CB1 and GPR55 Receptors Are Co-Expressed and Form Heteromers in Rat 3 and Monkey Striatum

YEXNR-11769; No. of pages: 9; 4C: Experimental Neurology xxx (2014) xxx–xxx

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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

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. ERK1/2 phosphorylation 156 was determined as described below. 157 107 Animals Fusion proteins and expression vectors 158 108 A total of three naïve adult male Macaca fascicularis primates (body 109 – weight 3.8 4.5 kg) were used in this study. Animal handling was con- The human cDNAs for the CB1, GPR55 and dopamine D4.2 receptors 159 110 ducted in accordance with the European Council Directive 2010/63/ cloned in pcDNA3.1 were amplified without their stop codons using 160 111 EU, as well as in agreement with the Society for Neuroscience Policy sense and antisense primers harboring either unique EcoRI and BamH1 161 112 on the Use of Animals in Neuroscience Research. The experimental de- sites (CB1R, GPR55) or Xho1 and EcoR1 (D4.2R). The fragments were 162 113 sign was approved by the Ethical Committee for Animal Testing of the then subcloned to be in-frame with Rluc into the EcoRI and BamH1 163 114 University of Navarra (ref: 009-12) as well as by the Department of (CB1R) restriction sites of an Rluc-expressing vector (pRluc-N1, 164 115 Health from the Government of Navarra (ref: NA-UNAV-09-12). PerkinElmer, Wellesley, MA), or into the BamH1 and EcoRI (GPR55) or 165 116 – – Sprague Dawley female rats, 7 9 weeks old and weighing Xho1 and EcoR1 (D4.2R) restriction sites of an EYFP expressing vector 166 117 200–250 g, were provided by the animal Service of the University (EYFP-N1; enhanced yellow variant of GFP; Clontech, Heidelberg, 167 118 of Navarra (Pamplona, Spain). The animals were maintained in positive Germany), to give the plasmids that express CB1, GPR55 or D4.2 receptors 168 119 pressure-ventilated racks at 25 ± 1 °C with a 12 h light/dark cycle, fed fused to Rluc or YFP on the C-terminal end of the receptor (CB1R–Rluc, 169 120Q3 ad libitum with a standard rodent pellet diet (Global Diet 2014; Harlan), GPR55–YFP or D4.2R–YFP). Expression of receptors was tested by confocal 170 121 and allowed free access toUNCORRECTEDfiltered and UV-irradiated water. Animal pro- microscopy and the PROOF receptor functionality was tested performing ERK1/2 171 122 cedures were conducted according to the Guidelines for the Care and activation assays. 172 123 Use of Mammals in Neuroscience and Behavioral Research (2003) and Cell line cultures and transfection 173 2 Abbreviations: 2AG, 2-arachidonoyl-glycerol; AEA, anandamide; BRET, bioluminescence resonance energy transfer; CB R, cannabinoid receptor 1; CB R, cannabinoid recep- 1 2 Human embryonic kidney 293T (HEK-293T) cells were grown in 174 tor 2; CLSM, confocal laser-scanning microscope; CNS, central nervous system; D4.2R, 175 dopamine D4.2 receptor; DEPC, diethylpyrocarbonate; DMEM, Dulbecco's modified eagle DMEM supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, medium; DMSO, dimethylsulfoxide; FBS, fetal bovine serum; GPCR, G-protein-coupled re- 100 units/ml penicillin/streptomycin, and 5% (v/v) heat-inactivated 176 ceptor; GPR55, G-protein-coupled receptor 55; HBSS, Hanks' balanced salt solution; HEK- fetal bovine serum (FBS) (all supplements were from Invitrogen, (Pais- 177 293T, human embryonic kidney 293T; L-DOPA, L-3,4-dihydroxyphenylalanine; LPI, L-α- ley, Scotland, UK)). Cells were maintained at 37 °C in a humidified atmo- 178 lysophosphatidylinositol; mBU, mili BRET units; NFAT, nuclear-factor of activated T cells; – fl 179 PB, phosphate buffer; PBS, phosphate buffered saline; PEI, polyethylenimine; PLA, proxim- sphere of 5% CO2 and were passaged when they were 80 90% con uent, ity ligation assay; RLU, relative light units; TBS, Tris buffered saline. i.e. approximately twice a week. 180

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 E. Martínez-Pinilla et al. / Experimental Neurology xxx (2014) xxx–xxx 3

181 HEK-293T cells growing in 35-mm-diameter wells of six-well plates Probemaker MINUS DUO92010, Sigma Chemical Co, USA) following 242 182 or 60-mm-diameter plates were transiently transfected with the corre- manufacturer's instructions. 243 183 sponding fusion proteins cDNAs by the ramiﬁed PEI (PolyEthylenImine, The PLA technique was carried out in 3 different HEK-293T cell 244 184 Sigma, St. Louis, MO, USA) method. Cells were incubated (4 h) with the preparations obtained by transfecting with the cDNAs coding for 245

