GPCRs, the Kir3.1 subunit, RGS3s, RGS4, and severaldeletion mutants and chimeras for co-immunoprecipitation experiments. Using anepitope-tagged degradation resistant RGS4 mutant RGS4(C2V), I detected coprecipitationof different GPCR-GIRK channel complexes with RGS4 but notRGS3s.The functional impact of RGS4 coupling to the GPCR-Kir3 channelcomplex versus uncoupled RGS3s was not apparent in recordings from CHO-K1cells presumably due to a high degree of RGS collision-coupling. Controlledexpression in Xenopus oocytes revealed a 30-fold greater potency for RGS4 inthe accelerating GIRK channel gating kinetics. In summary, these findings demonstrate that one of the ways for the cellto achieve signaling pathway specificity may be through selective coupling of thedifferent GPCR-effector-RGS protein complexes.

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26

Ranganathan, Anirudh. "The impact of GPCR structures on understanding receptor function and ligand binding". Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-129879.

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G protein-coupled receptors (GPCRs) form the largest superfamily of eukaryotic membrane proteins and are responsible for the action of nearly 30% of all marketed drugs. For a long period, efforts to study these receptors were limited by the paucity of atomic-resolution structural information. Numerous receptors spread across the GPCR superfamily have recently been crystallized, revealing crucial clues about receptor function and ligand recognition. The work in this thesis has primarily focused on using computational techniques to capitalize on this increasing amount of structural information. In papers I, II, and III protocols were developed to identify novel ligands for pharmaceutically important targets from in silico screens of large chemical libraries. In these papers, the fragment-based lead discovery (FBLD) approach was evaluated for GPCR targets using molecular docking screens. The high hit-rates obtained in these studies indicate promise for the use of computational approaches for fragment screening. In paper IV, molecular dynamics was used to identify a possible role for a conserved ionizable residue (Asp792.50) as a protonation switch during the activation process of the β2 adrenergic receptor. Analyses from this paper indicated that this residue could also perform a similar function in other class A GPCRs. Papers V and VI detail the modeling strategy followed during the GPCR Dock 2013 assessment to blindly predict the structure of two serotonin receptor subtypes (5-HT1B and 5-HT2B) bound to ergotamine. The developed ligand-steered homology modeling protocol was largely successful resulting in the best-ranked predictions for the 5-HT1B subtype. It is hoped that the work described in this thesis has highlighted the potential for structure-based computational approaches to identify novel ligands for important pharmaceutical targets and improve understanding of GPCR function.

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

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27

Bahena, Silvia. "Computational Methods for the structural and dynamical understanding of GPCR-RAMP interactions". Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-416790.

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Protein-protein interaction dominates all major biology processes in living cells. Recent studies suggestthat the surface expression and activity of G protein-coupled receptors (GPCRs), which are the largestfamily of receptors in human cells, can be modulated by receptor activity–modifying proteins (RAMPs). Computational tools are essential to complement experimental approaches for the understanding ofmolecular activity of living cells and molecular dynamics simulations are well suited to providemolecular details of proteins function and structure. The classical atom-level molecular modeling ofbiological systems is limited to small systems and short time scales. Therefore, its application iscomplicated for systems such as protein-protein interaction in cell-surface membrane. For this reason, coarse-grained (CG) models have become widely used and they represent an importantstep in the study of large biomolecular systems. CG models are computationally more effective becausethey simplify the complexity of the protein structure allowing simulations to have longer timescales. The aim of this degree project was to determine if the applications of coarse-grained molecularsimulations were suitable for the understanding of the dynamics and structural basis of the GPCRRAMP interactions in a membrane environment. Results indicate that the study of protein-proteininteractions using CG needs further improvement with a more accurate parameterization that will allowthe study of complex systems.

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Scarlin, Hugh. "Studies on the synthesis of novel PP2A inhibitors and a GPCR detergent". Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/7370/.

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Chapter 1 While targeting kinases in oncology research has been explored extensively, targeting protein phosphatases is currently in its infancy. However, a number of pharmaceutical companies are currently looking to expand their research efforts in this area. PP2A has been shown to down-regulate ERK5, a mitogen-activated protein kinase (MAPK) that has been shown to be important in driving the invasive phenotype of prostate cancer. Fostriecin and its related structural analogues PD 113,270 and 113,271 have been shown to inhibit a mitotic entry checkpoint in cell growth through the potent and selective inhibition of protein phosphatases PP1, PP2A, and PP4 (IC50 of 45 μM, 1.5 nM, and 3 nM respectively). Fostriecin is one of the most selective protein phosphatase inhibitors disclosed to date with a 104 fold selectivity for PP2A/PP4 versus PP1. Unfortunately, fostriecin and its analogues are very unstable, and this instability has effectively prevented them from being used as effective therapeutic leads. The microcystins and nodularins on the other hand, exhibit significant inhibitory activity against PP1 and PP2A (IC50 = 26 pM and 1.8 nM respectively), but their high toxicity has prevented any therapeutic application. Truncation of the ADDA chain from these polypeptides completely attenuates PP inhibitory activity. Simpler analogues incorporating the N-acylated ADDA chain and D-Ala retain moderate activity against PP1 and PP2A (IC50 = 1.0 μM and 0.17 μM respectively). The generation of a new series of fostriecin analogues to further expand its structure-activity relationship is envisaged with a view to creating new more stable PP2A inhibitors. It was hoped that by incorporating some of the more stable structural features of ADDA into fostriecin that stability and activity could be reconciled. With that in mind a series of PP2A inhibitors were synthesised and biologically evaluated. Chapter 2 GPCRs are an important area of research and are the targets of a quarter of the drugs on the market (2005). As a result, GPCRs continue to be at the forefront of research in both small and large drug companies. However one of the difficulties in studying this diverse class of membrane proteins is their tendency to denature in aqueous solution. As a result there is a pressing need to develop new detergents to solubilise, stabilise and crystallise GPCRs in their native form for further study. Cholesterol analogues have been shown to be important for stabilising membrane proteins and preventing their thermal inactivation. In addition the β2-adrenergic receptor, a GPCR membrane protein, has been crystallised in the active state with two cholesterol molecules bound between the I, II, III and IV helices of the protein. This appears to represent a distinct cholesterol binding pocket on the membrane protein that is speculated to be conserved across up to 44% of the rhodopsin class of GPCRs. CHOBIMALT is a cholesterol-based detergent that has been shown to exhibit promising GPCR-stabilising properties. When benchmarked against other cholesterol based detergents it was found to be superior to all others tested except for cholesteryl hemisuccinate.1 CHOBIMALT has an aggregation number of roughly 200 and forms 210 ± 30 kDa micelles, which are significantly larger than those of most detergents used for biological systems which is likely due to the packing constraints associated with CHOBMALT’s large polar headgroup.2 As a result, CHOBIMALT is used mostly as an additive to other commercially available detergents in order to decrease micelle size. A branched dimaltoside motif is common in recently synthesised detergents by Chae and co-workers. These detergents have shown promising detergent properties, for example the maltose neopentyl glycol (MNG) detergent synthesised by Chae. This branched dimaltoside detergent was shown to be able to solubilise and stabilise the very labile light harvesting complex I (LHI) from Rhodopsin capsulatus in its active form for 20 days with little loss of protein conformation.3 A cholesterol-based detergent was envisaged that combines the cholesterol framework of CHOBIMALT but replaces its linear tetrasaccharide with a branched dimaltoside. This detergent would then be investigated to assess its ability to solubilise, stabilise and crystallise GPCR proteins. This cholesterol-based detergent (shown below) was eventually synthesised in 9 linear steps from cholesterol.

