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Modulation of l-α-Lysophosphatidylinositol/GPR55 Mitogen-activated Protein Kinase (MAPK) Signaling by Cannabinoids *

Modulation of l-α-Lysophosphatidylinositol/GPR55 Mitogen-activated Protein Kinase (MAPK)... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 1, pp. 91–104, January 2, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Modulation of L--Lysophosphatidylinositol/GPR55 Mitogen-activated Protein Kinase (MAPK) Signaling by Cannabinoids Received for publication, August 23, 2011, and in revised form, October 12, 2011 Published, JBC Papers in Press, October 25, 2011, DOI 10.1074/jbc.M111.296020 ‡§1 ‡ ¶  ‡ ‡ Sharon Anavi-Goffer , Gemma Baillie , Andrew J. Irving ,Ju¨ rg Gertsch , Iain R. Greig , Roger G. Pertwee , and Ruth A. Ross From the School of Medical Sciences, Institute of Medical Sciences, Kosterlitz Centre for Therapeutics, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, United Kingdom, the Division of Medical Sciences, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom, the Institute of Biochemistry and Molecular Medicine, University of Bern, Bu¨hlstrasse 28, Bern CH-3012, Switzerland, and the Departments of Behavioral Sciences and Molecular Biology, Ariel University Center of Samaria, Ariel 40700, Israel Background: The endogenous L--lysophosphatidylinositol activates GPR55. Results: Structural analogues of SR141716A act both as agonists alone and as inhibitors of L--lysophosphatidylinositol. Certain CB receptor agonists also modulate GPR55 activity. Conclusion: Certain cannabinoids can both activate GPR55 and attenuate L--lysophosphatidylinositol-mediated phosphor- ylated ERK1/2 activation. This has mechanistic implications for the antinociceptive effects of certain CB agonists. Significance: Cannabinoid ligands have complex interactions with the L--lysophosphatidylinositol/GPR55 signaling system. GPR55 is activated by L--lysophosphatidylinositol (LPI) but that the endogenous lysophospholipid LPI activates GPR55 also by certain cannabinoids. In this study, we investigated the (3–10). In certain cancer cell lines, GPR55 is highly expressed, GPR55 pharmacology of various cannabinoids, including ana- and LPI mediates increased cell migration, invasion, and prolif- logues of the CB receptor antagonist Rimonabant,CB recep- eration (3, 8, 11). Moreover, increased circulating levels of LPI 1 2 tor agonists, and Cannabis sativa constituents. To test ERK1/2 have been found in cancer patients and are associated with a phosphorylation, a primary downstream signaling pathway that poor prognosis (12). These observations suggest that modula- conveys LPI-induced activation of GPR55, a high throughput tion of GPR55 may have therapeutic implications for the treat- system, was established using the AlphaScreen SureFire ment of pain, bone diseases, and cancer. assay. Here, we show that CB receptor antagonists can act both To date, certain arylpyrazole CB receptor antagonists such as agonists alone and as inhibitors of LPI signaling under the as Rimonabant (also known as SR141716A or Acomplia) and same assay conditions. This study clarifies the controversy sur- AM251 have been reported to be GPR55 agonists (5, 13, 14). rounding the GPR55-mediated actions of SR141716A; some However, other groups have suggested that Rimonabant is a reports indicate the compound to be an agonist and some report GPR55 antagonist (6, 8). Furthermore, reports on the behavior antagonism. In contrast, we report that the CB ligand of  -THC at GPR55 are also inconsistent. Lauckner et al. (6) GW405833 behaves as a partial agonist of GPR55 alone and have shown that  -THC is a GPR55 agonist capable of stimu- enhances LPI signaling. GPR55 has been implicated in pain lating calcium release, and Kapur et al. (5) did not detect -ar- transmission, and thus our results suggest that this receptor may restin-mediated activation of GPR55 with this phytocannabi- be responsible for some of the antinociceptive actions of certain noid. Another cannabis constituent, cannabidiol (CBD), is CB receptor ligands. The phytocannabinoids  -tetrahydro- reported to be an antagonist of GPR55 (15). The GPR55 phar- cannabivarin, cannabidivarin, and cannabigerovarin are also macology of many other Cannabis sativa (C. sativa) constitu- potent inhibitors of LPI. These Cannabis sativa constituents ents has still to be investigated. Furthermore, the pharmacology may represent novel therapeutics targeting GPR55. of various CB receptor-selective ligands at GPR55 has not been investigated. This is important because CB ligands are antino- ciceptive, a characteristic that may be shared by GPR55 ligands. The physiological roles of GPR55 and its possible involve- Activation of GPR55 by LPI, but also by certain cannabinoid ment in the pathophysiology of medical conditions are emerg- ligands, initiates multiple signaling pathways distinct from ing. Studies in mice lacking GPR55 have reported a reduction in inflammatory and neuropathic pain (1), and these mice have The abbreviations used are: LPI, L--lysophosphatidylinositol; AM281, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H- increased bone mass (2). Subsequently, studies have established pyrazole-3-carboxamide; AM251, 1-(2,4-dichlorophenyl)-5-(4-iodophe- nyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide; CBD, canna- * This work was supported, in whole or in part, by National Institutes of Health bidiol; CBDA, cannabidiolic acid; CBDV, cannabidivarin; CBG, cannabigerol; Grants DA-03672 and DA-09789 (to R. A. R. and R. G. P.). This work was also CBGA, cannabigerolic acid; CBGV, cannabigerovarin; GPR55, G protein- supported by a Knowledge Transfer Grant Award from the University of coupled receptor 55; SR141716A, 5-(4-chlorophenyl)-1-(2,4-dichloro- Aberdeen. R. G. P. and R. A. R. received funding from GW Pharmaceuticals. phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide;  -THC, □ S 9 9 9 This article contains supplemental Figs. 1 and 2.  -tetrahydrocannabinol;  -THCV,  -tetrahydrocannabivarin; GTPS, To whom correspondence should be addressed. E-mail: [email protected]. guanosine 5-3-O-(thio)triphosphate. JANUARY 2, 2012• VOLUME 287 • NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 91 This is an Open Access article under the CC BY license. Pharmacology of GPR55 Ligands those initiated by cannabinoid CB and CB receptors. GPR55 GPR55), preceded by the signal sequence from the human 1 2 is predominantly coupled to G leading to activation of growth hormone (residues 1–33), and subcloned into pcDNA 12/13 small G proteins (15). It has also been suggested to couple to 3.1 vector. The cells were maintained in Dulbecco’s modified G to promote the activation of phospholipase C and the Eagle’s medium DMEM/F-12 supplemented with 10% newborn increase of intracellular calcium release from inositol triphos- calf serum, 0.5 mg/ml G-418, 60 units of penicillin, 60 gof phate receptor-gated stores (6, 9). LPI-induced activation of streptomycin, and 2 mML-glutamine at 37 °C and 5% CO . GPR55 has been shown to lead to the recruitment of multiple Transfected cells and untransfected cells were plated on the nuclear transcription factors. Among these factors, the most same plates for comparison. investigated is ERK1/2 (5–9, 11, 15–17). Other nuclear tran- ERK1/2 MAPK Phosphorylation Assay—For experimental scription factors recruited by GPR55 activation are nuclear fac- studies of ERK1/2 MAPK phosphorylation, cells (40,000 cells/ tor of activated T cells (NFAT) (9, 