DOCSLIB.ORG

The Orphan G Protein-Coupled Receptor GPR17: Its Pharmacology

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Orphan G Protein-Coupled Receptor GPR17: Its Pharmacology The orphan G protein - coupled receptor GPR17: its pharmacology and function in recombinant and primary cell expression systems Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch - Naturwissenschaftlichen Fakultät der Rheinischen Friedrich - Wilhelms - Universität Bonn vorgelegt von Katharina Anna Maria Simon aus Düren Bonn 2016 Angefertigt mit Genehmigung der Mathematisch - Naturwissenschaftlichen Fakultät der Rheinischen Friedrich - Wilhelms Universität Bonn. 1. Gutachter: Prof. Dr. Evi Kostenis 2. Gutachter: Prof. Dr. Klaus Mohr Tag der Promotion: 19.01.2017 Erscheinungsjahr: 2017 Meiner Familie Abstract I Abstract The reconstitution of myelin sheaths, so called remyelination, represents an innovative therapeutic goal in multiple sclerosis (MS), the most common inflammatory - demyelinating disease of the central nervous system (CNS). Recently, the orphan G protein - coup led receptor (GPCR) GPR17, which is predominantly expressed in ol i- godendrocytes, has been identified as inhibitor of oligodendroglial differentiation, a r- resting oligodendrocytes in an immature, non - myelinating stage. Moreover, GPR17 expression is upregulat ed in human MS tissues, suggesting a key role of this receptor during the remyelination impairment that occurs in MS. However, the downstream si g- naling pathway connecting GPR17 to oligodendroglial maturation arrest is still poorly understood. The present work confirms that GPR17 activation by the small molecule agonist MDL29,9 51 results in a reduction of mature oligodendrocytes in vitro, evident by their decreased intracellular myelin basic protein (MBP) levels. The GPR17 - mediated mat u- ration block is cruci ally triggered by the Gα i/o pathway, which leads to an inhibition of two signaling cascades: (i) the adenylyl cyclase (AC) - cyclic adenosine monophosphate (cAMP) – protein kinase A (PKA) – cAMP response element - binding protein (CREB), and (ii) the AC - cAMP - excha nge protein directly activated by cAMP (EPAC). Remar k- ably, these findings demonstrate for the first time an involvement of EPAC on ol i- godendrocyte myelination. In conclusion, GPR17 but also its downstream signaling effectors as elucidated in this study pro vides several drug targets to influence oligode n- drocyte differentiation. Furthermore, knowledge about GPR17 pharmacology would benefit greatly from the identification of both its endogenous ligand(s) and/or selective antagonists. The activ a- tion of GPR17 by cysteinyl - leukotriene and purinergic ligands (UDP, UDP - gal, UDP - glc, LTC4 , and LTD4) is ten years after its initial publication still contr o versially di s- cussed. In line with other laboratories that did not validate GPR17 as dualistic receptor for both lig and families, the data of the present work obtained in (i) G protein rea r- rangement, (ii) β - arrestin recruitment , and (iii) ERK1/2 phosphorylation assays confirm that GPR17 must still be regarded as an orphan receptor. Additionally, the present work invalid ates the P2Y12 antagonists ticagrelor and cangrelor as GPR17 inhibitors. Ho w- ever, investigations on two structurally related CysLT2 receptor antagonists, HAMI3379 and Cay10633, strongly suggest that their basic chemical scaffolds will serve as valuable sta rting point for the generation of more potent and selective GPR17 antagonists to inhibit signaling of this orphan receptor in MS. II Abstract Keywords: GPR17, orphan GPCR, protein kinase A (PKA), exchange protein directly activated by cAMP (EPAC), cAMP response eleme nt - binding protein (CREB), label - free dynamic mass redistribution (DMR) Kurzfassung III Kurzfassung Die Regeneration von Myelinscheiden, die Remyelinisierung, repräsentiert ein innov a- tives therapeutisches Ziel bei Multipler Sklerose (MS), der häufigsten inflammator i- schen demyelinisierenden Erkrankung des zentralen Nervensystems (ZNS). Differe n- zierte Oligodendrozyten sind für die Bildung von Myelinscheiden verantwortlich. Der orphane G Protein - gekoppelte Rezeptor (GPCR) GPR17, der prädominant in Ol i- godendrozyten exprimiert ist, blockiert deren Differenzierung zu myelinisierenden Ol i- godendroz yten. GPR17 ist darüber hinaus im Gewebe von MS Patienten überexpr i- miert, was implizieren könnte, dass der Rezeptor eine zentrale Rolle als Inhibitor der Remy elinisierung bei MS spielt. Wie GPR17 Oligodendrozytendifferenzierung auf m o- lekularer Ebene blockiert, d.h. welche intrazellulären Signalwege involviert werden, ist jedoch kaum bekannt. Die vorliegende Arbeit bestätigt, dass die Aktivierung von GPR17 durch den synthet i- schen Agonisten MDL29,951 in vitro eine Reduktion der Reifung von Oligodendroz y- ten zur Folge hat, was durch eine verminderte