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Elenko, Eric. "Localization and fate of GAIP following G[alpha]i̳ linked GPCR stimulation /". Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2001. http://wwwlib.umi.com/cr/ucsd/fullcit?p3026385.

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30

Heuninck, Joyce. "Analysis of CXCR4 and ACKR3 oligomerisation". Thesis, Montpellier, 2019. http://www.theses.fr/2019MONTT018.

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Mon travail de thèse s’est focalisé sur l’étude des récepteurs CXCR4 et ACKR3, deux récepteurs aux chimiokines. Ceux-ci jouent des rôles majeurs dans différentes fonctions physiologiques notamment le chimiotactisme des cellules immunitaires. Le dérèglement de leur activité est souvent associé à différents pathologies, notamment des cancers. Outre le fait que ces récepteurs lient tous les deux la même chimiokine, CXCL12, il a été montré qu’ils étaient capables d’exercer l’un sur l’autre des régulations croisées. Les mécanismes sous-tendant celles-ci sont très mal connus. Ils pourraient être liés à une compétition entre les récepteurs pour lier le même ligand CXCL12 ou à des régulations au niveau des voies de signalisation activées lors de la liaison d’une chimiokine. Ces régulations croisées pourraient également résulter de la formation de complexes de récepteurs appelés oligomères, complexes qui disposeraient de propriétés pharmacologiques particulières. De tels complexes ont été décrits dès les années 1990 dans des systèmes d’expression hétérologues mais leur existence et leurs rôles dans des systèmes natifs reste très largement débattus. Une des raisons est la difficulté à élaborer des outils moléculaires permettant l’étude de ces oligomères dans les tissus natifs.Le premier objectif de ma thèse a été l’élaboration d’outils permettant d’étudier l’existence de ces oligomères dans les systèmes natifs. En collaboration avec des laboratoires d’Amsterdam, j’ai développé des nanobodies fluorescents, petits anticorps produits par le lama, afin de marquer spécifiquement les récepteurs endogènes exprimés à la surface des cellules. Pour rendre fluorescent ces nanobodies, nous avons utilisé une technique originale permettant de greffer un fluorophore sur l’extrémité C-terminale de la molécule. Ces nanobodies conservent des propriétés pharmacologiques remarquables puisqu’ils conservent de hautes affinité et spécificité pour leur cible. J’ai ainsi pu utiliser ces molécules et démontrer l’existence des oligomères CXCR4 dans des lignées cellulaires exprimant de façon endogène le récepteur CXCR4. Des analyses similaires sont en cours sur le récepteur ACKR3.Le second objectif de ma thèse a été de définir les rôles potentiels de ces oligomères. J’ai pu montrer que les hétéro-oligomères CXCR4 /ACKR3, complexes associant les deux types de récepteurs, possèdent des propriétés de liaison particulière. Ceux-ci semblent ne lier la chimiokine CXCL12 que sur le récepteur ACKR3 au sein du complexe. Cette asymétrie de liaison est très étonnante car le récepteur CXCR4 est capable de lier CXCL12 avec une cinétique bien supérieure à celle de ACKR3 pour ce même ligand. J’ai étudié les conséquences d’une telle asymétrie de liaison sur les propriétés de signalisation de ces récepteurs. Une analyse des différentes voies de couplage activées lors de la liaison de CXCL12 a été réalisée sur les récepteurs exprimés de façon isolée ou co-exprimés au sein d’une même cellule. Les résultats ne montrent pas de modifications majeures de leur propriété de couplage.Nous avons par ailleurs analysé la capacité de ces récepteurs à internaliser en absence ou en présence de ligand CXCL12. J’ai pu observer que les hétéro-oligomères CXCR4 / ACKR3 restaient vraisemblablement bloquer à la surface des cellules.Ces travaux ouvrent des perspectives intéressantes dans la mesure où elles constituent la première démonstration de l’existence d’oligomères CXCR4 dans des systèmes natifs.Par ailleurs l’observation d’une régulation différente de l’internalisation des hétéro-oligomères constitue une première piste conférant à ces complexes un rôle particulier dans les régulations croisées de l’activité que ces récepteurs exercent l’un sur l’autre
During my PhD, I focused on two chemokine receptors, CXCR4 and ACKR3. They have several important physiological functions, such as chemotaxis of immune cells. However, on the other hand, when their function is disturbed, they are involved in different immunological pathologies and cancer. Both receptors recognise the same chemokine, CXCL12 and many studies have reported a crosstalk between CXCR4 and ACKR3. However, the mechanisms behind this crosstalk are still poorly understood. This crosstalk can occur because both receptors are competing for CXCL12, at the level of signalling pathways or due to the formation of complexes between CXCR4 and ACKR3 receptors, called oligomers. The oligomers might have specific pharmacological properties different from the receptor monomers. Oligomeric complexes have been described since the nineties. Most of the studies on these oligomers were performed on heterologous expression systems, but still a lot of debate exists about their existence and their role in native tissues. One of the reasons behind this controversy is that studying oligomers in a native context is complicated, especially because we often lack the molecular tools for these studies.The first objective of my PhD was to generate efficient tools to study the existence of CXCR4 and ACKR3 oligomers in native systems. In collaboration with laboratories from Amsterdam and Ghent, we have developed fluorescent nanobodies, small antibodies produced by llamas. These specific tools allow the detection of receptors endogenously expressed at the cell surface. In order to fluorescently label these nanobodies, we have used an original strategy that can specifically attach the fluorophore to the C-terminus of the nanobody. Interestingly, the fluorescent nanobodies retain high affinity and specificity for their target. With these nanobodies, I have demonstrated the existence of CXCR4 oligomers in cell lines that endogenously express CXCR4. We are currently investigating the existence of ACKR3 oligomers.The second objective of my PhD consists of defining the functional roles of these oligomers. I have shown that CXCR4/ACKR3 hetero-oligomers have specific binding characteristics. It seems that CXCL12 is binding only to ACKR3 within this hetero-oligomer and that ACKR3 impairs the CXCL12 binding to CXCR4 within the hetero-oligomer. This is interesting, since we also have demonstrated that CXCL12 is binding much faster on CXCR4 than on ACKR3.In addition, I have studied the consequences of this negative cooperativity within the CXCR4/ACKR3 hetero-oligomer on different signalling pathways. We have compared conditions where the receptor was expressed alone or when receptors were co-expressed. No major modifications have been found on their signalling properties. However, when investigating the internalisation of CXCR4 and ACKR3, it seems that CXCR4/ACKR3 hetero-oligomers remain blocked on the cellular surface.This opens interesting perspectives, since it is the first time CXCR4 oligomers have been detected at an endogenous level. Moreover, the observation of a different internalisation pattern of the hetero-oligomer is a first step to further investigate the specific roles of these oligomers in the crosstalk between the receptors

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Brewer, Cynthia. "Use of PC12 cells to characterize PAFR-GPCR-mediated activity in neural precursors". Thesis, University of Ottawa (Canada), 2001. http://hdl.handle.net/10393/9170.