13), cAMP-response ele- well) were plated onto 96-well plates and serum-starved for ment-binding protein (CREB), nuclear factor -light chain 48 h. hGPR55-HEK293 cells were serum-starved in DMEM/ enhancer of activated T cells (NF-B) (4, 9, 13), p38 MAPK (18), F-12 medium supplemented with G-418 and 2 mML-glutamine. and Akt serine/threonine protein kinase (8, 11). HEK293 cells were serum-starved in DMEM/F-12 medium Here, we report on the GPR55 pharmacology of arylpyrazole supplemented with 2 mML-glutamine. Cells were assayed in analogues, CB agonists, and a number of C. sativa constituents DMEM/F-12 medium containing L-glutamine and incubated (for structures see Table 3). We show that arylpyrazole ana- for 20 or 60 min at 37 °C in a humidified atmosphere. Drugs logues act both as agonists alone and as inhibitors of LPI signal- were dissolved in DMSO, and stocks at a concentration of 10 ing. The compounds significantly decrease the E value for max mM were kept at 20 °C. LPI was stored at 80 °C for up to 3 the GPR55 endogenous agonist, LPI, which is characteristic months. Drugs were tested in the absence of LPI at a final con- of a noncompetitive mode of action; this may suggest allos- centration of 0.1% DMSO or in the presence of LPI at a final tery. These results provide a possible explanation for the concentration of 0.2% DMSO, unless stated otherwise. At the controversy surrounding the pharmacology of certain end of the assay, the medium was removed, and cells were lysed ligands at GPR55, which have been reported, by different with lysis buffer supplied in the AlphaScreen SureFire ERK groups, to behave as both agonists and antagonists. Here, we kit. demonstrate for the first time that a single ligand can display AlphaScreen SureFire ERK Assay—The assay was per- both behaviors in the same assay. Furthermore, we show that formed in 384-well white Proxiplates according to the manu- certain CB receptor-selective agonists also act as antago- nists of GPR55; this may have implications for the mecha- facturer’s instructions. Briefly, 4 l samples were incubated nism of action underlying the reported antinociceptive with 7 l of mixture containing the following: 1 part donor actions of these compounds (19). beads, 1 part acceptor beads, 10 parts activation buffer, 60 parts reaction buffer. Plates were incubated at room temperature and EXPERIMENTAL PROCEDURES read with the Envision system (PerkinElmer Life Sciences) 9 9 Materials—Cannabis constituents -THC,  -THCV, using AlphaScreen settings. ()CBD, CBDA, CBDV, CBG, CBGA, and CBGV were sup- Analysis—Raw data were presented as “Envision units.” Basal plied by GW Pharmaceuticals, and SR141716A was from level was defined as zero. Results were presented as means and Sanofi-Aventis (Montpellier, France). ()CBD, AM251, variability as S.E. or 95% confidence limits of the percent stim- AM281, CP55940, WIN55212-2, HU-308, GW405833, and ulation of phosphorylated ERK1/2 above the basal level (in the JWH-133 were from Tocris Cookson (Avonmouth, UK). PLX- presence of vehicle). Data were analyzed using nonlinear anal- 4720 was from Selleck, Houston, TX. ABD824 was synthesized ysis of log agonist versus response curve using GraphPad Prism using similar methodology to that described previously for 5.0 (GraphPad, San Diego). The results of this analysis were SR141716A (Iain R. Greig, University of Aberdeen). (E)-- presented as E  S.E. and pEC  S.E. (logEC )orEC max 50 50 50 Caryophyllene was gifted by Prof. Gertsch (Institute of Bio- 95% confidence limits (CL; where appropriate). In Fig. 6A, for chemistry and Molecular Medicine, University of Bern, Swit- each kinase inhibitor, the values for percent stimulation of zerland). PD98059 and AM1241 were from Cayman. G-418 was phosphorylated ERK1/2 were normalized to the mean value from PAA (United Kingdom), DMEM/F-12, DMEM, newborn produced by 10 M LPI (in the presence of vehicle) in matched calf serum, and penicillin/streptomycin solution were obtained experiments. Curves of LPI-induced response were not differ- from Fisher. L-Glutamine, LPI, LY294002, and Y27632 and all ent between experiments; therefore, the data were pooled. Data the other chemicals were obtained from Sigma-Aldrich (Dor- were presented as “pERK” stimulation as percent of LPI. The set, U.K.). AlphaScreen SureFire phospho-ERK kit (catalogue statistical significance of E  S.E. or logEC  pEC was TGRES10K) was from PerkinElmer Life Sciences. max 50 50 determined with an unpaired Student’s t test (95% confidence Cell Culture—Untransfected HEK293 cells were maintained interval). When curves could not be fitted on a nonlinear anal- in DMEM containing 2 mML-glutamine medium supple- ysis of log agonist versus response, the statistical significance of mented with 10% fetal bovine serum. The preparation of the stimulation was determined with an unpaired Student’s t HEK293 stably expressing the tagged human GPR55 receptor test at each specific concentration. Results were considered sig- (hGPR55-HEK293) has been published previously by Hen- nificant only when the F-test comparing the variance was not stridge et al. (4). Briefly, the GPR55 receptor was tagged with a triple hemagglutinin (HA epitope) at the N terminus (3HA- significantly different. 92 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 1 •JANUARY 2, 2012 Pharmacology of GPR55 Ligands FIGURE 1. Detection of LPI-induced ERK1/2 phosphorylation in hGPR55-HEK293 cells using the AlphaScreen SureFire ERK assay. A, mean log concentration-response curves for the effect of LPI on ERK1/2 phosphorylation in hGPR55-HEK293 cells after 20 min (n  4 each in triplicate) or 60 min (n  3 each 2–3 repeats) of incubation at 37 °C. Increasing the incubation time significantly (p  0.05) reduced the potency of LPI-induced ERK1/2 phosphorylation but did not affect LPI-induced maximal stimulation (E ) of the GPR55 receptor. B, compared with a 20-min incubation time at 37 °C, a 60-min incubation max significantly (p  0.05) reduced the basal level of ERK1/2 phosphorylation (n  4, each in 12 repeats). Raw data of AlphaScreen SureFire ERK assay are presented as Envision units, Student’s t test, ***, p 0.05. C, no significant differences were observed in basal levels of phosphorylated ERK1/2 in untransfected HEK293 and hGPR55-HEK293 incubated for 20 min in 0.1 or 0.5% DMSO. C, effect of DMSO on LPI-induced ERK1/2 phosphorylation was assessed at 0.1 or 0.5% DMSO in either hGPR55-HEK293 cells or untransfected HEK293 cells. In untransfected HEK293 cells, no stimulation of ERK1/2 phosphorylation was detected at any concentration of LPI at any concentration of DMSO (untransfected and hGPR55-HEK293 cells were seeded on the same plate). Each symbol represents the mean percentage change in bound phosphorylated ERK1/2 protein. RESULTS centration of DMSO (Fig. 1C). These results are similar to the results published by other groups who reported the Studying the Pharmacology of GPR55 Using AlphaScreen potency of LPI in ERK1/2 phosphorylation assays (5, 13) or Surefire pERK1/2 Assay—The phosphorylation of ERK1/2 other readouts (6). protein has been reported as one of the main signaling path- LPI Induces Sustained Activation of ERK1/2 Phosphorylation ways initiated upon stimulation of the GPR55 receptor; there- in hGPR55-HEK293 Cells—Sustained activation of ERK1/2 fore, we focused our research on phosphorylated ERK1/2 pro- phosphorylation has been implicated as a measure for cancer tein and established a high throughput system using the progression, increase in cell metastasis, and