Expression des intrazellulären Myelin Basic Proteins (MBP) detektierbar ist. Die GPR17 - vermittelte Entwicklungsblockie rung wird entscheidend durch den Gα i/o Signalweg beeinflusst. Dies beinhaltet die Hemmung von zwei Signalkaskaden: (i) die Adenylatzyklase (AC) - zyklischen Adenosinmono - phosphate (cAMP) – Proteinkinase A (PKA) – cAMP response element - binding protein (CREB) und (ii) die AC - cAMP - Exchange Protein directly acitvated by cAMP (EPAC) Kaskade. Interessanterweise, zeigen die erhaltenen Ergebnisse zum ersten Mal eine B e- teiligung des Guaninnukleotid - Austauschfaktors EPAC an der Myelinisierung durch Oligodendrozyten. Zu sammenfassend bieten sowohl GPR17 als auch seine intrazellul ä- ren Effektorproteine diverse Ansatzpunkte, um die Differenzierung von Oligodendroz y- ten therapeutisch zu beeinflussen. Zusätzlich würde die Identifizierung des endogenen Agonisten und/oder selekt iver A n- tagonisten einen wertvollen Beitrag zur bisher rudimentären GPR17 Pharmakologie leisten. Die Aktivierung von GPR17 durch Cysteinyl - Leukotriene und Purin Liganden (UDP, UDP - gal, UDP - glc, LTC4 und LTD4) wird zehn Jahre nach der ersten Postuli e- rung noc h immer kontrovers diskutiert. Im Einklang mit anderen Arbeitsgruppen, die nicht belegen konnten, dass GPR17 durch die genannten Substanzen aktiviert wird, b e- stätigen die Daten der vorliegenden Arbeit, mittels Assays zur (i ) G Protein - Umlagerung, (ii) β - A r restin Rekrutierung und (iii) ERK1/2 Phosphorylierung, dass GPR17 immer noch als orphaner Rezeptor anzusehen ist. Darüber hinaus konnten die P2Y12 Antagonisten, Ticagrelor und Cangrelor, nicht als GPR17 Inhibitoren bestätigt werden. Im Gegensatz dazu konnt en wir für zwei strukturell ähnliche CysLT2 - IV Kurzfassung Rezeptor - Antagonisten, HAMI3379 und Cay10633, eine Hemmung von GPR17 nac h- weisen, wodurch diese Substanzen als neue Ausgangspunkte für die Entwicklung von potenten und selektiven GPR17 Antagonisten angesehen werde n könnten, um somit den GPR17 Signalweg in MS zu blockieren. Table of contents V Table of conten t s Abstract ................................ ................................ ................................ ....................... I Kurzfassung ................................ ................................ ................................ .............. III Table of contents ................................ ................................ ................................ ........ V List of figures ................................ ................................ ................................ ............ IX List of tables ................................ ................................ ................................ .............. XI 1 Introduction ................................ ................................ ................................ ...... 1 1.1 Multiple sclerosis (MS): a severe neurological disease ................................ ....... 1 1.1.1 Remyelination: the challenging therapeutic goal ................................ ................. 1 1.2 GPCR signaling and their role in differentiation of oligodendrocytes .................. 3 1.2.1 Heterotrimeric G protein signalin g and its downstream effectors ........................ 5 1.2.2 β - arrestin recruitment and signaling ................................ ................................ .... 7 1.2.3 Extracellular signal - regulated kinases 1 and 2 (ERK1/2) signaling - mitogen - activated protein kinases ................................ ................................ ..................... 8 1.3 Orphan GPCRs ................................ ................................ ................................ ... 8 1.3.1 The G protein coupled receptor 17 (GPR17) ................................ ....................... 9 1.4 Aims of the study ................................ ................................ ............................. 13 2 Materials ................................ ................................ ................................ ......... 15 2.1 Cell culture ................................ ................................ ................................ ....... 15 2.1.1 Mammalian cell lines ................................ ................................ ....................... 15 2.1.2 Cell culture medium ................................ ................................ ......................... 17 2.2 Plasmids ................................ ................................ ................................ ........... 22 2.3 Antibodies ................................ ................................ ................................ ........ 23 2.3.1 Primary Antibodies ................................ ................................ ........................... 23 2.3.2 Secondary Antibodies ................................ ...............................