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Platelet activating factor (PAF: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active phospholipid mediator produced by neurons and glial cells. In adult central nervous system (CNS), physiological concentrations of PAF have been shown to mediate long-term potentiation, the purported cellular basis of mammalian learning and memory. Elevated levels of PAF are implicated in the pathophysiology of a number of neurodegenerative diseases, ischemia-reperfusion injury, human immunodeficiency virus-1 (HIV-1) associated dementia, and the developmental brain disorder Miller-Dieker Syndrome (MDS). Three neuropathologies are associated with sustained PAF exposure. It is not clear how these PAF-mediated effects are initiated in CNS. A seven transmembrane G-protein coupled receptor (PAFR-GPCR) has been cloned and mRNA is expressed in CNS. In addition, distinct intracellular receptor isoforms (iPAFRs) in rodent cortical extracts have been characterized pharmacologically. It is not known which of these PAF binding proteins initiate PAF-mediated neuropathology. Our ability to detect PAF receptors is further hindered by a lack of commercial PAF antibodies. To address these issues, in this thesis, I sought to: (1) Identify a neural precursor cell line capable of differentiating between PAFR-GPCR and PPAFR-initiated biological activity, (2) Determine the effect of PAFR-GPCR activation on differentiation of neural precursors to a neuronal phenotype, and (3) Characterize a new PAFR-GPCR antibody to facilitate localization of protein in CNS and CNS cell lines. (Abstract shortened by UMI.)

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Jackson, Verity. "Mechanistic studies of the adhesion-GPCR latrophilin and its interactions in neural guidance". Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:ecc6187a-3e20-4753-ada6-dc009ad7f6e2.

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The adhesion GPCRs are a poorly understood and evolutionarily ancient family of cell surface receptors, several of which have emerging functions in the development of the nervous system. aGPCRs comprise a large extracellular domain, providing binding sites for a variety of ligands, alongside a seven transmembrane domain characteristic of GPCRs. It has been proposed that aGPCRs may function as "context-recognisers", using their large ectodomains to bind different combinations of ligands depending on the molecular make-up of the environment. However there is a lack of direct evidence for this at a molecular level. The ectodomain of one subfamily of aGPCRs, the Latrophilins (Lphns) has been shown to directly interact with several ligands with roles in synaptogenesis and neural guidance. The best-validated of these interactions are those with Fibronectin Leucine-Rich Transmembrane (FLRT) proteins and the Teneurins. In addition, FLRT proteins, also interact with Uncoordinated5 (Unc5) proteins, mediating cell repulsion. Here I reveal that the FLRT-binding site of Lphn is bifunctional, mediating both cell adhesion and repulsion, and that Unc5 is capable of influencing the functional outcome of this interaction. Biophysics and structural studies show that fragments of the Lphn, FLRT and Unc5 ectodomains interact in an unusual and homologue-dependent stoichiometry. Despite the fact that Teneurin interacts with Lphn at a distinct site, Teneurin seems incapable of interacting with the Lphn-FLRT-Unc5 complex, but can form a ternary complex with Lphn and FLRT in the absence of Unc5. Alongside this I present a crystal structure of a large portion of a Teneurin ectodomain, revealing the ancient evolutionary origins of this receptor. Together these data provide strong molecular evidence for a role of Lphns as context-recognisers, by their abilities to bind diverse ligands in distinct combinations and variable stoichiometries.

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Bucco, Olgatina, i olgatina@gmail com. "Preparing, measuring and capturing G-protein coupled receptor (GPCR) signalling complexes for future development of cell-free assay technologies". Flinders University. medicine, 2006. http://catalogue.flinders.edu.au./local/adt/public/adt-SFU20060703.114912.

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G-protein coupled receptors (GPCRs) are integral membrane proteins which represent primary cellular targets for intracellular signalling. Many of these receptors are altered in disease states and hence are the target for over 50% of marketed drugs. Despite their physiological importance, high-throughput, cell-free assays which measure functional or signalling activity are only recently being investigated. The current approach by the pharmaceutical industry to initially screen compounds for functionality is to use heterologous cell-based assay formats. The aim of this work was to reconstitute a cell-free GPCR signalling system on an appropriate platform (surface) as a prototype for future rapid drug screening and other applications. The proof-of-concept approach involved using the ï¿½2A-adrenergic receptor (ï¿½2A-AR) containing cell membrane preparations as the model GPCR, reconstituted with a set of heterotrimeric G-proteins; Gï¿½i1 and ï¿½1ï¿½2 (the signal transducing complex being termed a ï¿½transductosomeï¿½). However, other receptors and G-proteins were also investigated. Receptors were initially obtained from natural (tissue) sources, however in the later stages they were expressed in a heterologous system (insect or mammalian expression system). G-proteins were expressed in Spodoptera frugiperida (Sf9) insect cells using the baculovirus expression system. Receptor expression was verified by radioligand binding assays and endogenous G-proteins were removed from membrane preparations using the chaotropic agent urea to allow for reconstitution with purified G-proteins. Signal transduction through the transductosome was measured using the [35S]GTPï¿½S binding assay. Receptor activated [35S]GTPï¿½S binding was used to determine functional reconstitution and to validate that the system was working in the normal physiological manner both on and off a surface (with surface attachment being via histidine attachment on the Gï¿½i1 (6xHIS) subunit). Using the captured (surface-attached) transductosomes, the IC50 values for Rauwolscine, Yohimbine (potent ï¿½2-AR antagonists), Prazosin (potent ï¿½1- AR antagonist) and Propranolol (ï¿½-AR antagonist) displayed the appropriate rank order for this class of receptor. This cell-free, surface-attached signalling complex prototype may have use in the future development of drug screening and discovery assay technologies as well as other applications as an alternative to cell-based assays which are not readily amendable to miniaturisation, long term storage and therefore stable robust microarray formats.

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Peyrassol, Xavier. "Développement et caractérisation d’anticorps de camélidés dirigés contre des récepteurs couplés aux protéines G et leur utilisation dans des approches structurales". Doctoral thesis, Universite Libre de Bruxelles, 2018. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/270870.