185 corresponding cDNA together with ramified PEI (5 ml of 10 mM PEI for CB1R, GRP55 and both receptors. Transfected cells were fixed in 4% 246 186 each mg of cDNA) and 150 mM NaCl in a serum-starved medium. After paraformaldehyde for 15 min, washed with PBS containing 20 mM 247 187 4 h, the medium was changed to a fresh complete culture medium. For glycine (to quench the aldehyde groups), and permeabilized with 248 188 ERK1/2 phosphorylation assay, HEK-293T-transfected cells were cultured PBS-glycine containing 0.05% Triton X-100. Cell cultures were incu- 249 189 in serum-free medium for 16 h before the addition of any agent. For bio- bated for 1 h at 37 °C with the blocking solution in a pre-heated hu- 250 190 luminescence resonance energy transfer (BRET) assay, 48 h after transfec- midity chamber, followed by an overnight incubation (4 °C) with the 251 191 tion, cells were washed twice in quick succession in Hanks' balanced salt PLA probe-linked antibodies described above (final concentration of 252

192 solution(HBSS;137mMNaCl;5mMKCl;0.34mMNa2HPO4 ×12H2O; 75 μg/ml). After washing with buffer A (Wash buffer A, DUO82047, 253 193 0.44 mM KH2PO4; 1.26 mM CaCl2 ×2H2O; 0.4 mM MgSO4 ×7H2O; Sigma Chemical Co, USA), samples were immersed for 1 h in a 254 194 0.5 mM MgCl2; and 10 mM HEPES pH 7.4) supplemented with 0.1% 1:400 solution of TO-PRO®-3 (Molecular Probes, T3605) for nuclear 255 195 glucose (w/v), detached by gently pipetting and resuspended in the staining. The presence/absence of receptor–receptor molecular in- 256 196 same buffer. To control the cell number, sample protein concentration teraction in these samples was detected using the Duolink II in situ 257 197 was determined using a Bradford assay kit (Bio-Rad, Munich, Germany) PLA detection kit (Duolink® In Situ Detection Reagents Red 258 198 and using bovine serum albumin (Sigma Chemical Co, St. Louis, MO, DUO92008, Sigma Chemical Co, USA). Following the detection proto- 259 199 USA) dilutions as standards. HEK-293T cell suspension (20 μgofprotein) col described by the manufacturer, cells were washed with buffer A 260 200 was distributed into 96-well microplates; black plates with a transparent at room temperature and incubated with the ligation solution for 1 261 201 bottom (Porvair, Leatherhead, UK) were used for fluorescence determina- h at 37 °C in a humidity chamber. Following washes with buffer A, 262 202 tions, whereas white opaque plates (Porvair, Leatherhead, UK) were used samples were incubated with the amplification solution for 100 263 203 for BRET experiments. min at 37 °C in humidity chamber and then washed with buffer B 264 (Wash buffer B, DUO82048, Sigma Chemical Co, USA), followed by 265 another wash with buffer B ×0.01. Samples were mounted using an 266 204 Bioluminescence Resonance Energy Transfer (BRET) assay aqueous mounting medium. Cell lines transfected only with either 267 CB R or GRP55 were used as appropriate negative control assays 268 205 HEK-293T cells were transiently co-transfected with the indicated 1 for the PLA technique to ensure lack of non-specificlabeling. 269 206 amounts of plasmid cDNAs corresponding to the indicated fusion Tissue sections containing the striatum (caudate and putamen nu- 270 207 proteins (see corresponding figure legends). To quantify receptor- cleus) were used to detect CB R–GPR55 heteromers by the PLA tech- 271 208 fluorescence expression, cells (20 μg protein) were distributed in 1 nique described above. Appropriate negative control assays were 272 209 96-well microplates (black plates with a transparent bottom; carried out to ensure that there was a lack of non-specificlabelingand 273 210 Porvair, Leatherhead, UK) and fluorescence was read using a Mithras amplification. 274 211 LB 940 (Berthold, Bad Wildbad, Germany) equipped with a high- 212 energy xenon flash lamp, using an excitation filter of 485 nm. 213 Receptor-fluorescence expression was determined as fluorescence 214 of the sample minus the fluorescence of cells expressing protein- 215 Rluc alone. For BRET measurements, the equivalent of 20 μgofcell 216 suspension was distributed in 96-well microplates (white opaque 217 plates; Porvair, Leatherhead, UK) and 5 μM Coelenterazine H (PJK 218 GMBH, Germany) was added. After 1 min of adding Coelenterazine 219 H, readings were collected using a Mithras LB 940 (Berthold, Bad 220 Wildbad, Germany) that allows the integration of the signals detect- 221 ed in the short-wavelength filter at 485 nm (440–500 nm) and the 222 long-wavelength filter at 530 nm (510–590 nm). To quantify 223 receptor-Rluc expression luminescence, readings were performed 224 after 10 min of adding 5 μM Coelenterazine H. Cells expressing 225 BRET donors alone were used to determine background. The net 226 BRET is defined as [(long-wavelength emission)/(short-wavelength 227 emission)]-Cf where Cf corresponds to [(long-wavelength emis- 228 sion)/(short-wavelength emission)] for the Rluc construct expressed 229 alone in the same experiment. BRET curves were fitted by using a 230 non-linear regression equation, assuming a single phase with 231 GraphPad Prism software (San Diego, CA, USA). BRET is expressed 232 as mili BRET units (mBU:UNCORRECTED 1000 × net BRET). PROOF