Load more

Recommended publications
  • Applying Screening Techniques to Two Orphan Gpcrs
    Universidade de Lisboa Faculdade de Farmácia Deorphanization of receptors: Applying screening techniques to two orphan GPCRs Ana Catarina Rufas da Silva Santos Mestrado Integrado em Ciências Farmacêuticas 2019 Universidade de Lisboa Faculdade de Farmácia Deorphanization of receptors: Applying screening techniques to two orphan GPCRs Ana Catarina Rufas da Silva Santos Monografia de Mestrado Integrado em Ciências Farmacêuticas apresentada à Universidade de Lisboa através da Faculdade de Farmácia Orientadora: Ghazl Al Hamwi, PhD Student Co-Orientadora: Professora Doutora Elsa Maria Ribeiro dos Santos Anes, Professora Associada com Agregação em Microbiologia 2019 Abstract G-Protein Coupled Receptors represent one of the largest families of cellular receptors discovered and one of the main sources of attractive drug targets. In contrast, it also has a large number of understudied or orphan receptors. Pharmacological assays such as β-Arrestin recruitment assays, are one of the possible approaches for deorphanization of receptors. In this work, I applied the assay system previously mentioned to screen compounds in two orphan receptors, GRP37 and MRGPRX3. GPR37 has been primarily associated with a form of early onset Parkinsonism due to its’ expression patterns, and physiological role as substrate to ubiquitin E3, parkin. Although extensive literature regarding this receptor is available, the identification of a universally recognized ligand has not yet been possible. Two compounds were proposed as ligands, but both were met with controversy. These receptor association with Autosomal Recessive Juvenile Parkinson positions it as a very attractive drug target, and as such its’ deorphanization is a prime objective for investigators in this area. Regarding MRGPRX3 information is much scarcer.
    [Show full text]
  • The Hypotensive Effect of Activated Apelin Receptor Is Correlated with Β
    This is the accepted (postprint) version of the following article: Besserer-Offroy É, et al. (2018), Pharmacol Res. doi: 10.1016/j.phrs.2018.02.032, which has been accepted and published in its final form at https://www.sciencedirect.com/science/article/pii/S1043661817313804 The hypotensive effect of activated apelin receptor is correlated with β-arrestin recruitment Élie Besserer-Offroya,c,ORCID ID, Patrick Bérubéa,c, Jérôme Côtéa,c, Alexandre Murzaa,c, Jean-Michel Longpréa,c, Robert Dumainea, Olivier Lesurb,c, Mannix Auger-Messierb,ORCID ID, Richard Leduca,c,ORCID ID, Éric Marsaulta,c,*,ORCID ID, Philippe Sarreta,c* Affiliations a Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, CANADA J1H 5N4 b Department of Medicine, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, CANADA J1H 5N4 c Institut de pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec, CANADA J1H 5N4 e-mail addresses [email protected] (ÉBO); [email protected] (PB); [email protected] (JC); [email protected] (AM); Jean- [email protected] (JML); [email protected] (RD); [email protected] (OL); [email protected] (MAM); [email protected] (RL); [email protected] (EM); [email protected] (PS) © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ This is the accepted (postprint) version of the following article: Besserer-Offroy É, et al. (2018), Pharmacol Res. doi: 10.1016/j.phrs.2018.02.032, which has been accepted and published in its final form at https://www.sciencedirect.com/science/article/pii/S1043661817313804 Corresponding Authors *To whom correspondence should be addressed: Philippe Sarret, Ph.D.; [email protected]; Tel.