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Les camélidés possèdent une caractéristique immunologique particulière parmi les mammifères. En plus des anticorps conventionnels tétramériques composés de 2 chaînes lourdes et de 2 chaînes légères, on retrouve dans des proportions variant de 25 à 50% des anticorps dépourvus de chaînes légères. Le paratope de ces anticorps est dès lors constitué de la partie variable monomérique des chaînes lourdes. Ce domaine d’environ 15 kDa représente le plus petit fragment capable de lier un antigène et est communément appelé nanobody de par sa petite taille. Les nanobodies possèdent des propriétés uniques considérables comparés aux anticorps conventionnels, comme leur capacité à reconnaître des épitopes cryptiques mais aussi la possibilité de les modifier et les assembler facilement afin d’améliorer leurs propriétés. Ces dernières années, les nanobodies ont connu un intérêt grandissant tant au niveau de la recherche fondamentale qu’au niveau du développement de nouvelles solutions diagnostiques et thérapeutiques. Grâce à leur utilisation, la biologie structurale des RCPGs a connu des avancées significatives avec notamment l’obtention de la structure du récepteur β2-adrénergique dans une conformation active et complexé à une protéine G hétérotrimérique. Les RCPGs représentent la plus grande famille de récepteurs membranaires avec près de 800 récepteurs différents. Ils sont exprimés dans toutes les cellules de l’organisme et répondent à une large variété de ligands, les rendant indispensables dans la régulation de nombreux processus physiologiques. Ce rôle central dans la modulation des fonctions biologiques fait des RCPGs des cibles thérapeutiques de premier choix, comme en atteste le pourcentage élevé (30 à 40%) de médicaments dirigés contre cette classe de récepteurs et actuellement sur le marché. Depuis quelques années maintenant, la biologie structurale des RCPGs a connu un essor sans précédent avec à ce jour, près de 190 structures tridimensionnelles expérimentales résolues. Ces avancées ont permis de mieux comprendre les mécanismes d’action de ces récepteurs ainsi que le mode de liaison de ligands, ouvrant notamment de nouvelles perspectives thérapeutiques par le développement rationnel de nouvelles molécules.Au cours de ce travail, nous nous sommes efforcés de développer des outils et une méthodologie nous permettant de résoudre la structure expérimentale de 2 récepteurs :ChemR23 et VPAC1. Pour cela, nous avons développé et caractérisé des nanobodies dirigés contre ces 2 récepteurs. Nous avons montré que les nanobodies dirigés contre le récepteur ChemR23 possèdent des propriétés antagonistes en inhibant partiellement la libération calcique de cellules CHO surexprimant ChemR23 ainsi que le chimiotactisme de cellules dendritiques induit par la chémérine. Profitant de la modularité offerte par les nanobodies, nous avons conçu un nanobody bivalent, dont les propriétés antagonistes sont significativement améliorées. Concernant le récepteur VPAC1, nous avons identifié que les nanobodies générés reconnaissent un épitope présent au niveau du large domaine amino-terminal et distinct du site orthostérique du peptide VIP. Bien que dépourvu de propriétés fonctionnelles, 2 de ces nanobodies voient leur affinité augmentée en présence d’un agoniste, et diminué en présence d’un agoniste inverse. Enfin, nous montrons qu’ils sont utilisables pour la détection du récepteur endogène présent à la surface de leucocytes mais également au niveau de coupes de tissus gastro-intestinaux sains.En parallèle, nous avons mis au point la production de ces récepteurs dans des cellules d’insecte, permettant de produire les quantités nécessaires à des études structurales. Nous avons également apporté et validé diverses modifications à la structure de ces récepteurs, en vue d’augmenter leur stabilité une fois extraits de leur environnement natif. Un processus itératif nous a permis de déterminer les conditions optimales de solubilisation de ces récepteurs afin de maximiser l’obtention d’une forme monomérique et de minimiser la présence de formes multimériques ou dégradées. Nos premiers essais de purification par chromatographie d’affinité sur colonnes de nickel, ainsi que par chromatographie d’exclusion de taille, nous ont permis d’isoler des récepteurs entiers. Cependant, les chromatogrammes issus des purifications par chromatographie d’exclusion de taille suggèrent la présence de récepteurs en partie agrégés. De plus, nous n’avons pu déterminer précisément à ce jour si les récepteurs purifiés maintenaient une conformation native, prérequis indispensable pour réaliser des études cristallographiques.Bien que nous n’ayons pas résolu la structure expérimentale de ces 2 récepteurs, le travail réalisé dans le cadre de notre thèse de doctorat a permis de développer des nanobodies qui représentent des outils innovants pour l’études des RCPGs ainsi que de mettre au point des protocoles de production et de purification préliminaire des récepteurs ChemR23 et VPAC1 en vue de leur étude cristallographique.
Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)
info:eu-repo/semantics/nonPublished

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do, Nascimento Júnior Francisco. "Classificação de Proteínas usando Máquinas de Aprendizagem e Descoberta de Padrões". Universidade Federal de Pernambuco, 2008. https://repositorio.ufpe.br/handle/123456789/1596.

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Máquinas de aprendizagem têm sido aplicadas em diferentes problemas em Bioinformática. Similarmente, algoritmos de descoberta de padrões também têm sido usados para descobrir motifs em seqüências de proteínas, contribuindo na definição de assinaturas (tais como impressões digitais) que caracterizam classes funcionais de proteínas. Como por exemplo, a classe de receptores acoplados a proteína-G (GPCR) que representam uma das maiores famílias no Genoma Humano. Esta família é um dos grandes alvos de pesquisa para a descoberta e desenvolvimento de novas drogas, conseqüentemente, de grande interesse para a indústria farmacêutica. O modelo proposto nesta dissertação combina máquinas de aprendizagem, como SVM (Support Vector Machine) e MLP (Multilayer Perceptron), e métodos de descoberta de padrões no desenvolvimento de um procedimento para predizer a relação entre uma seqüência primária de proteínas e sua classe funcional. Como caso de estudo, este trabalho apresenta experimentos com a superfamília GPCR, usando padrões em forma de expressões regulares desta família extraídos pelo SPEXS (Sequence Pattern EXhaustive Search), um algoritmo para descoberta de padrões

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36

Garcia, De Las Bayonas Alain. "Spatio-temporal and quantitative control of Rho1 activity by GPCR signaling during tissue morphogenesis". Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0548/document.