Fig. 1. CB1R forms heteromers with GPR55 in transfected cells. BRET saturation experi-

233 In situ proximity ligation assay (PLA) ments showing CB1R–GPR55 heteromerization were performed using cells transfected

with 0.5 μg of cDNA corresponding to CB1R–Rluc and increasing amounts of cDNA (0 to μ – 234 Proximity probes were consisted of affinity-purified antibodies 2 g cDNA) corresponding to GPR55 YFP (triangles). As negative control, cells were also transfected with cDNA corresponding to CB R–Rluc (0.5 μg) and to D R–YFP (0 to 4 μg 235 fi ′ 1 4,2 modi ed by covalent attachment of 5 end of various oligonucleotides cDNA) (squares). Both fluorescence and luminescence for each sample were measured be- 236 to each primary antibody. To create our PLA probes we conjugated a fore every experiment to confirm similar donor expressions (approximately 12,000 biolu- Q1 237 rabbit anti-CB1R (PA1-745, Thermo Scientific, Rockford, USA) with a minescence units) while monitoring the increase in acceptor expression (100 to 70,000 238 PLUS oligonucleotide (Duolink® In Situ Probemaker PLUS DUO92009, net fluorescence units). The relative amount of BRET is given as the ratio between the fluorescence of the acceptor minus the fluorescence detected in cells expressing only the 239 Sigma Chemical Co, USA) and a rabbit anti-GPR55 (10224, Cayman donor and the luciferase activity of the donor. BRET data are expressed as mean ± SEM 240 – Chemical, Michigan, USA), raised against the human 207 219 sequence (4–8 different experiments grouped as a function of the amount of BRET acceptor). 241 (ILLGRRDHTQDWV), with a MINUS oligonucleotide (Duolink® In Situ mBU: miliBRET units.

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 4 E. Martínez-Pinilla et al. / Experimental Neurology xxx (2014) xxx–xxx

275 Stained samples were inspected under a Zeiss 510 Meta confocal LI-COR Biosciences, Lincoln, Nebraska, USA) for 1 h at room temperature. 308 276 laser-scanning microscope (CLSM). To ensure appropriate visualization Bands were visualized by the Odyssey® Fc Imaging System (LI-COR Bio- 309 277 of the labeled elements and to avoid false positive results, the emission sciences, Lincoln, Nebraska, USA) and their densities quantified using 310 278 following excitation with the helium laser at 543 nm was filtered the Image Studio software 1.1 (LI-COR Biosciences, Lincoln, Nebraska, 311 279 through a band pass filter of 560–615 nm and color coded in light red. USA). The level of phosphorylated ERK1/2 isoforms was normalized for 312 280 Finally, a longpass filter of 650 nm was used to visualize the emission differences in loading using the total ERK1/2 protein band intensities. 313 281 from the helium laser at 633 nm and color coded in blue. Images were 282 acquired using the Zeiss software (Aim4). NFAT reporter gene assay 314