    [Show full text]
  • Nuclear Receptors — a Perspective from Drosophila
    REVIEWS NUCLEAR RECEPTORS — A PERSPECTIVE FROM DROSOPHILA Kirst King-Jones and Carl S. Thummel Abstract | Nuclear receptors are ancient ligand-regulated transcription factors that control key metabolic and developmental pathways. The fruitfly Drosophila melanogaster has only 18 nuclear-receptor genes — far fewer than any other genetic model organism and representing all 6 subfamilies of vertebrate receptors. These unique attributes establish the fly as an ideal system for studying the regulation and function of nuclear receptors during development. Here, we review recent breakthroughs in our understanding of D. melanogaster nuclear receptors, and interpret these results in light of findings from their evolutionarily conserved vertebrate homologues. CLADE Classical signal-transduction pathways originate with a A wide range of small, lipophilic molecules function as A group of organisms that membrane-bound receptor. On binding its cognate nuclear-receptor ligands, including steroids, thyroid includes common ancestor and ligand, the receptor initiates a cascade of events in the hormone, retinoic acid and vitamin D. Not all nuclear all of its descendants, cytoplasm, eventually affecting specific transcription receptors, however, have a known natural ligand, and at representing a distinct branch on a phylogenetic tree. factors in the nucleus. In contrast to these often complex least some of these so-called orphan receptors can and convoluted pathways, members of the nuclear- function in a ligand-independent fashion3,4. receptor superfamily
    [Show full text]
  • The Orphan Receptor GPR17 Is Unresponsive to Uracil Nucleotides and Cysteinyl Leukotrienes S
    Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2017/03/02/mol.116.107904.DC1 1521-0111/91/5/518–532$25.00 https://doi.org/10.1124/mol.116.107904 MOLECULAR PHARMACOLOGY Mol Pharmacol 91:518–532, May 2017 Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics The Orphan Receptor GPR17 Is Unresponsive to Uracil Nucleotides and Cysteinyl Leukotrienes s Katharina Simon, Nicole Merten, Ralf Schröder, Stephanie Hennen, Philip Preis, Nina-Katharina Schmitt, Lucas Peters, Ramona Schrage,1 Celine Vermeiren, Michel Gillard, Klaus Mohr, Jesus Gomeza, and Evi Kostenis Molecular, Cellular and Pharmacobiology Section, Institute of Pharmaceutical Biology (K.S., N.M., Ral.S., S.H., P.P., N.-K.S, L.P., J.G., E.K.), Research Training Group 1873 (K.S., E.K.), Pharmacology and Toxicology Section, Institute of Pharmacy (Ram.S., K.M.), University of Bonn, Bonn, Germany; UCB Pharma, CNS Research, Braine l’Alleud, Belgium (C.V., M.G.). Downloaded from Received December 16, 2016; accepted March 1, 2017 ABSTRACT Pairing orphan G protein–coupled receptors (GPCRs) with their using eight distinct functional assay platforms based on label- cognate endogenous ligands is expected to have a major im- free pathway-unbiased biosensor technologies, as well as molpharm.aspetjournals.org pact on our understanding of GPCR biology. It follows that the canonical second-messenger or biochemical assays. Appraisal reproducibility of orphan receptor ligand pairs should be of of GPR17 activity can be accomplished with neither the coapplica- fundamental importance to guide meaningful investigations into tion of both ligand classes nor the exogenous transfection of partner the pharmacology and function of individual receptors.
