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The Orphan G Protein-Coupled Receptor GPR17: Its Pharmacology

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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 ................................ ...............................
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