HEK-293T cells were seeded in 24-well plates (50,000 cells/well) and 315 283 ERK1/2 phosphorylation assays transiently transfected with pcDNA CB1R, pcDNA GPR55 or with both 316 pcDNA plasmids, at 500 ng/well using Lipofectamine 2000 (11668-027, 317 284 HEK-293T-transfected cells or striatal slices were treated or not with Thermo Scientific, Rockford, USA). For reporter gene assay, cells were ad- 318 285 the indicated ligands for the indicated time (see corresponding figure leg- ditionally transiently transfected (24 h after the first transfection) with a 319 286 ends), rinsed with ice-cold PBS and lysed by the addition of 100 μlor pNFAT-Luc reporter plasmid (PathDetect NFAT cis-Reporter; Stratagene) 320 287 150 μl, respectively, of an ice-cold lysis buffer (50 mM Tris–HCl pH 7.4; at 500 ng/well in combination with Renilla luciferase plasmid, an internal 321 288 50 mM NaF; 150 mM NaCl; 45 mM β-glycerophosphate; 1% Triton X- control in luciferase-based reporter gene assays, at 500 ng/well using 322 289 100; 0.4 mM NaVO ; and protease inhibitor mixture). The cellular debris 4 Lipofectamine 2000. 24 h later, cells were incubated with the indicated li- 323 290 was removed by centrifugation at 13,000 ×g for 5 min at 4 °C, and the gands (see corresponding figure legends) for 6 h in serum-free medium at 324 291 protein was quantified by the bicinchoninic acid method using bovine 37 °C. Luciferase activity was visualized by using the Dual-Luciferase® 325 292 serum albumin dilutions as standard. To determine the level of ERK1/2 Reporter Assay System kit (E1910, Promega) following manufacturer's in- 326 293 phosphorylation, equivalent amounts of protein (15 μg) were mixed structions. Luminescence was measured in a Microplate Luminometer 327 294 with 6× Laemli sample buffer, separated by electrophoresis on a denatur- (Orion II, Berthold, Bad Wildbad, Germany) for 5 s. Luminescence values 328 295 ing 10% SDS-polyacrylamide gel and transferred onto nitrocellulose are given as relative light units (RLU). 329 296 membranes (BioTrace™ NT Nitrocellulose Transfer Membrane, 66485, 297 PALL). After that, Odyssey blocking buffer (LI-COR Biosciences, Lincoln, 298 Nebraska, USA) was added, and the membranes were rocked for Statistical analysis 330 299 60 min. The membranes were then probed with a mixture of a mouse 300 anti-phospho-ERK1/2 antibody (1:1,000 dilution; M8159 Sigma Chemical Data result from at least 3 independent experiments. The data in the 331 301 Co., St. Louis, MO, USA) and rabbit anti-ERK1/2 antibody that recognizes graphs are presented as the mean ± SEM. Statistical analysis was per- 332 302 both phosphorylated and nonphosphorylated ERK1/2 (1:20,000; M5670 formed with SPSS 18.0 software. The test of Kolmogorov–Smirnov 333 303 Sigma Chemical Co., St. Louis, MO, USA) in blocking buffer for 2–3hat with the correction of Lilliefors was used to evaluate the fitofthedata 334 304 room temperature. After washed three times with TBS/Tween20, mem- to a normal distribution and the test of Levene to evaluate the homoge- 335 305 branes were incubated with a mixture of IRDye®CW 800 (anti-mouse) neity of variance. Significance was analyzed by one-way ANOVA test 336 306 antibody (1:15,000; 926-32210 LI-COR Biosciences, Lincoln, Nebraska, followed by Tukey's test for multiple comparisons. Significant differ- 337 307 USA) and IRDye® 680RD (anti-rabbit) antibody (1:15,000; 926-68071 ences were considered when p b 0.05. 338

UNCORRECTED PROOF

Fig. 2. PLA for CB1R and GPR55 in transfected cells. A) Schematic representation of the PLA technology. B–D) The speciﬁcity of PLA was tested by using HEK-293T cell lines transiently

transfected with the cDNAs of CB1R (B), GPR55 (C) or with both cDNAs receptors (D). Cells were processed for PLA stain according to the guidelines issued by the manufacturer. CB1R and GPR55 in close proximity are detectable as a punctate red ﬂuorescent signal by confocal microscopy (D). Cell nuclei were stained with TO-PRO®-3 (blue color). Scale bars B: 40 μm; C: 40 μm and D, D′,D″: 40, 20 and 4 μm, respectively. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

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 E. Martínez-Pinilla et al. / Experimental Neurology xxx (2014) xxx–xxx 5

339 Results increased indicated that the interaction between CB1R and GPR55 to 351 form CB1R–GPR55 heteromers was speciﬁc. Fitting the data (described 352 340 CB1R and GPR55 form heteromers in Materials and methods) allowed the calculation of the BRET parame- 353 ters: BRETmax =46±4mBU;BRET50 = 28 ± 7. Speciﬁcity of this inter- 354 341 To investigate the occurrence of heteromers and the consequent action was assessed by comparing the CB1R–Rluc and GPR55–YFP BRET 355 342 modulation of signaling cascades by co-expression of CB1R and GPR55, data with the low and linear ones obtained using the negative control, 356 343 transient transfection of HEK-293T cells was performed. The possible CB1R–Rluc and D4,2R–YFP, described by Callen et al. (2012). These re- 357 344 heteromerization of CB1R and GPR55 was explored by two complemen- sults indicate that CB1R and GPR55 interact in living HEK-293T cells 358 345 tary approaches. First, a direct interaction between CB1R and GPR55 was (Fig. 1). 359 346 assessed by a BRET biophysical approach. BRET studies in HEK-293T Second, we conducted a PLA in HEK-293T cells expressing GPR55 360