    [Show full text]
  • Genome-Wide Prediction of Small Molecule Binding to Remote
    bioRxiv preprint doi: https://doi.org/10.1101/2020.08.04.236729; this version posted August 5, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Genome-wide Prediction of Small Molecule Binding 2 to Remote Orphan Proteins Using Distilled Sequence 3 Alignment Embedding 1 2 3 4 4 Tian Cai , Hansaim Lim , Kyra Alyssa Abbu , Yue Qiu , 5,6 1,2,3,4,7,* 5 Ruth Nussinov , and Lei Xie 1 6 Ph.D. Program in Computer Science, The Graduate Center, The City University of New York, New York, 10016, USA 2 7 Ph.D. Program in Biochemistry, The Graduate Center, The City University of New York, New York, 10016, USA 3 8 Department of Computer Science, Hunter College, The City University of New York, New York, 10065, USA 4 9 Ph.D. Program in Biology, The Graduate Center, The City University of New York, New York, 10016, USA 5 10 Computational Structural Biology Section, Basic Science Program, Frederick National Laboratory for Cancer Research, 11 Frederick, MD 21702, USA 6 12 Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel 13 Aviv, Israel 7 14 Helen and Robert Appel Alzheimer’s Disease Research Institute, Feil Family Brain & Mind Research Institute, Weill 15 Cornell Medicine, Cornell University, New York, 10021, USA * 16 [email protected] 17 July 27, 2020 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.04.236729; this version posted August 5, 2020.
    [Show full text]
  • Receptor-Arrestin Interactions: the GPCR Perspective
    biomolecules Review Receptor-Arrestin Interactions: The GPCR Perspective Mohammad Seyedabadi 1,2 , Mehdi Gharghabi 3, Eugenia V. Gurevich 4 and Vsevolod V. Gurevich 4,* 1 Department of Toxicology & Pharmacology, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari 48471-93698, Iran; [email protected] 2 Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari 48167-75952, Iran 3 Department of Cancer Biology and Genetics, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA; [email protected] 4 Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-615-322-7070; Fax: +1-615-343-6532 Abstract: Arrestins are a small family of four proteins in most vertebrates that bind hundreds of different G protein-coupled receptors (GPCRs). Arrestin binding to a GPCR has at least three functions: precluding further receptor coupling to G proteins, facilitating receptor internalization, and initiating distinct arrestin-mediated signaling. The molecular mechanism of arrestin–GPCR interactions has been extensively studied and discussed from the “arrestin perspective”, focusing on the roles of arrestin elements in receptor binding. Here, we discuss this phenomenon from the “receptor perspective”, focusing on the receptor elements involved in arrestin binding and empha- sizing existing gaps in our knowledge that need to be filled. It is vitally important to understand the role of receptor elements in arrestin activation and how the interaction of each of these elements with arrestin contributes to the latter’s transition to the high-affinity binding state. A more precise knowledge of the molecular mechanisms of arrestin activation is needed to enable the construction of arrestin mutants with desired functional characteristics.
    [Show full text]
  • G-Protein-Coupled Receptor Gpr17 Regulates Oligodendrocyte
    www.nature.com/scientificreports OPEN G-Protein-Coupled Receptor Gpr17 Regulates Oligodendrocyte Diferentiation in Response Received: 11 May 2017 Accepted: 2 October 2017 to Lysolecithin-Induced Published: xx xx xxxx Demyelination Changqing Lu1,2, Lihua Dong2, Hui Zhou3, Qianmei Li3, Guojiao Huang3, Shu jun Bai3 & Linchuan Liao1 Oligodendrocytes are the myelin-producing cells of the central nervous system (CNS). A variety of brain disorders from “classical” demyelinating diseases, such as multiple sclerosis, stroke, schizophrenia, depression, Down syndrome and autism, are shown myelination defects. Oligodendrocyte myelination is regulated by a complex interplay of intrinsic, epigenetic and extrinsic factors. Gpr17 (G protein- coupled receptor 17) is a G protein-coupled receptor, and has been identifed to be a regulator for oligodendrocyte development. Here, we demonstrate that the absence of Gpr17 enhances remyelination in vivo with a toxin-induced model whereby focal demyelinated lesions are generated in spinal cord white matter of adult mice by localized injection of LPC(L-a-lysophosphatidylcholine). The increased expression of the activated form of Erk1/2 (phospho-Erk1/2) in lesion areas suggested the potential role of Erk1/2 activity on the Gpr17-dependent modulation of myelination. The absence of Gpr17 enhances remyelination is correlate with the activated Erk1/2 (phospho-Erk1/2).Being a membrane receptor, Gpr17 represents an ideal druggable target to be exploited for innovative regenerative approaches to acute and chronic CNS diseases. Oligodendrocytes are the myelin-producing cells of the central nervous system (CNS), and as such, wrap layers of lipid-dense insulating myelin around axons1. Mature oligodendrocytes have also been shown to provide met- abolic support to axons through transport systems within myelin, which may help prevent neurodegeneration2.