invasiveness of AlphaScreen SureFire phospho-ERK assay. We validated the tumor cells (20, 21). Importantly, GPR55-induced ERK1/2 assay by time (data not shown) and vehicle responses and using phosphorylation regulates human cancer cell migration in vitro untransfected HEK293 cells (Fig. 1 and Table 1). and proliferation in vivo (3, 8, 11). It also appears to govern the LPI (0.1% DMSO) produced a maximal stimulation of maintenance of persistent inflammatory pain (22, 23). There- 129.0  9.65% (E ) (Fig. 1A), which was not significantly max different from the maximal stimulation with 0.2% DMSO fore, we investigated the ability of LPI to maintain ERK1/2 (n  15 each in triplicate; Table 1; e.g. Fig. 2A). The potency phosphorylation after 20 and 60 min of incubation (Fig. 1A). of LPI was not different either (Table 1). In untransfected The potency of LPI after 20 min of incubation, with an EC of HEK293 cells, no stimulation of phosphorylated ERK1/2 was 0.27 M (0.10–0.76), was significantly (p  0.05) reduced to detected at any concentration of LPI nor in any given con- 4.61 M (0.75–28.3) after 60 min. Prolonged incubation JANUARY 2, 2012• VOLUME 287 • NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 93 Pharmacology of GPR55 Ligands reduced both basal and LPI-induced stimulation (Fig. 1B and Structural Analogues of SR141716A Inhibit LPI-induced Table 1) but did not affect the net effect of LPI-induced Activation of ERK1/2 Phosphorylation, Implications for Allos- maximal stimulation of ERK1/2 phosphorylation (Fig. 1A), teric Inhibition—There is still a major controversy surrounding showing that percent stimulation was sustained. This is in the profile of the arylpyrazoles at GPR55. In some studies, contrast, for example, to CBD (10 M) that significantly SR141716A is reported to be an inhibitor (6), and in other stud- increased the percent stimulation after 60 min (supplemen- ies, the compound is reported to be an agonist of GPR55 (5, 13, tal Fig. 2). 14). This is of major importance as SR141716A (Rimonabant) was widely prescribed to patients as an anti-obesity agent and was withdrawn because of mental health issues. We compared TABLE 1 Cannabinoid-mediated stimulation of ERK1/2 phosphorylation in the ability of AM251, AM281, SR141716A, and ABD824, an hGPR55-HEK293 cells AM251 analog in which the iodine was substituted with bro- Cells were treated with each drug for 20 min or for the indicated time at 37 °C. CL mine (Fig. 2, Table 1, and see Table 2 for structures), to induce means confidence limits, and NA means not applicable. ERK1/2 phosphorylation in hGPR55-HEK293 cells with that of Compound EC (95% CL) % E (S.E.) 50 max LPI. The efficacy of AM251 was not significantly different from LPI (0.1% DMSO) 20 min 0.27 (0.10–0.76) 129.0  9.65 that of LPI, but the compound was significantly less potent (Fig. LPI (0.2% DMSO) 20 min 1.12 (0.59–2.16) 126.0  9.44 a 2A and Table 1). It was not possible to obtain an accurate mea- LPI (0.1% DMSO) 60 min 4.61 (0.75–28.26) 139.8  45.70 AM251 2.34 (0.99–5.56) 153.2  22.10 surement of the E for SR141716A and AM281; however, the max ABD824 0.7 (0.11–4.44) ()34.16  22.4 (up to 1 M) compounds had efficacy that was not lower than that of LPI AM281 NA 106 (10 M) SR141716A NA 160 (10 M) (Fig. 2, A and B, and Table 1). Interestingly, ABD824 had a CP55940 NA 50 (10 M) bi-phasic response in hGPR55-HEK293 cells (Fig. 2C). JWH-133 0.16 (()30.98 to ()29.8  6.40 (up to 3 M) ()4.41) Arylpyrazole-induced stimulation appears to be GPR55-me- GW405833 1.87 (0.33 to 10.7) 54.0  13.04 diated as these ligands do not stimulate ERK1/2 phosphoryla- HU-308 NA 20 (10 M) AM1241 NA 15 (10 M) tion in non-expressing HEK293 cells (4), and CP55940 alone BCP NA 22 (10 M) did not induce a significant stimulation of ERK1/2 phosphor- p  0.05 versus LPI in 0.1% DMSO for 20 min (first raw). p  0.01 versus LPI in 0.1% DMSO for 20 min (first raw). ylation at any concentration (Fig. 2B). FIGURE 2. Structural analogues of SR141716A-induced ERK1/2 phosphorylation in hGPR55-HEK293 cells. A, mean log concentration-response curves for percent stimulation of ERK1/2 phosphorylation by LPI (n  4), AM251 (n  3), or AM281 (n  3) after a 20 min stimulation at 37 °C. B, mean log concentration- response curves of LPI (n 4), SR141716A (n 3), or CP55940 (n 3), a nonselective cannabinoid CB /CB agonist. C, mean log concentration-response curves 1 2 of LPI or ABD824, an AM251 analog in which iodine was substituted with a bromine (n  3, each in duplicate). The effect of SR141716A analogues on LPI induced ERK1/2 phosphorylation in hGPR55-HEK293 cells. D, LPI (n  4) in the presence or absence of 100 nM AM251 (n  3) or 1 M AM251 (n  4). E, effect of LPI in the presence or absence of 1 M SR141716A (n  4) or 1 M AM281 (n  3). F, effect of LPI in the presence or absence of 1 M and 3 M ABD824 (n 4, each in duplicate). Each symbol represents the mean percentage change in bound phosphorylated ERK1/2 S.E. over the basal level, and each independent experiment was performed in triplicate unless stated otherwise. 94 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 1 •JANUARY 2, 2012 Pharmacology of GPR55 Ligands TABLE 2 Effect of arylpyrazoles on LPI-mediated stimulation of ERK1/2 phosphorylation in hGPR55-HEK293 cells Cells were co-treated with LPI in the presence or absence of a given drug for 20 min at 37 °C. Final concentration of DMSO was 0.2%. Arylpyrazole LPI ± drug EC (µM) pEC ± S.E. % E ± S.E. % Inhibition 50 50 max Me Vehicle 1.15 5.94 ± 0.22 147.2 ± 17.76 AM251 100 nM 0.53 6.28 ± 0.07 108.6 ± 3.86 26% Cl AM251 1 µM 0.67 6.17 ± 0.29 68.70 ± 9.93 ** 53% Cl Me Vehicle 0.79 6.10 ± 0.20 161.9 ± 15.14 Cl Cl SR141716A 1 µM 0.56 6.25 ±0.29 108.3 ± 14.3* 33% Cl Me Vehicle 0.51 6.29 ± 0.24 107.4 ± 10.78 Br ABD824 1 µM 0.45 6.35 ± 0.37 70.39 ± 10.65 34% Cl ABD824 3 µM 0.42 6.38 ± 0.58 59.7 ± 11.7* 44% Cl O O Me Vehicle 0.89 6.05 ± 0.12 131.6 ± 8.9 Cl AM281 1 µM 0.25 6.59 ± 0.24 81.49 ± 8.71 * 45% Cl *, p  0.05; **, p  0.01 versus LPI in each experiment. % inhibition is percentage inhibition of E relative to LPI (100%). max In some studies, SR141716A was reported to inhibit GPR55 induced -arrestin trafficking and LPI-induced ERK1/2 phos- activity (6), and we therefore assessed if certain arylpyrazoles phorylation (5). For comparison, in this study, LPI-induced could inhibit LPI-induced GPR55 stimulation, in the same stimulation was 144.0  16.7% and CP55940 at 1 M reduced it experimental system. The arylpyrazoles AM251, AM281, by only 13% (124.9 16.0%), which was not significantly differ- SR141716A, and ABD824 reduced the maximal stimulation ent from LPI alone (supplemental Fig. 1C). (E Effect of CB ) by LPI of ERK1/2 phosphorylation but did not signifi- Agonists on LPI-induced Activation of ERK1/2 max 2 cantly alter the potency of this bioactive lipid (results are sum- Phosphorylation, Implications for Neuropathic Pain and Posi- marized in Fig. 2, E and F, and in Table 2). tive Allosteric Modulation—The antinociceptive effect of CB Importantly, these data provide the first demonstration of a receptor agonists has been extensively investigated (reviewed in dual action (both agonist and inhibitor) of these compounds in Ref. 24). Subsequently, CB receptor agonists have been devel- the same assay. The inhibition appears noncompetitive and oped by scientists and pharmaceutical companies as alternative may indicate allostery. Of the tested analogues, AM251 was the treatments aimed at alleviation of neuropathic pain. Interest- most effective as an inhibitor