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La constriction apicale des cellules du mésoderme et l'intercalation des cellules de l'ectoderme sont contrôlées par des réseaux contractiles d'acto-myosine dans l'embryon de Drosophile. Le niveau d'activation et la polarisation du cytosquelette d'acto-myosine détermine la nature des déformations cellulaires observées. Nous montrons que le GPCR Smog et les protéines G (Gα,Gβγ) en aval, activent la signalisation Rho1 et donc la Myosine-II dans les deux tissus. Dans l'ectoderme, Gα12/13 active Rho1 à la membrane apicale (aussi appelé compartiment médio-apical) tandis que les sous-unités Gβ13F-Gγ1 activent Rho1 en médio-apical et aux jonctions cellulaires. Les mécanismes contrôlant l’activation polarisée de Rho1 dans ce tissu demeurent incompris. Nous montrons ici que deux RhoGEFs, RhoGEF2 et une nouvelle RhoGEF Wireless/p114RhoGEF, activent Rho1 sous le contrôle des protéines G dans l’ectoderme. RhoGEF2 stimule Rho1 en médio-apical sous la dépendance de Gα12/13 alors que Wireless/p114RhoGEF contrôle l’activité de Rho1 aux jonctions avec Gβ13F-Gγ1. RhoGEF2 est présente aux jonctions et en médio-apical tandis que Wireless/p114RhoGEF est uniquement jonctionnelle où elle est recrutée par Gβ13F-Gγ1. Pour finir, Wireless/p114RhoGEF est absente des jonctions dans les cellules du mésoderme. En résumé, des GPCRs contrôlent l’activité spatio-temporelle de Rho1 au moyen de deux modules régulatoires dans l’ectoderme. Les protéines G transduisent le signal en recrutant et en activant deux RhoGEFs complémentaires en médio-apical et aux jonctions. Une variation dans la nature des GPCRs, protéines G ou des RhoGEFs détermine le contrôle tissu-spécifique de Rho1 au cours de la morphogenèse
Cell apical constriction in the mesoderm and cell intercalation in the ectoderm are controlled by contractile actomyosin networks in the developing Drosophila embryo. The extent of both actomyosin activation and polarization determines the nature of these cell deformations. We find that the GPCR Smog and the downstream G proteins (Gα,Gβγ) activate Rho1 signaling and thereby myosin-II in both tissues. In the ectoderm, Gα12/13 activates Rho1 at the apical membrane (also called medial-apical compartment) while Gβ13F-Gγ1 subunits promote Rho1 activity at the apical membrane and at cell junctions. How such a polarized activation of Rho1 is achieved remains unclear. Here, we show that two RhoGEFs, RhoGEF2 and a previously uncharacterized RhoGEF Wireless/p114RhoGEF, control Rho1 activity downstream of G proteins in the ectoderm. RhoGEF2 activates medial-apical Rho1 under control of Gα12/13 and Wireless/p114RhoGEF is required to mediate Gβ13F-Gγ1-dependent activation of Rho1 at junctions. RhoGEF2 is present both at junctions and at the apical membrane. In contrast, Wireless/p114RhoGEF only localizes at junctions together with Gβ13F-Gγ1 which recruit the GEF. Finally, we show that Wireless/p114RhoGEF is absent from junctions in the mesoderm. Collectively, GPCRs shape Rho1 activity through distinct biochemical modules in the ectoderm. Heterotrimeric G proteins transduce the signal by recruiting and activating two complementary RhoGEFs apically and at junctions. Variation in type of GPCRs, G proteins or RhoGEFs underlie the tissue-specific control of Rho1 signaling during morphogenesis

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Liebscher, Ines. "Die Physiologische Relevanz des G-Protein-gekoppelten Rezeptors GPR34". Doctoral thesis, Universitätsbibliothek Leipzig, 2011. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-64305.

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Die Familie der G-Protein-gekoppelten Rezeptoren (GPCRs) bildet die größte Gruppe von Membranrezeptoren im menschlichen Organismus. Für viele GPCRs sind bisher die physiologischen Funktionen nicht bekannt. Das biologische Verständnis der Funktionen im menschlichen Organismus dieser sogenannten „orphan“ GPCRs (oGPCRs) hat, aufgrund möglicher kausaler Beteiligung an der Pathogenese von Erkrankungen sowie deren therapeutische Beeinflussbarkeit, hohe medizinische Relevanz. Die GPCRs der P2Y12-ähnliche Rezeptorgruppe besitzen eine große physiologische Bedeutung bei der Thrombozytenaggregation und der Induktion der Migration von immunokompetenten Zellen in Schädigungsgebiete. Der ADP-Rezeptor P2Y12 kann durch verschiedene pharmakologische Wirkstoffe beeinflusst werden, was bereits klinisch-therapeutisch genutzt wird. Diese Gruppe von GPCRs enthält jedoch auch Mitglieder, deren Funktionen völlig unbekannt sind. Einer dieser oGPCRs ist der GPR34. Ziel dieser Arbeit war es, mittels verschiedener in-vitro-Methoden und anhand eines GPR34-defizienten Mausstamms die physiologische Relevanz dieses P2Y12-ähnlichen Rezeptors zu analysieren. Dazu wurde ein GPR34-Knockout-Mausmodell etabliert. Die GPR34-Defizienz hatte keinen wesentlichen Einfluss auf die Entwicklung, Morphologie, das Wachstum oder die Fertilität bei Mäusen. Die Ergebnisse aus Immunisierungs– und Infektionsstudien zeigten jedoch, dass dieser evolutionär hoch konservierte Rezeptor eine wichtige Funktion in der Feinkontrolle der zellulären Immunabwehr ausübt. Neben einer verstärkten Antwort im Delayed-type Hypersensitivity (DTH)-Test war die Abwehr einer Cryptococcus-Infektion in diesem GPR34-defizienten Tiermodell beeinträchtigt. Signifikant erhöhte Zytokinspiegel nach Antigen- bzw. Pathogenexposition deuteten auf eine gestörte Immunregulation in GPR34-defizienten Mäusen hin. Weiterführende Untersuchungen sollten sich der Identifizierung des endogenen Agonisten und der Funktion des GPR34 bei der Koordinierung der zellulären Immunreaktion widmen.

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Akkuzu, Selin. "The Functional Assessment Of Fluorecently Tagged Adenosine A2a And Dopamine D2 Receptors And Qualitative Analysis Of Dimerization Of Adenosine A2a And Dopamine D2 Receptor By Using Fret". Master's thesis, METU, 2013. http://etd.lib.metu.edu.tr/upload/12615474/index.pdf.

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Recently, several studies have demonstrated that G protein coupled receptors exist as homo/heterodimers or oligomers. Adenosine A2A receptors and dopamine D2 receptors are present as both homo- and heterodimer. In the GABAergic striatopallidal neurons A2AR are co- localized with D2 receptors (D2R), and establish functional A2AR-D2R heteromers, which modulates dopaminergic activity. Due to be involved in physiological processes, these receptors bear critical roles. Dopamine receptors play critical role in dopaminergic pathways in regulation of memory, food intake and psychomotor activity, etc. On the other hand, adenosine A2A receptors are involved in the regulations of neurotransmission, immune response and cardiovascular systems. Dopamine D2R andadenosine A2AR have been shown to interact in striatum and modulate dopaminergic activity The purpose of this study is to assess the functionality of EGFP (enhanced green fluorescent protein) and mCherry (a red fluorescent protein) tagged adenosine A2A and dopamine D2 receptors and to detect homo/ hetero-dimerization of these receptors in live cells via Fluorescence Resonance Energy Transfer (FRET). Understanding the mechanisms of the interaction between adenosine and dopamine signaling will help us to figure out some molecular mechanism of neurophysiological disorders. Furthermore, the fluorescence based live cell model could be used to observe the effects of potential anti-psychotic drugs on the interaction of these two receptors.

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Sheng, Yinglun. "G protein signaling and G protein coupled receptor (GPCR) pathway in Xenopus oocyte maturation". Thesis, University of Ottawa (Canada), 2005. http://hdl.handle.net/10393/29262.