347 cells transfected with CB1R–Rluc and GPR55–YFP indicated a strong en- and/or CB1R that permits the direct detection of molecular interactions 361 348 ergy transfer, i.e. close proximity of the donor and acceptor in the C- between two proteins. Labeling heterodimers by PLA requires both re- 362 349 terminus of the two fusion proteins (Fig. 1). The saturation of the ceptors to be sufﬁciently close to allow the two antibody-DNA probes 363 350 BRET signal when the amount of the acceptor (GPR55–YFP) was to form double stranded segments (b17 nm), a signal that is further 364

UNCORRECTED PROOF

Fig. 3. Cross-talk between CB1R and GPR55 on ERK1/2 phosphorylation in HEK-293T transfected cells. A) Cells co-transfected with the cDNA corresponding to CB1R (1.5 μg) and GPR55

(1.5 μg). B) Cells transfected with the cDNA corresponding to CB1R (1.5 μg). C) Cells transfected with the cDNA corresponding to GPR55 (1.5 μg). In all cases, cells were treated with

the CB1R agonist, ACEA (100 nM), with the GPR55 agonist, CID1792197 (1 μM),orbothfor10minorpretreatedfor10minwithCB1R antagonist SR141716 (250 nM) prior to agonist treatment. ERK1/2 phosphorylation was determined as described under “Materials and methods”. The immunoreactive bands from 4 to 5 independent experiments were quantiﬁed, and the values represent the mean ± SEM of the percentage of the ratio pERK1/2 versus ERK1/2 relative to the basal levels found in untreated cells (100%). Signiﬁcant differences were analyzed by a one-way ANOVA followed by post-hoc Tukey's test (##p b 0.01, ###p b 0.001 compared with control; &&p b 0.01, &&&p b 0.001 compared with treated with ACEA alone; **p b 0.01 compared with treated with CID1792197 alone). A representative Western blot is shown in each panel (top).

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 6 E. Martínez-Pinilla et al. / Experimental Neurology xxx (2014) xxx–xxx

365 amplified in the presence of fluorescent oligonucleotides. Thus, the de- CB1R–CB2R heteromers in which antagonist binding to CB1R blocks the 391 366 tection of a punctate fluorescent signal by confocal microscopy is de- signaling mediated by the partner receptor (Callen et al., 2012). Identi- 392 367 pendent on the close proximity of the receptors (Soderberg et al., fication of the fingerprint could be later used to detect heteromer ex- 393

368 2008; Trifilieff et al., 2011)(Fig. 2A). In these experiments, specificpri- pression in the CNS. In cells co-expressing CB1R and GPR55, the 394 369 mary antibodies directed against each of the two receptors were used: a specificCB1R agonist, ACEA (100 nM), led to a significant increase in 395 370 rabbit anti-CB1R antibody whose specificity has been described previ- ERK1/2 phosphorylation (p b 0.001) that was reverted by the selective 396 371 ously (Carriba et al., 2007), and a rabbit anti-GPR55 antibody that has al- antagonist SR141716 (250 nM) (p b 0.001). ERK1/2 phosphorylation 397 372 ready been tested in transfected cells and neurons (Lauckner et al., was also significant in cells treated with 1 μM CID1792197 (p b 0.001), 398 373 2008). By incubating cells with these antibodies we observed, in a selective GPR55 agonist (Fig. 3A). As there are not commercially avail- 399

374 double-transfected cells, the formation of CB1R–GPR55 heteromers, im- able antagonist the speciﬁcity of the effect of CID1792197 was proven 400 375 munoreactivity is visible as red spots in neurons with TO-PRO®-3- by using single transfected cells; the agonist had no effect on CB1R- but 401 376 stained nuclei (Fig. 2D). Single transfected cells did not display red in GPR55-expressing cells (Figs. 3B–C). The effect of ACEA was evident 402

377 spots while they were not able to express both receptors (Figs. 2B–C). in cells single transfected with the cDNA for CB1R and was blocked by 403 378 These results demonstrate the occurrence of heteromers in cells co- the CB1R antagonist. Remarkably, the increase in pERK1/2 due to the 404 379 expressing the receptors. No red spots were observed in double- GPR55 agonist in cotransfected cells was blocked by pretreatment with 405

380 transfected cells treated with only one primary antibody and secondary SR141716, the CB1R antagonist (p b 0.001) (Fig. 3A). In untransfected 406 381 antibody-DNA probes (not shown). cells none of the agonist led to any signiﬁcant signal (data not shown). 407 Moreover, SR141716 by itself was not able to neither decrease the basal 408 409 382 CB1R–GPR55 heteromer ﬁngerprint in HEK-293T cells pERK1/2 levels nor increase them (Fig. 3A). These results indicate that the cross-antagonism detected in double-transfectants may be consid- 410