of LPI (Fig. 2D). The E of LPI of ingly, a comparison of the behavioral responses of wild type max M and to mice with GPR55 knock-out mice in pain models revealed the 147.2  17.8% was reduced to 108.6  3.86% by 100 n 68.7  9.93% (p  0.01) by 1 M AM251. involvement of GPR55 in the conduction of neuropathic pain We continued to explore the effects of high concentrations of (1). These results suggest that the analgesic effects of certain AM251 and SR141716A. The maximal stimulation by LPI in the CB receptor-selective ligands may be mediated by GPR55. We presence of high concentrations of AM251 or SR141716A was compared several CB agonists that have been intensively not significantly different from that of LPI only. This would be investigated in the past and thus form a core of active structures expected because the compounds are agonists alone at these for GPR55-mediated ERK1/2 phosphorylation. JWH-133 (up concentrations (supplemental Fig. 1, A and B). to 3 M) significantly reduced basal pERK levels. GW405833 Previous studies reported that CP55940, a nonselective can- behaved as a partial agonist of GPR55 (E 54  13.0% versus max nabinoid receptor agonist, is an inactive ligand at GPR55 that LPI 106.0  10.5%; p  0.05), with a similar potency to that of could not increase intracellular calcium level (6) but behaves as LPI (1.9 M (0.3–10)) (Fig. 3A). In contrast to the arylpyrazoles, a competitive antagonist in the presence of LPI (4); another GW405833 enhanced LPI-induced ERK1/2 phosphorylation at group showed it antagonized LPI, AM251, and SR141716A- a concentration that alone had no effect on pERK (Fig. 3B). JANUARY 2, 2012• VOLUME 287 • NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 95 Pharmacology of GPR55 Ligands FIGURE 3. Effect of CB receptor agonists on ERK1/2 phosphorylation in hGPR55-HEK293 cells. A, mean log concentration-response curves for percent stimulation of ERK1/2 phosphorylation by LPI (n 7), GW405833 (n 4), or JWH-133 (n 3) after 20 min of stimulation at 37 °C. JWH-133 significantly reduced basal pERK levels, *, p  0.05; **, p  0.01, one-sample t test. B, GW405833 at 10, 30, and 100 nM enhanced the LPI-induced ERK1/2 phosphorylation (n  4). C, JWH-133 at 1 and 3 M inhibited the LPI-induced ERK1/2 phosphorylation (n 4). D, percent stimulation of ERK1/2 phosphorylation by LPI (n 3), AM1241 (n 4), or HU-308 (n 4) and (E)--caryophyllene (BCP)(n 3) after 20 min of stimulation at 37 °C. E, (E)--caryophyllene at 1, 3, and 10 M (n 3) did not alter the LPI-induced ERK1/2 phosphorylation nor did AM1241 (F, n 3). Each symbol represents the mean percentage change in bound phosphorylated ERK1/2 S.E. over the basal level, and each independent experiment was performed in duplicate. Conversely, JWH-133 at 1 and 3 M (Fig. 3C), but not at 10 M We then assessed the modulation of LPI-induced stimula- 9 9 (results not shown), inhibited the LPI-induced response. These tion with 1 M  -THC or 1 M  -THCV (produced stimula- results further support the data obtained with the arylpyrazoles, tion of 22.2  11.1% (not significant from zero) and 31.4 indicating that cannabinoids can act as noncompetitive inhibi- 10.0% (p  0.01 versus 0), respectively). Incubation of LPI with 9 9 tors of LPI signaling. Of the tested compounds, (E)--caryo- 1 M of  -THC or  -THCV significantly inhibited LPI-in- phyllene (25), HU-308 (data not shown), and AM1241 had lit- duced stimulation (Fig. 4, B and C, respectively, and Table 3). tle/no effect up to a concentration of 10 M (Fig. 3D) and did Neither ligand affected the potency of LPI (Table 3). These results not significantly alter the LPI-induced effect on GPR55 (Fig. 3, show for the first time that the phytocannabinoids  -THC and 9 9 E and F).  -THCV are inhibitors of LPI. Moreover,  -THCV elicited a Effect of Phytocannabinoids on LPI-induced Activation of downward shift in the log concentration-response curve of LPI, ERK1/2 Phosphorylation, Comparison of  -THC and such that LPI-induced pERK level was lower in the presence of 9 9 9 -THCV—Similarly to arylpyrazoles, the effect of  -THC,  -THCV, and this resembles negative cooperativity. the main psychoactive constituent of C. sativa, on GPR55 is also Structural Analogues of Cannabidiol—We have recently controversial. It has been shown to increase calcium release (6) shown that CBD antagonizes LPI-induced stimulation of but not to mobilize -arrestin (5) in GPR55-expressing cells. In [ S]GTPS binding in breast cancer MDA-MB-231 cells, this study, we compared the structurally related cannabis con- which highly express GPR55 (3). CBD also inhibits the metastasis 9 9 stituents,  -THC and  -THCV (Fig. 4A and Table 3). Both and aggressiveness of brain and breast cancer cells (26, 27) and compounds stimulated ERK1/2 phosphorylation. However, for LPI-induced calcium mobilization in prostate cancer cells (8). neither compound could the stimulation be fitted to a concen- Therefore, we have evaluated two cannabis constituents that are tration-response curve. Compared with the maximal stimula- structurally related to CBD, alone or in combination with LPI. tion of LPI (129.0  9.65%; Fig. 1A), these results suggest that Cannabidiolic acid (CBDA) has an acid group (COOH) on the 9 9 -THC is a low affinity partial agonist, whereas  -THCV is a benzene ring, whereas cannabidivarin (CBDV) has a shorter side low affinity, high efficacy, agonist of GPR55. The levels of chain compared with that of CBD (see Table 3 for structures). ERK1/2 phosphorylation after prolonged incubation (60 min) We compared the effects of CBD from two sources (Tocris 9 9 with  -THC or  -THCV were not significantly different and GW Pharmaceuticals). The effect of CBD on ERK1/2 phos- from those obtained after 20 min. phorylation after 20 min was not significantly different between 96 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 1 •JANUARY 2, 2012 Pharmacology of GPR55 Ligands 9 9 FIGURE 4. Effect of  -THC and  -THCV on ERK1/2 phosphorylation in hGPR55-HEK293 cells. Mean log concentration-response curves for ERK1/2 9 9 phosphorylation after 20 or 60 min of stimulation at 37 °C with -THC (n  3) or  -THCV (n  4) (A). B shows the effect of LPI in the presence or absence of 1 9 9 M -THC after 20 min of stimulation at 37 °C (n 4). C shows the effect of LPI in the presence or absence of 1 M -THCV after 20 min of stimulation at 37 °C (n 3). Each symbol represents the mean percentage change in bound phosphorylated ERK1/2 S.E. over the basal level, and each independent experiment was performed in triplicate. the two sources (supplemental Fig. 2A); However, after a 60 min gest that the previously reported CBD antagonism of LPI-in- incubation, CBD significantly increased ERK1/2 phosphoryla- duced stimulation of ERK1/2 phosphorylation can be further tion (100.5 11.9% and 220.9 33.2% each at 10 M, Tocris or enhanced by shortening its alkyl side chain from pentyl to pro- GW Pharmaceuticals, respectively). Neither CBD nor CBDA pyl, although the additional acid group mainly affects the inhi- affected basal pERK after a 20 min incubation, whereas CBDV bition of ERK1/2 phosphorylation at a low concentration of produced a maximal stimulation of 43.0 23.0% and an EC of LPI. 1.9 M (confidence intervals 0.13–27.9 M)(supplemental Fig. Structural Analogues of Cannabigerol—Following these find- 2, B and C, respectively). ings, we sought to evaluate CBG and two structurally related The effects of CBD, CBDA, and CBDV on LPI-induced cannabis constituents that either contain an acid group on the ERK1/2 phosphorylation