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Xenopus laevis oocytes are physiologically arrested at the first meiotic prophase. Progesterone reinitiates meiosis (maturation) through inhibition of an oocyte adenylyl cyclase (AC) and reduction of intracellular cAMP. However, the mechanism by which progesterone regulates AC activity and cAMP level still remains unclear. In this thesis, I summarize work I conducted that collectively helps elucidate how high levels of cAMP might be achieved in G2 arrested oocytes. In Chapter 2, I describe our finding that inhibiting endogenous G-protein betagamma subunits, through the use of two structurally distinct Gbetagamma scavengers, causes hormone-independent oocyte maturation. In contrast, overexpression of Xenopus Gbeta1, alone or together with bovine Ggamma2, inhibits progesterone-induced oocyte maturation. These results for the first time implicate that an endogenous G protein coupled receptor system releases a Gbetagamma complex as the dominant meiosis inhibitor. Chapter 3 describes my research aiming to reveal the identity of the oocyte AC responsible for generating meiosis-inhibiting cAMP. I provide further evidence here that the ability of Gbetagamma to inhibit meiosis is attributed to the activation of an endogenous AC, rather than other possible Gbetagamma effectors. Through molecular cloning and biochemical characterization, I discovered that the likely AC candidate is Xenopus AC7, an isoform that is activated by Gbetagamma, but only in the presence of GTP-bound Gsalpha. The identification of xAC7 suggests that the maintenance of high levels of cAMP may require the cooperation of Gsalpha and Gbetagamma. Finally, in Chapter 4, I describe our efforts in identifying the GPCR(s) responsible for activating the cAMP signaling in prophase-arrested oocytes. A screening of known antagonists of GPCR(s) led to the identification of ritanserin, a potent antagonist of serotonin receptors, as a potent maturation inducer in Xenopus oocytes. Pharmacological and molecular studies, however, have ruled out the involvement of a known serotonin receptor in meiosis arrest. Instead, the most likely candidate is a "constitutively activated" GPCR that bears structural similarities to Xenopus serotonin receptor 7.

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Ellaithy, Amr. "Metabotropic Glutamate Receptor 2 Activation: Computational Predictions and Experimental Validation". VCU Scholars Compass, 2018. https://scholarscompass.vcu.edu/etd/5319.

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G protein-coupled receptors (GPCRs) are the largest family of signaling proteins in animals and represent the largest family of druggable targets in the human genome. Therefore, it is of no surprise that the molecular mechanisms of GPCR activation and signal transduction have attracted close attention for the past few decades. Several stabilizing interactions within the GPCR transmembrane (TM) domain helices regulate receptor activation. An example is a salt bridge between 2 highly conserved amino acids at the bottom of TM3 and TM6 that has been characterized for a large number of GPCRs. Through structural modeling and molecular dynamics (MD) simulations, we predicted several electrostatic interactions to be involved in metabotropic glutamate receptor 2 (mGlu2R) activation. To experimentally test these predictions, we employed a charge reversal mutagenesis approach to disrupt predicted receptor electrostatic intramolecular interactions as well as intermolecular interactions between the receptor and G proteins. Using two electrode voltage clamp in Xenopus laevis oocytes expressing mutant receptors and G-proteins, we revealed novel electrostatic interactions, mostly located around intracellular loops 2 and 3 of mGlu2R, that are critical for both receptor and G-protein activation. These studies contribute to elucidating the molecular determinants of mGluRs activation and conformational coupling to G-proteins, and can likely be extended to include other classes of GPCRs.

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MOLTENI, LAURA. "PHARMACOLOGICAL CHARACTERIZATION OF THE INTRACELLULAR SIGNALLING PATHWAY ACTIVATED BY TLQP-21". Doctoral thesis, Università degli Studi di Milano-Bicocca, 2017. http://hdl.handle.net/10281/158161.