383 There have been several reports on the heteromerization of GPCRs, ered a ﬁngerprint for CB1R–GPR55 heteromers. 411 384 which result in the formation of novel ligand binding properties and HEK–CB1R–GPR55 cells were also used to investigate whether activa- 412 385 heteromer-speciﬁc signaling (Renner et al., 2012; Rivero-Muller et al., tion of the nuclear-factor of activated T cells (NFAT), a transcription factor 413 386 2010; Sedej et al., 2012; Waldhoer et al., 2005). A common characteris- downstream GPR55 (Henstridge et al., 2009), is affected by the co- 414

387 tic of receptor heteromers is that ligand binding to one receptor can expression of CB1R. Activation of NFAT results in its translocation to the 415 388 modulate ligand binding to the partner receptor in the heteromer nucleus, where it regulates the expression of a variety of genes (Im 416 389 (Ferre et al., 2009; Gonzalez et al., 2012). This biochemical cross-talk and Rao, 2004). The results show that 1 μM CID1792197 (GPR55 ago- 417

390 constitutes a ﬁngerprint of the heteromer and has been described for nist)-induced activation of NFAT was blocked by the selective CB1R 418

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Fig. 4. The speciﬁcCB1R antagonist SR141716 modulates GPR55 transcription factor activation in HEK-293T co-transfected cells. A) Cells co-transfected with the cDNA corresponding to CB1R

(500 ng) and GPR55 (500 ng). B) Cells transfected with the cDNA corresponding to CB1R (500 ng). C) Cells transfected with the cDNA corresponding to GPR55 (500 ng). In all cases, 24 h after

ﬁrst transfection cells were transfected with 500 ng of NFAT-luciferase reporter plasmid and 24 h after cells were stimulated with the CB1R antagonist, SR141716 (250 nM), and with the GPR55 agonist, CID1792197 (1 μM), for 6 h in a serum-free medium. Luciferase activity was measured as described under “Materials and methods”. Data are mean ± SEM from three independent experiments performed in quadruplicate, normalized and expressed as percentage of activation relative to the basal levels found in untreated cells (100%), relative light units (RLU). Signiﬁcant differences were analyzed by a one-way ANOVA followed by post-hoc Tukey's test (#p b 0.05, ##p b 0.01 compared with control; *p b 0.05 compared with treated with CID1792197 alone).

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 E. Martínez-Pinilla et al. / Experimental Neurology xxx (2014) xxx–xxx 7

cells, the signiﬁcant ERK1/2 phosphorylation obtained in slices treated 432 with 1 μM of the selective GPR55 agonist, CID1792197, was blocked by 433

the CB1R antagonist (SR141716) (p b 0.05) that was not able by itself 434 to signiﬁcantly affect the basal pERK1/2 levels (Fig. 5). The cross- 435

antagonism detected may be considered a ﬁngerprint for CB1R–GPR55 436 heteromers in striatum. 437

Monkey striatum displays CB1R–GPR55 heteromers 438

Further proof of heteromer expression in the CNS was attempted 439 using PLA. PLA was not possible in rat slices as the anti-GPR55 antibody 440 did not provide good images; probably this was due to relatively small ho- 441 mology between the human and the rat sequences in the fragment used 442 to raise the anti-peptide antibody (ILLGRRDHTQDWV in human and 443

ILLsiqgD___DqWV in rat). As we have experience with CB1R-containing 444 heteromer identiﬁcation by PLA in the basal ganglia of non-human pri- 445 mates, assays were performed in striatal slices from Macaca fascicularis. 446 The high homology in protein sequence between GPCRs in this primate 447 model and humans (near 100%) should be noted, and this was a further 448 reason to select this model whose receptors are therefore similar to the 449 human version of the receptors transfected in HEK-293T cells (see 450 above). By incubating slices with the above-described antibodies tested 451