are summarized in Table 3 and Fig. 5, benzene ring (CBGA) or a propyl side chain (CBGV). After a 20 A–C. CBD did not significantly alter LPI efficacy or potency at min incubation, CBG did not produce a significant stimulation 1or3 M (Table 3). However, the inhibition of 32% at 3 M (supplemental Fig. 2D). CBGA decreased basal pERK with an showed a trend toward antagonism. In fact, we have found sim- E of ()79.9 27.2% (20 min) and after a 60 min incubation, max ilar results using the [ S]GTPS binding assay with MDA-MB- it was ()31.4  7.34% with an EC of 5.26 M (confidence 231 breast cancer cells (3). In these endogenously expressing intervals 0.99–27.9) and 0.83 M (confidence intervals 0.01– GPR55 receptor cells, LPI stimulates [ S]GTPS binding, 4.79), respectively (supplemental Fig. 2E). whereas 1 M CBD produces a significant downward shift We then assessed the effect of each compound on LPI-in- (6.6 5.35% versus ()17.5 3.69 with CBD) but did not affect duced stimulation of GPR55 (Fig. 5, D and E, and Table 3). The the maximal stimulation (E ) of LPI-induced [ S]GTPS effect of each compound at 1 M was not significantly different max binding (LPI, 50.1  7.5%; 48.3  21.2%). However, these cells versus zero (stimulation of ()7.7% for CBG; ()7.4% for express a spectrum of receptors making them not ideal for fur- CBGA; ()6.2% for CBGV)). At 1 M, CBGA (Fig. 5E) and ther characterization of the pharmacology of GPR55. Taken CBGV (Fig. 5F) but not CBG (Fig. 5D) significantly reduced the together, these results support the hypothesis that CBD is an maximal stimulatory effect of LPI on ERK1/2 phosphorylation inhibitor of GPR55. (p  0.05), suggesting that these compounds are also inhibitors Of the tested analogues, CBDV (9.99% stimulation at 1 M; of LPI-induced activation of the GPR55 receptor. We observed not significant versus zero) significantly reduced the maximal a similar structure-activity relationship to that found for CBD stimulation of LPI by 56% (p  0.05) (Fig. 5C and Table 3). analogues, and thus the rank order of LPI inhibition was CBGV Increasing the CBDV concentration to 3 M did not inhibit or (69%) CBGA (56%) CBG (16%). Furthermore, CBGV dis- increase LPI stimulation (data not shown). CBDA did not sig- played negative cooperativity with LPI, whereby basal pERK nificantly affect the efficacy or potency of LPI but produced a was significantly lower in the presence of both compounds. significant downward shift of its response (11.15 10.1% versus GPR55 Signaling Involves Cross-talk between MAPK and Rho ()24.1  5.81% with CBDA; p  0.05) (Fig. 5B and Table 3); GTPases Signaling Pathways—We then tested if LPI-induced this may be indicative of negative cooperativity. In summary, stimulation of ERK1/2 phosphorylation can be inhibited by the the three compounds structurally related to CBD significantly MEK1/2 noncompetitive inhibitor PD98059. LPI (10 nM to 10 inhibit LPI-induced ERK1/2 phosphorylation with a rank order M) did not stimulate the phosphorylation of ERK1/2 proteins of CBDV (56%) CBDA (16%) CBD (9%). Furthermore, both in the presence of PD98059, which significantly inhibited basal CBDA and CBDV displayed negative cooperativity with LPI. pERK alone (Fig. 6, A and B). These findings were in line with Basal pERK was significantly lower when either of these com- the receptor-independent mechanism of action of PD98059 by pounds was co-incubated with LPI. These results underlie a binding the inactive form of MEK1 and inhibiting its activation new structure-activity relationship of GPR55 ligands and sug- by upstream activators such as Raf kinases. However, this could JANUARY 2, 2012• VOLUME 287 • NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 97 Pharmacology of GPR55 Ligands TABLE 3 Effect of phytocannabinoids and CB receptor compounds on LPI-mediated stimulation of ERK1/2 phosphorylation in hGPR55-HEK293 cells Cells were co-treated with LPI in the presence or absence of a given drug at 1 M for 20 min at 37 °C. Final concentration of DMSO was 0.2%. not explain previous observations showing that GPR55 is pre- Y27632 significantly inhibited the response to LPI under these dominantly coupled to G . To test our hypothesis for conditions. 12/13 cross-talk between MAPK and Rho GTPases signaling path- Enhancement of GPR55 Signaling by PLX-4720 Oncogene ways, we co-incubated LPI with Y27632, a p160ROCK (Rho- B-Raf Inhibitor—B-Raf is a member of Raf kinase family and associated kinase) inhibitor. After 20 min, 10 M Y27632 signif- plays a major role in regulating ERK1/2 phosphorylation. icantly inhibited LPI-induced stimulation of ERK1/2 Importantly, inherited and acquired mutations, such as V600E, phosphorylation (Fig. 6A; p  0.01), without inducing a signif- in B-Raf are associated with various diseases of which most icant change in LPI potency. Although 10 M Y27632 or 10 M are cancers (28). Therefore, B-Raf inhibitors have been LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), developed as anti-cancer therapeutics. PLX-4720, a potent could equally stimulate ERK1/2 phosphorylation (Fig. 6B), only and selective B-Raf inhibitor (29), induced a concentration- 98 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 1 •JANUARY 2, 2012 Pharmacology of GPR55 Ligands TABLE 3—continued *, p  0.05 versus LPI in each experiment. % inhibition is percentage inhibition of E relative to LPI (100%). **, p  0.01 versus LPI in each experiment. max FIGURE 5. Effect of CBD and CBDA and CBDV on ERK1/2 phosphorylation in hGPR55-HEK293 cells. Mean log concentration-response curves for ERK1/2 phosphorylation after 20 min of stimulation at 37 °C. The effect of LPI in the presence or absence is shown as follows: 1 M CBD (n  5) (A); 1 M CBDA (n  3) (B); 1 M CBDV (n  3) (C); 1 M CBG (n  4) (D); 1 M CBGA (n  4) (E); 1 M CBGV (n  3) (F). CBDV and CGV pronouncedly inhibited LPI compared with their analogues. Each symbol represents the mean percentage change in bound phosphorylated ERK1/2  S.E. over the basal level (n 3). CBDA, CBDV, and CBGV significantly reduced basal pERK levels, *, p  0.05; **, p  0.01; ***, p  0.001 one-sample t test. dependent stimulation of ERK1/2 phosphorylation at a mag- gests an additive relationship between intracellular inhibi- nitude similar to that induced by LPI (Fig. 6C), opposing the tion of B-Raf and extracellular stimulation of LPI-induced of inhibitory effects of the MEK signaling pathway in ERK1/2 phosphorylation, supporting a cross-talk between BRAF(V600E)-expressing cells. In the presence of LPI, MAPK and Rho GTPases signaling pathways. increasing concentrations of PLX-4720 (100, 300, 1000 nM) DISCUSSION significantly increased the E of LPI (119.2  10.53%) max reaching 220.6  13.97% at 1000 nM (p  0.01, one-way The lack of appropriate radiolabeled ligands for GPR55 pre- analysis of variance) but did not alter its potency; this sug- cludes the characterization of the binding of novel small mole- JANUARY 2, 2012• VOLUME 287 • NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 99 Pharmacology of GPR55 Ligands FIGURE 6. Effect of kinase inhibitors. A, LPI-induced stimulation of ERK1/2 phosphorylation was attenuated by 10M PD98059, a MEK1 inhibitor, and inhibited by 10 M Y27632, a Rho-associated protein kinase inhibitor (n 5 each in duplicate), but not by 10 M LY294002, a phosphatidylinositol 3-kinase inhibitor (n 3 each in duplicate). B, effect of each inhibitor alone on phosphorylated ERK1/2 level (n  4 for PD98059; n  7 for Y27632; and n  3 for LY294002, each in duplicate). PD98059 significantly