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L’obesità è una condizione patologica per la quale non è stato ancora possibile identificare un trattamento farmacologico efficace. Il controllo dell’appetito è regolato da diversi fattori, la cui integrazione da parte dell’ipotalamo risulta nella generazione di specifiche risposte che regolano il bilancio energetico. TLQP-21 è un neuro-peptide coinvolto nella regolazione di diverse funzioni fisiologiche, incluso metabolismo, diabete, dolore e funzioni gastriche. Nonostante la recente identificazione di due potenziali recettori per TLQP-21, ad oggi, il meccanismo d’azione con cui agisce questo peptide rimane largamente sconosciuto. TLQP-21 viene attualmente proposto come un nuovo target per la cura di diverse patologie. Pertanto, lo scopo del nostro studio è stato quello di studiare il pathway intracellulare attivato dal legame di TLQP-21 col suo recettore, al fine di creare in futuro nuovi modelli utili allo sviluppo di farmaci che agiscano su questo sistema. Abbiamo inizialmente caratterizzato la capacità di TLQP-21 di indurre, in maniera dose-dipendente, un aumento di Ca2+ intracellulare nelle cellule CHO, N9, e RAW264.7. In particolare, l’aumento di Ca2+ intracellulare osservato dopo stimolazione con TLQP-21 è dovuto al rilascio di Ca2+ dal reticolo endoplasmatico, come dimostrato dalla capacità della tapsigargina di inibire l’effetto di TLQP-21. È noto che il rilascio di Ca2+ dal reticolo endoplasmatico è regolato dall’attivazione della PLC e dalla conseguente produzione di IP3 che si lega a specifici recettori presenti sul reticolo endoplasmatico. Nelle cellule CHO, N9 e RAW264.7 il trattamento con l’inibitore della PLC U73122 e con l’antagonista dei recettori IP3 2-APB riduce l’attività di TLQP-21, confermando per questo peptide un meccanismo d’azione PLC-dipendente. Inoltre, nelle cellule CHO, TLQP-21 induce una rapida defosforilazione della PLCγ1, suggerendo che l’aumento di Ca2+ osservato in seguito a stimolazione con TLQP-21 sia mediato dal legame del peptide con un recettore accoppiato a una proteina di tipo Gq, che a sua volta attiverebbe la PLCβ. Il rilascio di Ca2+ dal reticolo indotto da TLQP-21 attiva inoltre un flusso di Ca2+ dall’ambiente extracellulare, come dimostrato dal trattamento con SKF-96365 e YM-58483, due specifici inibitori dello store-operated calcium entry process. Nelle cellule CHO, TLQP-21 induce un aumento della fosforilazione della PKC e, di conseguenza, di ERK1/2. Inoltre, l’aumento di Ca2+ intracellulare indotto da TLQP-21 stimola l’attivazione di Akt/PKB. I risultati di questa ricerca suggeriscono quindi che il recettore stimolato da TLQP-21 appartenga alla famiglia dei recettori accoppiati a proteine Gq. Il legame di TLQP-21 con questo recettore, attivando la PLC, stimola la produzione di secondi messaggeri che a loro volta inducono il rilascio di Ca2+ dal reticolo endoplasmatico e il conseguente flusso in entrata di Ca2+ dall’ambiente extracellulare. In conclusione, la nostra ricerca fornisce nuove evidenze riguardo il meccanismo di azione di TLQP-21, e fornisce nuove indicazioni sul recettore specifico espresso in alcune linee cellulari che rispondono a TLQP-21. La futura caratterizzazione molecolare di tale recettore potrà permettere lo sviluppo di modelli sperimentali utili per la ricerca di farmaci per il trattamento di diverse patologie, inclusi obesità e diabete.
Obesity is a global epidemic for which the current weight loss therapies are relatively ineffective. Many central and peripheral factors are involved in the mechanisms controlling eating behaviour, and the integration of these signals within the hypothalamus results in the generation of specific responses aimed at regulating energy balance. TLQP-21 is a novel neuropeptide that has been implicated in the regulation of energy homeostasis, nociception, gastric function and several other physiologic functions. Although recent studies identified different receptors as the targets for TLQP-21, its molecular mechanisms of action at the cellular level remain largely unknown. Thus, since TLQP-21 is emerging as a novel target for obesity-associated disorders, diabetes, neuropathic pain, and other human pathologies, the purpose of this study was to better investigate the intracellular signalling pathway activated by the peptide-receptor interaction. Here, using intracellular calcium mobilization assay and western blot analysis, we have pharmacologically characterized the intracellular signalling pathway activated by TLQP-21 in ovary, macrophage and microglial cells. TLQP-21 dose-dependently stimulated a rapid and transient intracellular Ca2+ increase in CHO, RAW264.7 and N9 cells, and repeated exposure to the peptide resulted in a reduced response, indicating a possible desensitization mechanism of TLQP-21 receptor. In particular, TLQP-21 stimulation induced an increase of cytoplasmic Ca2+ levels that was sustained by Ca2+ release from the ER, since treatment of the cells with thaspigargin reduced the TLQP-21-mediated increase of intracellular Ca2+. The release of Ca2+ from the ER store is regulated by the activation of PLCs and the subsequent production of IP3 that binds to its receptors on the surface of the ER. In our cellular systems, TLQP-21 activity was reduced by the treatment with the PLC inhibitor U73122 and the IP3R antagonist 2-APB, confirming a PLC-dependent mechanism of action for the peptide. Furthermore, TLQP-21 induced a rapid dephosphorylation of PLCγ1 in CHO cells, suggesting that Ca2+ response to TLQP-21 is mediated by the binding of the peptide to a Gq-coupled receptor that in turn activates PLCβ. Ca2+ release from the ER activated Ca2+ entry from the extracellular environment, as demonstrated by the treatment of the cells with SKF-96365 and YM-58483, two specific inhibitors of the SOCE pathway. In CHO cells, TLQP-21 induced also an increase of PKC phosphorylation and, afterwards, of ERK1/2 phosphorylation. Moreover, the increase of cytosolic Ca2+ concentration following TLQP-21 administration, stimulated the activation of Akt/PKB. Our results suggest that the receptor stimulated by TLQP-21 belongs to the family of the Gq-coupled receptors, that activates membrane-lipid derived second messengers which thereby induce Ca2+ mobilization from the ER followed by a slower store-operated Ca2+ entry from outside the cell. In conclusion, our research provides additional evidences about the molecular mechanisms of action of TLQP-21, and could be useful to open new approaches to improve the treatment of several human disorders, including obesity and diabetes.

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Holmes, Steven P. "The characterization, functional expression, and localization of the first arthropod myokinin receptor from the southern cattle tick, Boophilus microplus (Acari: ixodidae)". Diss., Texas A&M University, 2003. http://hdl.handle.net/1969.1/60.

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Myokinins are invertebrate neuropeptides with myotropic and diuretic activity. The lymnokinin receptor from the snail Lymnaea stagnalis was the only previously identified myokinin receptor. A cDNA encoding a neuropeptide receptor was cloned from the southern cattle tick, Boophilus microplus. The deduced amino acid sequence was 40 % identical to the lymnokinin receptor. The receptor transcript is present in all tick life stages as determined by semiquantitative RT-PCR. When expressed in mammalian CHO-K1 cells, myokinins at nanomolar concentrations induced increases in intracellular calcium as measured by fluorescent cytometry. The rank order of potency for peptides tested was FFFSWS-NH2≥FFFSWG-NH2≥FFSWG-NH2>FYSWG-NH2>muscakinin>lymnokinin>>APTGFFGVR-NH2. The receptor coupled to a pertussis toxin insensitive G protein. Absence of extracellular calcium did not inhibit the calcium response, indicating the release of Ca2+ from intracellular stores. Receptor transcript was detected by RT-PCR in the dissected synganglia, ovaries, salivary glands, guts and Malpighian tubules of partially engorged adult female ticks. It is concluded that the B. microplus receptor is the first myokinin receptor cloned from an arthropod, and the first neuropeptide receptor known from the Acari. The presence of this receptor transcript in multiple tissues and all life stages suggests a multifunctional role in ticks.

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Sohn, Johann. "IDENTIFICATION AND CHARACTERIZATION OF CONTACT SITES BETWEEN HUMAN FOLLICLE STIMULATING HORMONE AND THE FOLLICLE STIMULATING HORMONE RECEPTOR". UKnowledge, 2005. http://uknowledge.uky.edu/gradschool_diss/270.

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Follicle stimulating hormone (FSH) comprises an ?? subunit and a ?? subunit,whereas the FSH receptor consists of two halves with distinct functions, the N-terminalextracellular exodomain and C-terminal membrane associated endodomain. FSH initiallybinds to exodomain, and the resulting FSH/exodomain complex modulates the endodomainand generates signal. However, it has been difficult to determine which subunit of FSHcontacts the exodomain or endodomain, and in what orientation FSH interacts with them.To address these crucial issues, the receptor was Ala-scanned and the hormone subunitswere probed with photoaffinity labeling with receptor peptides corresponding to the Nterminalregion of the exodomain and exoloop 3 of the endodomain. The results show thatboth regions of the receptors are important for hormone binding and signal generation. Inaddition, the FSH ?? subunit is specifically labeled with the N-terminal peptide, whereas the?? subunit is labeled with the exoloop 3 peptide. These contrasting results show that the FSH?? subunit is close to the N-terminal region and the ?? subunit is projected toward exoloop 3in the endodomain. The results raise the fundamental question whether the ?? subunit,common among the glycoprotein hormones, plays a major role in generating the hormonesignal common to all glycoprotein hormones.

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McCaffrey, Rebecca. "IDENTIFICATION AND CHARACTERIZATION OF CONTACT SITES BETWEEN HUMAN CHORIONIC GONADOTROPIN AND THE AMINO TERMINAL REGION OF THE LUTEINIZING HORMONE/CHORIOGONADOTROPIN RECEPTOR". UKnowledge, 2002. http://uknowledge.uky.edu/gradschool_theses/204.

