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. GPR17 is receptors nucleotide purinergic G protein–coupled receptor, cys- an orphan receptor characterized by some as a dualistic uracil teinyl leukotriene 1, to reconstitute the elusive pharmacology. nucleotide/cysteinyl leukotriene receptor and by others as in- Moreover, our study does not support the inhibition of GPR17 by active toward these stimuli altogether. Whereas regulation of the marketed antiplatelet drugs cangrelor and ticagrelor, previously central nervous system myelination by GPR17 is well estab- suggested to antagonize GPR17. Whereas our data do not lished, verification of activity of its putative endogenous ligands disagreewitharoleofGPR17perseasanorchestratorof has proven elusive so far. Herein we report that uracil nucleotides central nervous system functions, they challenge the utility of at ASPET Journals on September 27, 2021 and cysteinyl leukotrienes do not activate human, mouse, or rat the proposed (ant)agonists as tools to imply direct contribution GPR17 in various cellular backgrounds, including primary cells, of GPR17 in complex biologic settings. Introduction receptors are known as orphan GPCRs. Deorphanization and identification of their in vivo roles are essential to clarify novel G protein–coupled receptors (GPCRs) constitute the largest regulatory mechanisms and, consequently, to disclose novel family of membrane receptors in the cell. By functioning as drug targets. Whether GPR17 is such an orphan receptor is sensors for extracellular stimuli, they are involved in a broad still a matter of debate (Davenport et al., 2013; Harden, 2013; variety of physiologic phenomena and, therefore, belong to the Qi et al., 2013) (http://www.guidetopharmacology.org/GRAC/ most common targets of pharmaceutical drugs, although only ObjectDisplayForward?objectId588). a small percentage of GPCRs are targeted by current thera- The original deorphaning report introduced the possibility pies (Rask-Andersen et al., 2011). For about 25% of the more that GPR17 might represent a dualistic receptor responding to than 400 nonolfactory GPCRs, a defined physiologically two classes of endogenous signaling molecules: cysteinyl relevant ligand is still lacking (Chung et al., 2008). These leukotrienes and uracil nucleotides (Ciana et al., 2006). Whereas this receptor ligand assignment agrees well with This work was funded by a grant from the German Federal Ministry for the phylogenetic classification of GPR17, which is located Education and Research within the BioPharma Initiative “Neuroallianz” intermediate between the nucleotide purinergic G protein– [Grant 1615609B]. K.S. and E.K. are members of the German Research Foundation (DFG)-funded Research Training Group RTG1873. coupled receptors (P2Y) and the cysteinyl leukotriene recep- Note in memoriam: We dedicate this manuscript to our coworker, colleague, tors, it is not yet accepted by the scientific community (Bläsius and friend Lucas Peters, who deceased while this study was in progress. et al., 1998; Heise et al., 2000; Maekawa et al., 2009; Benned- K.S., N.M., and Ral.S. contributed equally to this work. 1Current affiliation: CNS Discovery Research, UCB Pharma, Chemin du Jensen and Rosenkilde, 2010; Wunder et al., 2010; Davenport Foriest R4, B-1420 Braine-l’Alleud, Belgium. et al., 2013; Harden, 2013; Hennen et al., 2013; Qi et al., 2013; https://doi.org/10.1124/mol.116.107904. s This article has supplemental material available at molpharm. Köse et al., 2014). More precisely, discordant findings have aspetjournals.org. been reported on the nature of its cognate endogenous ligands ABBREVIATIONS: BRET, bioluminescence resonance energy transfer, CysLT1, cysteinyl leukotriene 1; CysLT1R, cysteinyl leukotriene 1 receptor; DMEM, Dulbecco’s modified Eagle’s medium; DMR, dynamic mass redistribution; DMSO, dimethyl sulfoxide; ERK1/2, extracellular signal-regulated kinase 1 and 2; GPCR or GPR, G protein–coupled receptor; IP, inositol phosphate; LTC4, leukotriene C4, LTD4, leukotriene D4; MDL29,951, 2-carboxy-4,6-dichloro-1H-indole-3-propionic acid; P2Y receptors, purinergic G protein–coupled receptors. 