reduced basal pERK levels; *, p  0.05; **, p  0.01, one-sample t test. C, diagram representing the putative GPR55/LPI signaling cascade. D, selective B-Raf inhibitor, PLX-4720, stimulated hGPR55-HEK293-expressing cells in a similar manner to LPI (n  3 each in duplicate). E, increasing concentrations of PLX-4720 significantly increased the efficacy of LPI-induced response (100 nM, p 0.05; 300 nM, p 0.01; 1000 nM, p 0.01) and the bottom of the curve (300 nM, p  0.05), (n  3 each in duplicate). cules to this receptor. In this study, we established a new rapid phosphorylation is also associated with a variety of human and sensitive AlphaScreen SureFire assay as a strategy for pathologies (38). In addition, many mutations of components studying the pharmacology of GPR55. This method has been upstream to ERK1/2 alter this signaling pathway and have been used to explore ligands of the cytokine receptors (30), LDL associated with increased cancer metastasis and invasiveness endothelial receptor (31), and potassium channel TREK-1 (20, 38). The combination of strength and duration of ERK1/2 receptors (32) and to develop antagonists for other intracellular signaling determines the distinct outcomes, ranging from sus- targets (33–35). tained high activation that can lead to apoptosis or differentia- LPI and Certain Cannabinoids Induce Sustained GPR55-me- tion to sustained lower levels of activation that are correlated diated ERK1/2 Phosphorylation—Studies have shown that with cell proliferation (39). In our case, it appears that LPI- GPR55 induces maximal ERK1/2 phosphorylation response induced activation parallels the latter scenario, supporting the after 10–20 min (this study and see Refs. 4, 5, 7); here we show role of GPR55 in cancers. Therefore, it will be interesting to that although both basal and LPI-induced stimulation levels are determine whether this mechanism involves the endogenous reduced at 60 min (Fig. 1 compared with 20 min) the percent mitogen-activated protein kinase phosphatase 1 (MKP-1) that stimulation is sustained, but the potency of LPI is significantly controls the constitutive activation of ERK1/2 (21). decreased after 60 min. GPR55 Pharmacology of Arylpyrazoles—A key finding of this Sustained ERK1/2 activation in injured neurons has been study is the demonstration that arylpyrazoles, e.g. SR141716A, suggested to reflect alterations in the intracellular feedback reg- can act both as agonists of GPR55 and inhibitors of LPI-induced ulators that normally function to terminate signaling responses activation of pERK. Thus, we demonstrate here for the first (36) and has been associated with brain ischemia (37). ERK1/2 time that both types of behavior (agonism and inhibition) as 100 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 1 •JANUARY 2, 2012 Pharmacology of GPR55 Ligands FIGURE 7. Pharmacological mechanisms for the modulation of GPR55. LPI is suggested to primarily bind the GPR55 orthosteric binding site. GPR55 may also contain an allosteric binding site. These observations raise at least two possibilities as follows. Left, one possibility is that certain arylpyrazole ligands actually represent bitopic ligands of GPR55. These ligands may have the capacity to modulate both the orthosteric (agonists) and the allosteric site through different pharmacophores. The second possibility is that AM251 and certain arylpyrazole analogues are only allosteric ligands that are ago-allosteric alone. Right,inthe presence of LPI, arylpyrazoles can also behave as allosteric inhibitors and GW405833 as an allosteric enhancer. In addition, a number of Cannabis sativa constituents appear to inhibit ERK1/2 phosphorylation in an allosteric manner. reported by previous studies from different laboratories can distinct binding sites at GPR55. Nevertheless, this study pro- exist in the same model system. Our data suggest that there may vides the first potential explanation for the considerable con- be two topographically distinct binding sites on GPR55 (Fig. 7). troversy in the pharmacology of cannabinoid ligands at GPR55 In the first scenario, certain ligands (e.g. AM251 and and has important therapeutic implications. GW405833) could act as agonists alone (either via the The arylpyrazoles belong to a subgroup of ligands that inter- orthosteric (LPI) binding site or a distinct (allosteric) binding act with both GPR55 and CB receptors. In this study, arylpyra- site (40). In the second scenario, the same ligands could act as zole analogues induced activation of pERK with a rank order of modulators of LPI-induced pERK activation. This suggests that potency LPI AM251 SR141716A AM281. This rank the compounds may bind to an allosteric site to induce a con- order of potency is in agreement with the study by Henstridge et formational change in the orthosteric (LPI) binding site such al. (4) using the same hGPR55-expressing HEK293 cells and is that the efficacy of LPI is reduced (e.g. AM251) or enhanced (e.g. similar to the study by Kapur et al. (5) that used hGPR55-ex- GW405833). These observations raise the possibility that cer- pressing U2OS cells and a -arrestin mobilization assay. tain arylpyrazole ligands may represent bitopic ligands of Although in the latter study arylpyrazoles could not induce the GPR55. Bitopic ligands have the capacity to interact with both phosphorylation of ERK1/2, the difference between this and the orthosteric site (as agonists) and the allosteric site (as mod- our study may be due to differences in method sensitivity ulators) through distinct chemical structures (41). (Western blot versus AlphaScreen) or may reflect cell type Allosteric binding sites have been described for other lipid differences. receptors, including allosteric modulators of anandamide at the Collectively, these results suggest that AM251, SR141716A, cannabinoid CB receptor (42) and ago-allosteric ligands for and AM281 are weak agonists of GPR55 as measured by their ability to induce ERK1/2 activation. Interestingly, ABD824, an short chain free fatty acids at FFA2 and FFA3 receptors (43). This study demonstrates the modulation of GPR55 by certain AM251 analog, decreased basal pERK at concentrations below ligands, possibly by an allosteric mechanism. The first evidence 3 M. This suggests a role for the chemical group on the aryl is the reduction in E of LPI observed in the presence of ring in shifting the GPR55 receptor between inactive and active max receptor conformations. As for the efficacy for the observed various compounds. However, it is important to note that this study does not provide direct evidence of allostery (e.g. dissoci- antagonism of LPI by arylpyrazoles, the rank order is AM251 ation kinetics), and there is a possibility that the compounds AM281 ABD824  SR141716A, which does not correspond may inhibit LPI signaling downstream of the receptor to disrupt to the relative potencies of these ligands as GPR55 agonists (as the pERK signaling cascade. The second evidence is the appar- detailed above). This suggests that the pharmacophore for inhi- ent negative cooperativity between various ligands and LPI bition of GPR55 by these pyrazoles may be different from that such that LPI-induced pERK level is significantly decreased in responsible for activation of GPR55. the presence of both ligands, an effect not observed with either SR141716A was developed as a cannabinoid CB receptor ligand alone. antagonist and progressed to clinical studies as a drug treat- Clearly, this evidence is circumstantial, and dissociation ment for