    [Show full text]
  • When 7 Transmembrane Receptors Are Not G Protein–Coupled Receptors
    When 7 transmembrane receptors are not G protein–coupled receptors Keshava Rajagopal, … , Robert J. Lefkowitz, Howard A. Rockman J Clin Invest. 2005;115(11):2971-2974. https://doi.org/10.1172/JCI26950. Commentary Classically, 7 transmembrane receptors transduce extracellular signals by coupling to heterotrimeric G proteins, although recent in vitro studies have clearly demonstrated that they can also signal via G protein–independent mechanisms. However, the physiologic consequences of this unconventional signaling, particularly in vivo, have not been explored. In this issue of the JCI, Zhai et al. demonstrate in vivo effects of G protein–independent signaling by the angiotensin II type 1 receptor (AT1R). In studies of the mouse heart, they compare the physiologic and biochemical consequences of transgenic cardiac-specific overexpression of a mutant AT1R incapable of G protein coupling with those of a wild-type receptor. Their results not only provide the first glimpse of the physiologic effects of this newly appreciated mode of signaling but also provide important and previously unappreciated clues as to the underlying molecular mechanisms. Find the latest version: https://jci.me/26950/pdf commentaries gene, spastin, regulates microtubule stability to 16. Wittmann, C.W., et al. 2001. Tauopathy in Dro- 21. Chan, Y.B., et al. 2003. Neuromuscular defects in modulate synaptic structure and function. Curr. sophila: neurodegeneration without neurofibril- a Drosophila survival motor neuron gene mutant. Biol. 14:1135–1147. lary tangles. Science. 293:711–714. Hum. Mol. Genet. 12:1367–1376. 12. Sherwood, N.T., Sun, Q., Xue, M., Zhang, B., and 17. Shapiro, W.R., and Young, D.F.
    [Show full text]
  • Aptamers and Antisense Oligonucleotides for Diagnosis and Treatment of Hematological Diseases
    International Journal of Molecular Sciences Review Aptamers and Antisense Oligonucleotides for Diagnosis and Treatment of Hematological Diseases Valentina Giudice 1,2,* , Francesca Mensitieri 1, Viviana Izzo 1,2 , Amelia Filippelli 1,2 and Carmine Selleri 1 1 Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana”, University of Salerno, Baronissi, 84081 Salerno, Italy; [email protected] (F.M.); [email protected] (V.I.); afi[email protected] (A.F.); [email protected] (C.S.) 2 Unit of Clinical Pharmacology, University Hospital “San Giovanni di Dio e Ruggi D’Aragona”, 84131 Salerno, Italy * Correspondence: [email protected]; Tel.: +39-(0)-89965116 Received: 30 March 2020; Accepted: 2 May 2020; Published: 4 May 2020 Abstract: Aptamers or chemical antibodies are single-stranded DNA or RNA oligonucleotides that bind proteins and small molecules with high affinity and specificity by recognizing tertiary or quaternary structures as antibodies. Aptamers can be easily produced in vitro through a process known as systemic evolution of ligands by exponential enrichment (SELEX) or a cell-based SELEX procedure. Aptamers and modified aptamers, such as slow, off-rate, modified aptamers (SOMAmers), can bind to target molecules with less polar and more hydrophobic interactions showing slower dissociation rates, higher stability, and resistance to nuclease degradation. Aptamers and SOMAmers are largely employed for multiplex high-throughput proteomics analysis with high reproducibility and reliability, for tumor cell detection by flow cytometry or microscopy for research and clinical purposes. In addition, aptamers are increasingly used for novel drug delivery systems specifically targeting tumor cells, and as new anticancer molecules. In this review, we summarize current preclinical and clinical applications of aptamers in malignant and non-malignant hematological diseases.
