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Elucidating Agonist-Induced Signaling Patterns of Human G Protein-Coupled Receptor GPR17 and Uncovering Pranlukast As a Biased Mixed Agonist-Antagonist at GPR17

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Elucidating Agonist-Induced Signaling Patterns of Human G Protein-Coupled Receptor GPR17 and Uncovering Pranlukast As a Biased Mixed Agonist-Antagonist at GPR17 Elucidating agonist-induced signaling patterns of human G protein-coupled receptor GPR17 and uncovering pranlukast as a biased mixed agonist-antagonist at GPR17 Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Stephanie Monika Hennen aus Saarburg Bonn 2011 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: 15.09.2011 Erscheinungsjahr: 2011 Die vorliegende Arbeit wurde in der Zeit von April 2008 bis März 2011 am Institut für Pharmazeutische Biologie der Rheinischen Friedrich-Wilhelms Universität Bonn unter der Leitung von Frau Prof. Dr. Evi Kostenis durchgeführt. Meinen Eltern Abstract I Abstract The progress of human genome sequencing has revealed the existence of several hundred orphan G protein-coupled receptors (GPCRs), whose endogenous ligands are not yet identified, thus their deorphanization and characterization is fundamental in order to clarify their physiological and pathological role as well as their relevance as new drug targets. Recently, the orphan GPCR GPR17 that is phylogenetically and structurally related to the known P2Y and CysLT receptors has been identified as a dual uracil nucleotide/cysteinyl-leukotriene receptor. In spite of this, these deorphanization efforts could not be verified yet by independent laboratories, thus this classification remains a controversial matter and GPR17 most likely still represents an orphan GPCR. Additionally, a subsequent study revealed a ligand-independent regulatory role for GPR17 suppressing CysLT1 receptor function via GPCR-GPCR interactions. By means of a high throughput pharmacogenomic approach our group has identified a small molecule agonist for GPR17 that is used as pharmacological tool for characterization of ligand-dependent behaviors triggered by this receptor. As a consequence, in the present thesis, evidence is provided that GPR17 does not lack the common features of GPCR signaling. Upon agonist challenge GPR17 induces signaling via promiscuous G protein-coupling (Gαi/o, Gαq and Gαs) in an agonist-concentration-dependent manner, as determined by means of traditional second messenger assays (cAMP and IP1) and by the label-free dynamic mass redistribution (DMR) technology in two different cellular backgrounds (CHO and HEK293 cells) engineered to stably express the short isoform of human GPR17. The use of ELISA and immunofluorescence techniques revealed that activation of GPR17 with the agonist is also linked with a time-dependent reduction of cell surface expression. Furthermore, GPR17 recruits β-arrestin2 upon ligand-stimulation in a G protein-dependent and –independent manner as monitored by use of bioluminescence resonance energy transfer (BRET2) analyses. The GPR17-mediated signaling can be efficiently abrogated by the CysLT1 antagonist pranlukast in a non-competitive mode of action, but not by montelukast, zafirlukast and MK571, as investigated by use of DMR analyses, traditional second messenger assays and BRET2 approach. Additionally, evidence is provided that pranlukast acts as a mixed agonist/antagonist at GPR17, differentially modulating individual signaling pathways. Furthermore, it is demonstrated that β-arrestin2, known as scaffolding protein involved in desensitization and trafficking processes of GPCRs, exhibits the capability to fine-tune G protein signaling specifity of GPR17 via shifting the preference to the most preferred G protein subunit (here Gαi/o), a phenomenon that has not been described before. Keywords: G protein-coupled receptor (GPCR), GPR17, pranlukast, β-arrestin2, dynamic mass redistribution, multiplicity Kurzbeschreibung II Kurzbeschreibung Die Sequenzierungsfortschritte des menschlichen Erbgutes offenbarten die Existenz von mehreren hundert orphanen G Protein-gekoppelten Rezeptoren (GPCRs), dessen endogene Liganden noch nicht identifiziert wurden. Ihre Deorphanisierung sowie Charakterisierung sind elementar um die physiologische und pathologische Rolle aufzuklären und sie können als neue potenzielle Arzneimittel Targets betrachtet werden. Kürzlich ist der orphane GPCR GPR17, der phylogenetisch und strukturell mit P2Y und CysLT Rezeptoren verwandt ist, als ein Uracil Nukleotid/Cysteinyl-Leukotrien Rezeptor identifiziert worden. Diese Deorphanisierung konnte jedoch noch nicht durch unabhängige Labore verifiziert werden, somit bleibt diese Klassifikation eine umstrittene Postulierung und GPR17 muss weiterhin als orphaner GPCR betrachtet werden. Weiterhin zeigte eine nachfolgende Studie