obesity-related disorders (44). Interestingly, kinetic analysis (which is not currently feasible because of the SR141716A has been shown to reduce neuropathy associated lack of appropriate radioligand) or extensive mutation studies, with type-2 diabetes in patients (45). This has also been sup- will be required to confirm the existence of topographically ported by in vivo studies in which SR141716A reduced neuro- JANUARY 2, 2012• VOLUME 287 • NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 101 Pharmacology of GPR55 Ligands pathic pain in a murine model for obesity (46) and in a rodent  -THC have so far been considered to be mediated by activa- model for nerve injury (47). This is unexpected for an inverse tion of cannabinoid receptors. Recently, GPR55 has been linked agonist of CB because CB receptor agonists are effective in to inhibition of the migration and proliferation of cancer cells 1 1 neuropathic pain. Collectively, these studies suggest that by CBD (3, 8). The results warrant further investigation into the SR141716A may act on a target other than the CB receptor to potential therapeutic use of various phytocannabinoid. CBDA inhibit pain. As GPR55 knock-out mice are less sensitive to the and CBG have been shown to activate TRPA1- and TRPV1- development of chronic pain (1), our findings that LPI-induced expressing cells and to antagonize TRPM8, implying a role in activation of GPR55 is inhibited by SR141716A suggests a novel analgesia and prostate cancer for these compounds (58, 59). mechanism of action for the regulation of pain and possibly for Our study suggests that GPR55 is an additional target for these metabolic syndrome by this compound. This effect may be compounds; inhibition of GPR55 would therefore further sup- mediated by a putative allosteric binding site on GPR55. port their potential role in the treatment of carcinoma and pain GPR55 Pharmacology of CB Agonists—This study has iden- (51). tified GW405833 as a GPR55 partial agonist. JWH-133 signifi- GPR55-mediated MAPK Signaling—Importantly, our find- cantly reduced basal pERK and as such may be an inverse ago- ings demonstrate that LPI-induced ERK1/2 phosphorylation is nist of GPR55. In line with our results, JWH-133 has been controlled, at least in part, by ROCK, supporting the role for previously studied for its analgesic effects and has been shown G in LPI signaling and indicating cross-talk between 12/13 to produce antinociception via nicotinic-dependent pathways MAPK and Rho GTPases signaling, which is in line with (48). However, its effect could not always be reversed Andradas et al. (11). Such cross-talk may be communicated via by SR144528, a CB receptor antagonist (49). Similarly, the serine/threonine kinase Raf family, of which the expression GW405833 was found to promote a pro-antinociceptive level of the oncogene B-Raf in turn controls the stimulation of response in a rat model of osteoarthritis in contrast to its behav- ROCK (60). In line with Hatzivassiliou et al. (16), the selective ior as a CB receptor partial agonist (50). These studies suggest B-Raf inhibitor PLX-4720 unregulated ERK1/2 phosphoryla- that JWH-133 and GW405833 have an additional target(s), and tion in hGPR55-HEK293 cells that do not carry the our results suggest that GPR55 signaling may be one of their BRAF(V600E) mutation; interestingly, the magnitude of the targets. response closely resembled that of LPI. Furthermore, ERK1/2 Phytocannabinoids That Inhibit LPI-mediated Activation of phosphorylation was unregulated following the co-incubation GPR55—The pharmacological actions of several C. sativa con- of PLX-4270 with LPI in an additive manner, supporting a stituents have been recently reviewed; both the psychoactive cross-talk between B-Raf and ROCK signaling pathways in LPI- and nonpsychoactive compounds have multiple targets and mediated GPR55 signaling. Further support for this interaction convey a range of pharmacological actions (51). In this study, comes from B-Raf knock-out mice in which ROCKII expression we have evaluated a range of C. sativa constituents for their is reduced (61). ability to modulate GPR55. Although  -THC activates CB Conclusions—We have established a rapid and sensitive and CB receptors and GPR55 and behaves as a partial agonist method to study the pharmacology of the GPCR GPR55 using at these receptors,  -THCV is a CB antagonist and a CB the AlphaScreen SureFire ERK assay. It is important to note 1 2 partial agonist. In this study, we found that  -THCV is a weak that this study exclusively measures ERK1/2 phosphorylation agonist of GPR55 that can significantly inhibit LPI-induced as a readout of GPR55. This readout may not allow the distinc- stimulation of pERK in GPR55-expressing cells. Thus, the anal- tion between various signaling pathways; thus, this work might gesic actions of -THCV in vivo (52) might, at least in part, be be limited by the fact that the signaling cascade leading to mediated by modulation of GPR55. Expression of GPR55 ERK1/2 activation may vary with the compound used. That mRNA has been detected in adipose tissue (15), and a polymor- said, to our knowledge this study provides the first evidence phism in the GPR55 gene has been found in females with ano- that certain cannabinoids can display both activation of GPR55 rexia nervosa (53). Although the precise role of GPR55 in food and inhibition of LPI-mediated pERK stimulation in the same intake and metabolism remains to be elucidated, it is possible assay. Further studies should lead to a resolution of the contro- that the inhibition of food intake reported for  -THCV (54) versy regarding the pharmacology of cannabinoids at GPR55. and other cannabinoids may be mediated, at least in part, by Furthermore, we show that certain ligands previously thought modulation of GPR55. to be selective for the CB receptor also modulate GPR55. Our Here, we report that the little investigated cannabis constit- findings also suggest that GPR55 may be a new pharmacologi- uents CBDV, CBGA, and CBGV are potent inhibitors of LPI- cal target for the following C. sativa constituents:  -THCV, induced GPR55 signaling. CBD is the most investigated non- CBDV, CBGA, and CBGV. Combined mutagenesis and phar- psychotropic constituent of cannabis and has a wide range of macological investigations will enable us to determine the pharmacological targets (51); together with a safe profile in pharmacophores responsible for cannabinoid binding to humans, this made it highly attractive for the development of GPR55 to facilitate rational drug design (62). This study has drugs for neuropathic pain associated with multiple sclerosis implications for developing new therapeutics for the treatment (55) and cancer (56). Several studies have also shown that CBD of cancer, pain, and metabolic disorders. is effective against proliferation, migration, and invasion of a variety of breast cancer, glioblastoma, prostate cancer, and REFERENCES human cervical cancer cell lines (3, 8, 26, 27, 57). In cancer cells, 1. Staton, P. C., Hatcher, J. P., Walker, D. J., Morrison, A. D., Shapland, E. M., the target for CBD remains elusive, whereas the actions of Hughes, J. 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Modulation of l-α-Lysophosphatidylinositol/GPR55 Mitogen-activated Protein Kinase (MAPK) Signaling by Cannabinoids *

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American Society for Biochemistry and Molecular Biology
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Copyright © 2012 Elsevier Inc.