518 GPR17 Is Unresponsive to its Assigned Endogenous Ligands 519 with an ongoing controversy over their true identity (Bläsius work by Qi and coworkers (Qi et al., 2013), we report herein et al., 1998; Heise et al., 2000; Ciana et al., 2006; Maekawa et al., that all proposed endogenous ligands are completely in- 2009; Benned-Jensen and Rosenkilde, 2010; Wunder et al., active on GPR17 orthologs from humans, rats, and mice. 2010; Hennen et al., 2013; Qi et al., 2013; Köse et al., 2014). To Unfortunately, this also extends to the surrogate antago- date, 10 years after the original deorphaning report (Ciana nists cangrelor and ticagrelor, two marketed antiplatelet et al., 2006), only a single independent study has confirmed medicines (Ingall et al., 1999; Nicholas, 2001; Qamar and the activation of GPR17 by uracil nucleotides and yet failed Bhatt, 2016) that were previously reported to inhibit GPR17 to recapitulate activation by cysteinyl leukotrienes (Benned- (Ciana et al., 2006; Gelosa et al., 2014). Jensen and Rosenkilde, 2010). Although these authors did In view of these results, we strongly recommend reassess- indeed note statistically significant signaling upon chal- ment of their value as pharmacologic tools to probe the lenge of GPR17 with uracil nucleotides, they questioned the physiology and function of orphan GPR17, as well as re- biologic relevance of this receptor-ligand pairing because of striction of their use for in vitro/ex vivo/in vivo applications the low-efficacy responses observed in their functional that do not intend to elaborate on the cellular consequences assays (Benned-Jensen and Rosenkilde, 2010). Regardless, that arise from stimulation or inhibition of this orphan GPR17 deorphanization must be considered tentative at this receptor. point because the number of contradictory reports (Bläsius et al., 1998; Heise et al., 2000; Maekawa et al., 2009; Downloaded from Benned-Jensen and Rosenkilde, 2010; Wunder et al., 2010; Materials and Methods Hennen et al., 2013; Qi et al., 2013; Köse et al., 2014) clearly Materials. Tissue culture media and reagents were purchased and continuously outnumbers the independent follow-up from Thermo Fisher Scientific (Karlsruhe, Germany). MDL29,951 was studies (Benned-Jensen and Rosenkilde, 2010) and, there- from Maybridge (Loughborough, UK). Ticagrelor was a gift from Astra fore, leaves the original deorphaning report unconfirmed. Zeneca London, UK). Other laboratory reagents were obtained from In principle, pairings of orphan GPR17 with its endogenous Sigma-Aldrich (Steinheim, Germany), unless stated otherwise. molpharm.aspetjournals.org agonists are findings that should be considered particularly Cell Culture. Cell lines were kept in a humidified atmosphere significant because they supply the scientific community with with 5% CO2 at 37°C. All media were supplemented with 10% (v/v) tools to explore the pharmacology and function of this re- fetal calf serum, penicillin (100 U/ml), and streptomycin (100 mg/ml). Native and recombinant human embryonic kidney (HEK)293 and ceptor. Endogenous agonists may also be used to identify 1321N1 cells were maintained in Dulbecco’s modified Eagle’s antagonist ligands that provide starting points for studying medium (DMEM). For mGPR17-HEK293, rGPR17-HEK293, receptor pharmacology and physiology. With endogenous and hP2Y14-HEK293, and FLAG-hCysLT1-HEK293 (carrying an ad- surrogate (ant)agonist ligands at hand, fundamental ques- ditional hemagglutinin signaling sequence and hereafter referred tions related to GPR17 biology may in principle be addressed to as hCysLT1-HEK293), the medium was supplemented with in various in vitro/in vivo