YEXNR-11769; No. of pages: 9; 4C: Experimental Neurology xxx (2014) xxx–xxx Contents lists available at ScienceDirect Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr 1 Regular Article 2 CB1 and GPR55 receptors are co-expressed and form heteromers in rat 3 and monkey striatum 4 E. Martínez-Pinilla a,⁎, I. Reyes-Resina e, A. Oñatibia-Astibia a,M.Zamarbidea,A.Ricobarazad,G.Navarroe, 5 E. Moreno e,I.G.Dopeso-Reyesb,c, S. Sierra b,c, A.J. Rico b,c,E.Rodab,c,J.L.Lanciegob,c,1,R.Francoa,e,1 6 a Laboratory of Cell and Molecular Neuropharmacology, Neurosciences Division, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain 7 b Laboratory of Basal Ganglia Neuroanatomy, Neurosciences Division, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain 8 c Network Center for Biomedical Research in Neurodegenerative Diseases (CIBERNED), Spain 9 d Laboratoire de Plasticité du Cerveau, ESPCI-ParisTech, Paris, France 10 e Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, Barcelona, Spain 11 article info abstract 12 Article history: Heteromerization of G-protein-coupled receptors is an important event as they integrate the actions 22 13 Received 6 May 2014 of extracellular signals to give heteromer-selective ligand binding and signaling, opening new ave- 23 14 Revised 13 June 2014 nues in the development of potential drug targets in pharmacotherapy. A further aim of the present 24 15 Accepted 17 June 2014 paper was to check for cannabinoid CB –GPR55 receptor heteromers in the central nervous system 25 16 Available online xxxx 1 (CNS), specifically in striatum. First, a direct interaction was demonstrated in cells transfected with 26 27 17 Keywords: thecDNAforthehumanversionofthereceptors,using bioluminescence resonance energy transfer fi 28 18 GPCR heteromerization and in situ proximity ligation assays (PLA). In the heterologous system, a biochemical ngerprint 19 Cannabinoids consisting on cross-antagonism in ERK1/2 phosphorylation was detected. The cross-antagonism was 29 20 PLA also observed on GPR55-mediated NFAT activation. Direct identification of GPR55 receptors in stria- 30 21 BRET tum is here demonstrated in rat brain slices using a specific agonist. Moreover, the heteromer finger- 31 print was identified in these rat slices by ERK1/2 phosphorylation assays whereas PLA assays showed 32 results consistent with receptor–receptor interaction in both caudate and putamen nuclei of a non- 33 human primate. The results indicate not only that GPR55 is expressed in striatum but also that CB1 34 35 and GPR55 receptors form heteromers in this specificCNSregion. 36 © 2014 Published by Elsevier Inc. 373840 39 41 Introduction protein, leading to decreases in the intracellular levels of the second 53 messenger cAMP upon its activation (Howlett, 2005; Pertwee et al., 54 42 Endocannabinoids are neuromodulators that act on the canna- 2010). 55 43 binoid receptors which currently represent potential therapeutic G-protein-coupled receptor 55 (GPR55) has been for many 56 44 targets for pain and for alterations to the central nervous system years considered an orphan receptor, i.e. a receptor for which no 57 45 (CNS). 2-Arachidonoyl-glycerol (2AG) and anandamide (AEA) are endogenous agonist was identified. As some CB1R ligands, AM251 58 46 two main endocannabinoids that exert their function via specific and rimonabant (SR141617) were able to bind to GPR55 (Brown 59 47 cannabinoid receptors 1 (CB1R) and 2 (CB2R) (Pacher and Kunos, and Robin Hiley, 2009; Hiley and Kaup, 2007; Johns et al., 2007; 60 48 2013). CB1R is activated by endocannabinoids by the main psycho- Lauckner et al., 2008; Pertwee, 2007; Petitet et al., 2006; Ryberg 61 49 active constituent of Cannabis sativa, Δ9-tetrahydrocannabinol et al., 2007), it was for some time assumed to be a third cannabi- 62 50 (Matsuda et al., 1990; Sugiura et al., 1999). It is the most highly noid receptor. In 2007, Oka and coworkers reported L-α- 63 51 expressed G-protein-coupled receptor (GPCR) in the brain. It has been lysophosphatidylinositol (LPI) as endogenous agonist of GPR55. 64 52 UNCORRECTED PROOF 65 clearly demonstrated that CB1R is coupled to a Gi heterotrimeric G Duringthelastdecadeithasbeendemonstrated that GPR55 does not couple to the adenylate cyclase system but its activation in- 66 duces intracellular calcium release via a Gα13/RhoA-mediated 67 pathway (Henstridge et al., 2009; Oka et al., 2007, 2009), regulates 68 the neutrophils cytoskeletal rearrangement and migration 69 ⁎ Corresponding author at: Laboratory of Cell and Molecular Neuropharmacology, (Balenga et al., 2011; Obara et al., 2011), and promotes prolifera- 70 Center for Applied Medical Research (CIMA), University of Navarra, Pio XII 55, 31008 tion via ERK1/2 MAPK activation (Andradas et al., 2011; 71 Pamplona, Spain. E-mail address: [email