in HEK-293T cells, CB1R–GPR55 heteromers were identified as red spots 452 in neurons with TO-PRO®-3-stained nuclei (Fig. 6). No red spots were ob- 453 – fi Fig. 5. CB1R GPR55 heteromer ngerprint was found in rat striatum. Striatal rat slices served in slices treated with only one primary antibody and secondary 454 were treated with the CB R agonist, ACEA (100 nM), with the GPR55 agonist, 1 antibody-DNA probes (not shown). These positive results were obtained 455 CID1792197 (1 μM), or both for 10 min or pretreated for 10 min with CB1R antagonist SR141716 (250 nM) prior to agonist treatment. ERK1/2 phosphorylation was determined in both caudate (Fig. 6A) and putamen (Fig. 6B) and indicate the overall 456 “ ” as described under Materials and methods . The immunoreactive bands from two differ- presence of CB1R–GPR55 heteromers in the primate striatum. 457 ent experiments performed in triplicate were quantified, and the values represent the mean ± SEM of the percentage of the ratio pERK1/2 versus ERK1/2 relative to the basal 458 levels found in untreated slices (100%). Significant differences were analyzed by a one- Discussion way ANOVA followed by post-hoc Tukey's test (#p b 0.05 compared with control; *p b 0.01 compared with treated with CID1792197 alone). A representative Western blot is The signaling pathways and function of GPR55 have been under in- 459 shown (top). vestigation in heterologous expression systems such as CHO cells 460 (Lauckner et al., 2008) and HEK-293T cells (Henstridge et al., 2009; 461 Ryberg et al., 2007), in neurons (Lauckner et al., 2008; Obara et al., 462 419 b antagonist SR141716 (p 0.05) (Fig. 4A). The cross-antagonism on 2011), in cancer cells (Hu et al., 2011; Paul et al., 2014; Perez-Gomez 463 420 GPR55-mediated NFAT activation was not found in single transfected et al., 2013; Pineiro et al., 2011), in platelets (Kargl et al., 2013), in neu- 464 421 – – cells (Figs. 4B C) thus suggesting that was due to CB1R GPR55 co- trophils (Balenga et al., 2011) and in cells of the immune system (Oka 465 422 expression. et al., 2010). There are difficulties in investigating the pharmacology of 466 the receptor as it is able to bind cannabinoid-receptor ligands, many 467 423 Striatal slices display a biochemical fingerprint of the heteromer of which are of lipidic nature such as LPI itself. Anavi-Goffer et al. 468 (2012) have shown that MAP kinase signaling due to the activation of 469 424 First, to look for heteromers in the CNS, rat striatal slices were used to GPR55 is modulated by cannabinoids. It seems therefore that 470 425 detect expression of GPR55. The specific GPR55 agonist, CID1792197, led GPR55 signaling and function may depend on the cell context and 471 426 to an increase in the phosphorylation of ERK1/2 (p b 0.05). Also in the on the extracellular signals (endocannabinoids included) impacting 472

427 same samples ACEA, a selective CB1R agonist, led to an increase in the on the GPR55-expressing cell (Anavi-Goffer et al., 2012). Reasons 473 428 phosphorylation of ERK1/2 (p b 0.05) thus indicating that both CB1R for such diversity are differential expression of G proteins and 474 429 and GPR55 are expressed and are functional in rat striatum. Interestingly, other relevant components of the signaling machinery but it may 475 430 the same ﬁngerprint detected in co-transfected cells was found in rat be also due to receptor heteromerization. Increasing evidence, 476 431 slices. As it would be predicted if heteromers are expressed in striatal points to heteromers as key players in the regulation of cell events; 477

UNCORRECTED PROOF

Fig. 6. CB1R forms heteromers with GPR55 in Macaca fascicularis striatum. Illustrative PLA examples of neurons taken from caudate (A) and putamen monkey nucleus (B). Sections were

processed for PLA stain according to the guidelines issued by the manufacturer, and CB1R and GPR55 in close proximity are detectable as a punctate red ﬂuorescent signal by confocal microscopy. Cell nuclei were stained with TO-PRO®-3 (blue color). Scale bars A and B: 50 μm; A′ and B′:5μm. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

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 8 E. Martínez-Pinilla et al. / Experimental Neurology xxx (2014) xxx–xxx