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The luteinizing hormone / choriogonadotropin receptor (LH/CG-R) is a member of theG protein-coupled receptor family. The LH/CG-R has seven transmembrane helices, threeexoloops, three cytoloops, a C-terminal tail, and an extensive N-terminal exodomain. Theexodomain is capable of binding hormone with high affinity without hormone action. Previousstudies have shown that the amino-terminal region of the LH/CG receptor contacts both subunitsof human chorionic gonadotropin (hCG). In particular, three residues (Leu20, Cys22, and Gly24)were found to be crucial for hormone binding. In this thesis work, benzoylphenylalanine (Bpa),a photoactivatable reagent, was used to continue investigating the interactions of the N-terminalregion of the LH/CG-R with hCG. Bpa has been directly incorporated at a defined position intopeptides representing amino acids 17-36 of the LH/CG-R. These peptides were radiolabeledwith 125I and used in photoaffinity labeling studies to identify and characterize the contact site(s)between the N-terminal region of the LH/CG-R and hCG. Results suggest that Cys22 is theprimary contact residue in this region. Peptide and hormone concentration dependent as well asUV duration dependent photoaffinity labeling experiments confirm that the photolabeling ofhCG by hLHR17-36(C22Bpa) is specific. Competition of labeling studies indicate that the hLHR17-36(C22Bpa) peptide is a good mimic of the wild type N-terminal portion of the receptor. In-geldigestions of photolabeled hCG ?? and photolabeled hCG ?? with CNBr indicate that the Nterminalregions of both hCG ?? and hCG ?? were photoaffinity labeled by hLHR17-36(C22Bpa).Based on the fact that the N-terminal regions of each subunit are located on the convex side ofthe heterodimer, these results provide evidence that the N-terminal portion of the receptor wrapsaround the back of hCG, contacting the convex face of the hormone.

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Preißler, Julia. "Die gliale Relevanz des G-Protein-gekoppelten Rezeptors 34". Doctoral thesis, Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-167010.

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In der vorliegenden Arbeit wurde die Funktion des G-Protein-gekoppelten Rezeptors 34 (GPR34) in Mikroglia untersucht. Dieser Rezeptor weist eine hohe Expression auf Gliazellen auf, jedoch ist über dessen Aufgabe innerhalb dieser Zellpopulation bisher nichts bekannt. In bisherigen Arbeiten wurde dem GPR34 eine Rolle in der Immunantwort zugeschrieben. Knock-out (ko)-Mäuse, welche mit Cryptococcus neoformans infiziert wurden, zeigten im Vergleich zum infizierten Wildtyp (wt) eine deutlich höhere Pathogenlast in verschiedenen Geweben u.a. im Gehirn, was für eine inadäquate Immunantwort spricht. In dieser Arbeit konnte mittels morphologischer Studien gezeigt werden, dass eine GPR34- Defizienz zu einer veränderten Gestalt der Mikroglia im Cortex sowie der Retina führt. Mikrogliazellen aus ko-Mäusen sind kleiner und deutlich weniger ramifiziert. Mit Hilfe von Transkriptomanalysen wurde eine große Vielfalt an unterschiedlich exprimierten Genen zwischen ko- und wt-Tieren identifiziert. Hierunter befanden sich Gene, die die Motilität, aber auch die Phagozytose der Mikroglia beeinflussen. Um den Einfluss der GPR34-Defizienz auf diese Vorgänge zu untersuchen, wurden zahlreiche funktionelle Untersuchungen an murinen Mikrogliazellen durchgeführt. Mittels basalen Motilitätsstudien aber auch unter Stimulation durch Laserläsion und Läsion des entorhinalen Cortex konnten keine Unterschiede in der Beweglichkeit von Mikroglia aufgedeckt werden. Jedoch zeigten ko- Mikrogliazellen des Cortex und der Retina eine deutlich geringere Phagozytoseaktivität. Dies ist ein möglicher Erklärungsansatz für die beschriebene erhöhte Pathogenlast in den GPR34- defizienten Tieren. Da die Phagozytoseaktivität von Mikroglia in neurodegenerativen Erkrankungen wie Multipler Sklerose oder der Alzheimer´schen Demenz eine bedeutende Rolle spielt, sollte zukünftig die Relevanz des GPR34 bei diesen Erkrankungen untersucht werden.

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Bittencourt, Fabiola M. "Examination of the Function of the Murine Cytomegalovirus Encoded G Protein-Coupled Receptor M33 in vivo". University of Cincinnati / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1397234044.

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Jin, Hongjun. "Structural and functional investigation of human chemokines and applications of human chemokines in blocking HIV-1 entry". [College Station, Tex. : Texas A&M University, 2007. http://hdl.handle.net/1969.1/ETD-TAMU-2430.

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Alice, Brown. "Allosteric Coupling in the Dimeric Calcium Sensing Receptor". Thesis, The University of Sydney, 2017. http://hdl.handle.net/2123/17738.

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The Calcium Sensing Receptor (CaSR) is a Class C GPCR that supports systemic calcium homeostasis by sensing and responding to small fluctuations in serum calcium (Ca2+o). It was hypothesised Cys-236 in the Venus Fly Trap Domain (VFTD) and Cys-561 in the Cysteine-Rich Domain (CRD) participate in an interdomain disulfide bond that is required for signal transmission. C236S, C561S, and C236S/C561S CaSR constructs were generated to eliminate the predicted disulfide and were analysed. While the mutant receptors failed to respond to VFTD-binding agonists and modulators, receptor activation was observed in the presence of cinacalcet, which binds in the HHD thereby bypassing the loss of disulfide-dependent allosteric coupling between the domains. To assess the presence of a physical link between the residues, constructs with an engineered thrombin cleavage site between the two residues were evaluated, following thrombin digestion, by western blotting. The results demonstrate that Cys-236 and Cys-561 are essential for signal transmission and provide coupling between the VFTD and the HHD through an interdomain disulfide bond. The CaSR signals as a homodimer and forms functional heterodimers with other Class C GPCRs. A trafficking system was developed wherein CaSR/GABAB chimeras were generated to exploit the ability of the GABAB C-termini to control receptor expression. Co-immunoprecipitation and FRET-based analyses confirmed expression of CaSR-B1/CaSR-B2 heterodimers at the cell surface. The system was then employed to study the subunit requirements for signalling. The VFTD and HHD requirements for modulation by PAMS were investigated using receptor chimeras with domains selectively disabled with mutations known to impair PAM-sensing. The mechanism of VFTD-activation coupling to intracellular signalling was also assessed. The results demonstrate that two, functional CaSR subunits are required for full PAM-sensing and VFTD-activation couples to the HHD in a trans direction.

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Kunisue, Sumihiro. "Roles of the Orphan Receptor Gpr176-mediated G-protein Signaling in the Central Circadian Clock". Kyoto University, 2019. http://hdl.handle.net/2433/242672.

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Ghimire, Ganga D., ガンガＤ. ギミレ, Kenichiro Imai, 賢一郎今井, Fumitsugu Akazawa, 史嗣赤沢, Toshiyuki Tsuji i in. "Physicochemical properties of amino acid sequences of G-proteins for understanding GPCR-G-protein coupling". Chem-Bio Informatics Society, 2006. http://hdl.handle.net/2237/9277.

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