protected] (E. Martínez-Pinilla). Henstridge et al., 2010; Perez-Gomez et al., 2013). The pharmacol- 72 1 These authors contributed equally to this paper. ogy and the broad profile of tissue expression (Henstridge et al., 73 http://dx.doi.org/10.1016/j.expneurol.2014.06.017 0014-4886/© 2014 Published by Elsevier Inc. Please cite this article as: Martínez-Pinilla, E., et al., CB1 and GPR55 receptors are co-expressed and form heteromers in rat and monkey striatum, Exp. Neurol. (2014), http://dx.doi.org/10.1016/j.expneurol.2014.06.017 2 E. Martínez-Pinilla et al. / Experimental Neurology xxx (2014) xxx–xxx 74 2011), make GPR55 an attractive choice for further studies.2 De- approved by the Ethical Committee for Animal Testing of the University 124 75 spite its expression in mammalian brain (Ryberg et al., 2007), the of Navarra and the Department of Health of the Government of Navarra. 125 76 significant levels of LPI in nervous tissues (Oka et al., 2009), and 77 the active lipid metabolism in neural cells, the role of GPR55 in Monkey perfusion and tissue processing 126 78 the CNS is largely unknown. Recently, however, a knockout 79 mouse for GPR55 has been characterized by Wu et al. (2013) who Animals were anesthetized with an overdose of 10% chloral hydrate 127 80 report that the receptor plays a role in motor coordination. and perfused transcardially. The perfusate consisted of a saline Ringer's 128 81 Expression of dimers/oligomers constituted by different GPCRs solution followed by 3000 ml of a fixative solution containing 4% para- 129 fi 82 is a common nding from evidence in the last 15 years. Therefore formaldehyde and 0.1% glutaraldehyde in 0.125 M phosphate buffer 130 83 the occurrence of dimers/oligomers constituted by cannabinoid re- (PB), pH 7.4. Perfusion was continued with 1000 ml of a cryoprotectant 131 fi 84 ceptors and other GPCRs further diversi es the physiological ac- solution containing 10% glycerin and 2% dimethylsulfoxide (DMSO) in 132 85 tions of endocannabinoids. Interestingly, CB1Rs form homodimers 0.125 M PB, pH 7.4. Once perfusion was completed, the skull was 133 86 (Wager-Miller et al., 2002) and also heterodimers with CB2Rs opened, the brain was removed and stored for 48 h in a cryoprotectant 134 87 (Callen et al., 2012). In addition, Kargl et al. (2012) have reported solution containing 20% of glycerin and 2% DMSO in 0.125 M PB, pH 7.4. 135 88 that GPR55 co-immunoprecipitates with CB1R when co-expressed Finally, frozen serial sagittal sections (40 μm thick) were obtained on a 136 89 in heterologous cells. Based on the pleiotropic signaling of both sliding microtome and collected in 0.125 M PB, pH 7.4, as 10 series of ad- 137 90 cannabinoid receptors coupled with the atypical pharmacology of jacent sections. 138 91 the GPR55, the aim of this study was to know whether GPR55 is 92 expressed in the CNS and forms heteromers with CB R. In samples 1 Rat brain slices preparation 139 93 from striatum the CB1R–GPR55 heteromer fingerprint was detect- 94 ed and the interaction was further investigated by the use of the Rats were anesthetized with 4% isoflurane (2-chloro-2- 140 95 recently-developed in situ proximity ligation assay (PLA). (difluoromethoxy)-1,1,1-trifluoro-ethane) and decapitated with a 141 guillotine; brain was rapidly removed and placed in ice-cold oxy- 142 − 143 96 Materials and methods genated (O2/CO2: 95/5%) Krebs-HCO3 buffer (124 mM NaCl; 4mMKCl;1.25mMNaH2PO4;1.5mMMgCl2;1.5mMCaCl2; 144 145 97 Drugs 10 mM glucose; and 26 mM NaHCO3 pH 7.4). Brains were sliced at 4 °C in a brain matrix (Zivic Instruments, Pittsburgh, PA) into 146 147 98 N-(2-Chloroethyl)-5Z,8Z,11Z,14Z-eic­osatetraenamide (ACEA) 0.5 mm coronal slices and the striatum area was dissected. Slices − 148 99 was from Tocris Bioscience, 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)- were kept at 4 °C in Krebs-HCO3 buffer during the dissection of 149 100 4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (SR141716) was the striatum. Each slice was transferred into an incubation tube − 150 101 from Cayman Chemical and CID1792197 was from PubChem containing 1 ml of ice-cold Krebs-HCO3 buffer. The temperature 151 102 (Kotsikorou et al., 2011). ACEA was supplied as pale yellow oil that wasraisedto23°Candafter30minthemediumwasreplacedby − 152 103 was diluted in ethanol in a 10 mM concentration stock solution. 2mlKrebs-HCO3 buffer (23 °C). The slices were incubated under – 153 104 SR141617 (10 mM) and CID1792197 (10 mM) stock solutions were constant oxygenation (O2/CO2:95/5%)at30°Cfor4 5hinan 154 105 prepared in DMSO. Aliquots of these stock solutions were kept frozen Eppendorf Thermomixer (5 Prime, Inc., Boulder, CO). The media μ − 155 106 at −20 °C until use. were replaced by 200 l of fresh Krebs-HCO3 buffer and incubated for 30 min before the addition of ligands.