478 in fact heteromers are considered novel entities with differential Bonaventura, J., Rico, A.J., Moreno, E., Sierra, S., Sanchez, M., Luquin, N., Farre, D., Muller, C. 539 E., Martinez-Pinilla, E., Cortes, A., Mallol, J., Armentero, M.T., Pinna, A., Canela, E.I., 540 479 GPCR trafficking, pharmacology, and function (Rozenfeld and Devi, Lluis, C., McCormick, P.J., Lanciego, J.L., Casado, V., Franco, R., 2014. L-DOPA-treatment 541 480 2011; Tadagaki et al., 2012). Little is known about the expression in primates disrupts the expression of A2A adenosine-CB1 cannabinoid-D2 dopamine 542 481 and role in brain. Here, we detected functional GPR55 in striatum receptor heteromers in the caudate nucleus. Neuropharmacology 79, 90–100. 543 544 482 Brown, A.J., Robin Hiley, C., 2009. Is GPR55 an anandamide receptor? Vitam. Horm. 81, and tested the hypothesis that GPR55 heteromerizes with CB1Rs in 111–137. 545 483 this specific CNS region. Callen, L., Moreno, E., Barroso-Chinea, P., Moreno-Delgado, D., Cortes, A., Mallol, J., Casado, V., 546 547 484 Kargl et al. (2012) demonstrated that CB1R modulates the signaling Lanciego, J.L., Franco, R., Lluis, C., Canela, E.I., McCormick, P.J., 2012. Cannabinoid recep- – 548 485 properties of GPR55. Working in HEK-293T cells, the authors showed tors CB1 and CB2 form functional heteromers in brain. J. Biol. Chem. 287, 20851 20865. Carriba, P., Ortiz, O., Patkar, K., Justinova, Z., Stroik, J., Themann, A., Muller, C., Woods, A.S., 549 486 co-localization and also co-immunoprecipitation in cells co-expressing Hope, B.T., Ciruela, F., Casado, V., Canela, E.I., Lluis, C., Goldberg, S.R., Moratalla, R., 550 487 both receptors. The results suggest heteromer formation that correlated Franco, R., Ferre, S., 2007. Striatal adenosine A2A and cannabinoid CB1 receptors 551 552 488 with findings that each receptor modifies the signaling of the partner re- form functional heteromeric complexes that mediate the motor effects of cannabinoids. Neuropsychopharmacology 32, 2249–2259. 553 489 – ceptor. The hypothesis is that CB1R GPR55 heteromers are unique enti- Ferre, S., Baler, R., Bouvier, M., Caron, M.G., Devi, L.A., Durroux, T., Fuxe, K., George, S.R., 554 490 ties that may “play an important physiological and/or pathophysiological Javitch, J.A., Lohse, M.J., Mackie, K., Milligan, G., Pfleger, K.D., Pin, J.P., Volkow, N.D., 555 556 491 role in tissues endogenously co-expressing both receptors” (Kargl et al., Waldhoer, M., Woods, A.S., Franco, R., 2009. Building a new conceptual framework for receptor heteromers. Nat. Chem. Biol. 5, 131–134. 557 492 2012). Our results are a good complement of those by Kargl et al. Gonzalez, S., Moreno-Delgado, D., Moreno, E., Perez-Capote, K., Franco, R., Mallol, J., 558 493 (2012) as a direct interaction between CB1R and GPR55 is demonstrated Cortes, A., Casado, V., Lluis, C., Ortiz, J., Ferre, S., Canela, E., McCormick, P.J., 2012. 559 494 by BRET, which is a powerful technique to unambiguously detect Circadian-related heteromerization of adrenergic and dopamine D(4) receptors mod- 560 561 495 ulates melatonin synthesis and release in the pineal gland. PLoS Biol. 10, e1001347. heteromers in living cells. PLA assays in HEK-293T cells co-expresssing Henstridge, C.M., Balenga, N.A., Ford, L.A., Ross, R.A., Waldhoer, M., Irving, A.J., 2009. The 562 2+ 496 the two receptors provided both confirmation of heteromer expression GPR55 ligand L-alpha-lysophosphatidylinositol promotes RhoA-dependent Ca sig- 563 497 and the technical basis to detect heteromers- by an imaging technique- naling and NFAT activation. FASEB J. 23, 183–193. 564 565 498 Henstridge, C.M., Balenga, N.A., Schroder, R., Kargl, J.K., Platzer, W., Martini, L., Arthur, S., in striatal samples. The cross-antagonism (see below) at both the MAP Penman, J., Whistler, J.L., Kostenis, E., Waldhoer, M., Irving, A.J., 2010. GPR55 ligands pro- 566 499 kinase and NFAT-reporter assays also confirms heteromer expression mote receptor coupling to multiple signalling pathways. Br. J. Pharmacol. 160, 604–614. 567 500 and functional cross-talk. Henstridge, C.M., Balenga, N.A., Kargl, J., Andradas, C., Brown, A.J., Irving, A., Sanchez, C., 568 569 501 Detection of heteromers in natural sources is still a challenge. One suc- Waldhoer, M., 2011. Minireview: recent developments in the physiology and pathol- ogy of the lysophosphatidylinositol-sensitive receptor GPR55. Mol. Endocrinol. 25, 570 502 cessful approach is the detection of cross-antagonism (Callen et al., 2012), 1835–1848. 571 503 i.e. the blockade of a receptor-mediated signaling when the partner re- Hiley, C.R., Kaup, S.S., 2007. GPR55 and the vascular receptors for cannabinoids. Br. J. 572 – 573 504 ceptor in the heteromer binds an antagonist. First of all the cross- Pharmacol. 152, 559 561. Howlett, A.C., 2005. Cannabinoid receptor signaling. Handb. Exp. Pharmacol. 53–79. 574 505 antagonism was searched in HEK-293T cells in which signaling to ERK1/ Hu, G., Ren, G., Shi, Y., 2011. The putative cannabinoid receptor GPR55 promotes cancer 575 – 576 506 2 kinase pathway, induced by GPR55 agonist, was blocked by a CB1Ran- cell proliferation. Oncogene 30, 139 141. 2+ 577 507 tagonist. In rat striatal slices the occurrence of functional GPR55 was con- Im, S.H., Rao, A., 2004. Activation and deactivation of gene expression by Ca /calcine- urin-NFAT-mediated signaling. Mol. 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