eine Ligand-unabhängige Funktion für GPR17 über GPCR-GPCR Wechselwirkungen als negativer Regulator der CysLT1 Rezeptor-vermittelten Signalwegsaktivierung. Dennoch hat unsere Arbeitsgruppe einen synthetischen GPR17-Agonisten identifiziert, der als pharmakologisches Werkzeug für die Charakterisierung von Ligand-induzierten Rezeptor-nachgeschalteten Ereignissen verwendet wird. Entgegen der bisher postulierten Thesen wird in der gegenwärtigen Arbeit gezeigt, dass GPR17 allgemeine Eigenschaften der GPCR-Signaltransduktion aufweist. Agonist-Stimulierung des GPR17 induziert Signaltransduktion über Kopplung mit promiskuitiven G Proteinen (Gαi/o, Gαq und Gαs) in einer konzentrationsabhängigen Weise, wie mittels der klassischen funktionellen GPCR Analysen (cAMP und IP1) und durch die Technologie der dynamischen Massenumverteilung (DMR) gezeigt werden konnte, unter Verwendung von zwei verschiedenen rekombinanten Zelllinien (CHO und HEK293), welche die kurze Isoform des humanen GPR17 stabil exprimieren. Der Gebrauch von ELISA und Immunfluoreszenz Techniken offenbarte, dass die Aktivierung von GPR17 mit dem Agonisten auch mit einer zeitabhängigen Verminderung des Zelloberflächenexpression verbunden ist. Außerdem wird unter Verwendung der Biolumineszenz Resonanz Energietransfer (BRET2) Methode gezeigt, dass GPR17 β-Arrestin2 nach Ligand-Stimulierung rekrutiert, und zwar in einer G Protein- abhängigen als auch -unabhängigen Weise. Die GPR17-vermittelte Signaltransduktion kann vom CysLT1 Antagonisten Pranlukast in einer nicht-kompetitiven Weise gehemmt werden, aber nicht durch Montelukast, Zafirlukast und MK571. Weiterhin wird gezeigt, dass Pranlukast als ein gemischter Agonist/Antagonist am GPR17 wirkt, der partial und funktionell selektiv die Gαi/o- vermittelte Signalwegstransduktion aktiviert. Weiterhin wird dargelegt, dass β-Arrestin2, bekannt als Gerüst-Protein, das an der Desensibilisierung und Internalisierung von GPCRs beteiligt ist, die Fähigkeit besitzt die gemischte G Protein-Kopplung von GPR17 zu modulieren, und zwar über die Verschiebung der Kopplungspräferenzen zur bevorzugtesten G Protein-Untereinheit (hier Gαi/o). Dies stellt ein Phänomen dar, das zuvor nicht beschrieben worden ist. Schlüsselwörter: G Protein-gekoppelte Rezeptoren (GPCR), GPR17, Pranlukast, β-Arrestin2, Dynamische Massenumverteilung (DMR), Multiplizität Table of contents III Table of contents Abstract ............................................................................................................................... I Kurzbeschreibung ................................................................................................................ II Table of contents ............................................................................................................... III List of figures ..................................................................................................................... VII List of tables ....................................................................................................................... XI 1 Introduction .................................................................................................................. 1 1.1 Structural architecture and classification of GPCRs ...................................................... 1 1.2 Classical model of G protein-dependent signaling ........................................................ 3 1.2.1 Guanine nucleotide binding proteins and their major signaling characteristics................................................................................................... 3 1.2.2 Multiplicity in G protein-coupling and functional selectivity ........................... 5 1.3 Classical and new roles of β-arrestin ............................................................................. 7 1.3.1 Desensitization ................................................................................................. 7 1.3.2 Internalization .................................................................................................. 9 1.3.3 Signaling ........................................................................................................... 9 1.4 Orphan GPCRs ............................................................................................................. 11 1.5 G protein-coupled receptor 17 .................................................................................... 11 1.5.1 Classification of GPR17 as dual nucleotide/lipid receptor is a controversial matter ............................................................................................................. 11 1.5.2 Expression patterns of GPR17 ........................................................................ 13 1.5.3 Current thoughts on the physiological role of GPR17
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