    [Show full text]
  • GPCR Regulation of ATP Efflux from Astrocytes
    G PROTEIN-COUPLED RECEPTOR REGULATION OF ATP RELEASE FROM ASTROCYTES by ANDREW EDWARD BLUM Thesis advisor: Dr. George R. Dubyak Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Department of Physiology and Biophysics CASE WESTERN RESERVE UNIVERSITY May, 2010 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of _____________________________________________________ candidate for the ______________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Dedication I am greatly indebted to my thesis advisor Dr. George Dubyak. Without his support, patience, and advice this work would not have been possible. I would also like to acknowledge Dr. Robert Schleimer and Dr. Walter Hubbard for their encouragement as I began my research career. My current and past thesis committee members Dr. Matthias Buck, Dr. Cathleen Carlin, Dr. Edward Greenfield, Dr. Ulrich Hopfer, Dr. Gary Landreth, Dr. Corey Smith, Dr. Jerry Silver have provided invaluable guidance and advice for which I am very grateful. A special thanks to all of the past and present
    [Show full text]
  • G Protein-Coupled Receptors: What a Difference a ‘Partner’ Makes
    Int. J. Mol. Sci. 2014, 15, 1112-1142; doi:10.3390/ijms15011112 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Review G Protein-Coupled Receptors: What a Difference a ‘Partner’ Makes Benoît T. Roux 1 and Graeme S. Cottrell 2,* 1 Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK; E-Mail: [email protected] 2 Reading School of Pharmacy, University of Reading, Reading RG6 6UB, UK * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +44-118-378-7027; Fax: +44-118-378-4703. Received: 4 December 2013; in revised form: 20 December 2013 / Accepted: 8 January 2014 / Published: 16 January 2014 Abstract: G protein-coupled receptors (GPCRs) are important cell signaling mediators, involved in essential physiological processes. GPCRs respond to a wide variety of ligands from light to large macromolecules, including hormones and small peptides. Unfortunately, mutations and dysregulation of GPCRs that induce a loss of function or alter expression can lead to disorders that are sometimes lethal. Therefore, the expression, trafficking, signaling and desensitization of GPCRs must be tightly regulated by different cellular systems to prevent disease. Although there is substantial knowledge regarding the mechanisms that regulate the desensitization and down-regulation of GPCRs, less is known about the mechanisms that regulate the trafficking and cell-surface expression of newly synthesized GPCRs. More recently, there is accumulating evidence that suggests certain GPCRs are able to interact with specific proteins that can completely change their fate and function. These interactions add on another level of regulation and flexibility between different tissue/cell-types.
    [Show full text]
  • 1 Presenilin-Based Genetic Screens in Drosophila Melanogaster Identify
    Genetics: Published Articles Ahead of Print, published on January 16, 2006 as 10.1534/genetics.104.035170 Presenilin-based genetic screens in Drosophila melanogaster identify novel Notch pathway modifiers Matt B. Mahoney*1, Annette L. Parks*2, David A. Ruddy*3, Stanley Y. K. Tiong*, Hanife Esengil4, Alexander C. Phan5, Panos Philandrinos6, Christopher G. Winter7, Kari Huppert8, William W. Fisher9, Lynn L’Archeveque10, Felipa A. Mapa11, Wendy Woo, Michael C. Ellis12, Daniel Curtis11 Exelixis, Inc., South San Francisco, California, 94083 Present addresses: 1Department of Discovery, EnVivo Pharmaceuticals, Watertown, Massachusetts, 02472 2Biology Department, Boston College, Chestnut Hill, Massachusetts, 02467 3Oncology Targets and Biomarkers, Novartis Institutes for BioMedical Research, Inc., Cambridge, Massachusetts, 02139 4Department of Molecular Pharmacology, Stanford University, Stanford, California, 94305 5Department of Biostatistics, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, 21205 6ITHAKA Academic Cultural Program in Greece, San Francisco, California, 94102 7Merck Research Laboratories, Boston, Massachusetts, 02115 8Donald Danforth Plant Science Center, St. Louis, Missouri, 63132 1 9Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, 94720 10Biocompare, Inc., South San Francisco, California, 94080 11Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Inc., Cambridge, Massachusetts, 02139 12Renovis, Inc., South San Francisco, California, 94080
    [Show full text]