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0021-9258
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DOI
10.1074/jbc.m111.296020
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 1, pp. 91–104, January 2, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Modulation of L--Lysophosphatidylinositol/GPR55 Mitogen-activated Protein Kinase (MAPK) Signaling by Cannabinoids Received for publication, August 23, 2011, and in revised form, October 12, 2011 Published, JBC Papers in Press, October 25, 2011, DOI 10.1074/jbc.M111.296020 ‡§1 ‡ ¶  ‡ ‡ Sharon Anavi-Goffer , Gemma Baillie , Andrew J. Irving ,Ju¨ rg Gertsch , Iain R. Greig , Roger G. Pertwee , and Ruth A. Ross From the School of Medical Sciences, Institute of Medical Sciences, Kosterlitz Centre for Therapeutics, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, United Kingdom, the Division of Medical Sciences, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom, the Institute of Biochemistry and Molecular Medicine, University of Bern, Bu¨hlstrasse 28, Bern CH-3012, Switzerland, and the Departments of Behavioral Sciences and Molecular Biology, Ariel University Center of Samaria, Ariel 40700, Israel Background: The endogenous L--lysophosphatidylinositol activates GPR55. Results: Structural analogues of SR141716A act both as agonists alone and as inhibitors of L--lysophosphatidylinositol. Certain CB receptor agonists also modulate GPR55 activity. Conclusion: Certain cannabinoids can both activate GPR55 and attenuate L--lysophosphatidylinositol-mediated phosphor- ylated ERK1/2 activation. This has mechanistic implications for the antinociceptive effects of certain CB agonists. Significance: Cannabinoid ligands have complex interactions with the L--lysophosphatidylinositol/GPR55 signaling system. GPR55 is activated by L--lysophosphatidylinositol (LPI) but that the endogenous lysophospholipid LPI activates GPR55 also by certain cannabinoids. In this study, we investigated the (3–10). In certain cancer cell lines, GPR55 is highly expressed, GPR55 pharmacology of various cannabinoids, including ana- and LPI mediates increased cell migration, invasion, and prolif- logues of the CB receptor antagonist Rimonabant,CB recep- eration (3, 8, 11). Moreover, increased circulating levels of LPI 1 2 tor agonists, and Cannabis sativa constituents. To test ERK1/2 have been found in cancer patients and are associated with a phosphorylation, a primary downstream signaling pathway that poor prognosis (12). These observations suggest that modula- conveys LPI-induced activation of GPR55, a high throughput tion of GPR55 may have therapeutic implications for the treat- system, was established using the AlphaScreen SureFire ment of pain, bone diseases, and cancer. assay. Here, we show that CB receptor antagonists can act both To date, certain arylpyrazole CB receptor antagonists such as agonists alone and as inhibitors of LPI signaling under the as Rimonabant (also known as SR141716A or Acomplia) and same assay conditions. This study clarifies the controversy sur- AM251 have been reported to be GPR55 agonists (5, 13, 14). rounding the GPR55-mediated actions of SR141716A; some However, other groups have suggested that Rimonabant is a reports indicate the compound to be an agonist and some report GPR55 antagonist (6, 8). Furthermore, reports on the behavior antagonism. In contrast, we report that the CB ligand of  -THC at GPR55 are also inconsistent. Lauckner et al. (6) GW405833 behaves as a partial agonist of GPR55 alone and have shown that  -THC is a GPR55 agonist capable of stimu- enhances LPI signaling. GPR55 has been implicated in pain lating calcium release, and Kapur et al. (5) did not detect -ar- transmission, and thus our results suggest that this receptor may restin-mediated activation of GPR55 with this phytocannabi- be responsible for some of the antinociceptive actions of certain noid. Another cannabis constituent, cannabidiol (CBD), is CB receptor ligands. The phytocannabinoids  -tetrahydro- reported to be an antagonist of GPR55 (15). The GPR55 phar- cannabivarin, cannabidivarin, and cannabigerovarin are also macology of many other Cannabis sativa (C. sativa) constitu- potent inhibitors of LPI. These Cannabis sativa constituents ents has still to be investigated. Furthermore, the pharmacology may represent novel therapeutics targeting GPR55. of various CB receptor-selective ligands at GPR55 has not been investigated. This is important because CB ligands are antino- ciceptive, a characteristic that may be shared by GPR55 ligands. The physiological roles of GPR55 and its possible involve- Activation of GPR55 by LPI, but also by certain cannabinoid ment in the pathophysiology of medical conditions are emerg- ligands, initiates multiple signaling pathways distinct from ing. Studies in mice lacking GPR55 have reported a reduction in inflammatory and neuropathic pain (1), and these mice have The abbreviations used are: LPI, L--lysophosphatidylinositol; AM281, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H- increased bone mass (2). Subsequently, studies have established pyrazole-3-carboxamide; AM251, 1-(2,4-dichlorophenyl)-5-(4-iodophe- nyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide; CBD, canna- * This work was supported, in whole or in part, by National Institutes of Health bidiol; CBDA, cannabidiolic acid; CBDV, cannabidivarin; CBG, cannabigerol; Grants DA-03672 and DA-09789 (to R. A. R. and R. G. P.). This work was also CBGA, cannabigerolic acid; CBGV, cannabigerovarin; GPR55, G protein- supported by a Knowledge Transfer Grant Award from the University of coupled receptor 55; SR141716A, 5-(4-chlorophenyl)-1-(2,4-dichloro- Aberdeen. R. G. P. and R. A. R. received funding from GW Pharmaceuticals. phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide;  -THC, □ S 9 9 9 This article contains supplemental Figs. 1 and 2.  -tetrahydrocannabinol;  -THCV,  -tetrahydrocannabivarin; GTPS, To whom correspondence should be addressed. E-mail: [email protected]. guanosine 5-3-O-(thio)triphosphate. JANUARY 2, 2012• VOLUME 287 • NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 91 This is an Open Access article under the CC BY license. Pharmacology of GPR55 Ligands those initiated by cannabinoid CB and CB receptors. GPR55 GPR55), preceded by the signal sequence from the human 1 2 is predominantly coupled to G leading to activation of growth hormone (residues 1–33), and subcloned into pcDNA 12/13 small G proteins (15). It has also been suggested to couple to 3.1 vector. The cells were maintained in Dulbecco’s modified G to promote the activation of phospholipase C and the Eagle’s medium DMEM/F-12 supplemented with 10% newborn increase of intracellular calcium release from inositol triphos- calf serum, 0.5 mg/ml G-418, 60 units of penicillin, 60 gof phate receptor-gated stores (6, 9). LPI-induced activation of streptomycin, and 2 mML-glutamine at 37 °C and 5% CO . GPR55 has been shown to lead to the recruitment of multiple Transfected cells and untransfected cells were plated on the nuclear transcription factors. Among these factors, the most same plates for comparison. investigated is ERK1/2 (5–9, 11, 15–17). Other nuclear tran- ERK1/2 MAPK Phosphorylation Assay—For experimental scription factors recruited by GPR55 activation are nuclear fac- studies of ERK1/2 MAPK phosphorylation, cells (40,000 cells/ tor of activated T cells (NFAT) (9, 13), cAMP-response ele- well) were plated onto 96-well plates and serum-starved for ment-binding protein (CREB), nuclear factor -light chain 48 h. hGPR55-HEK293 cells were serum-starved in DMEM/ enhancer of activated T cells (NF-B) (4, 9, 13), p38 MAPK (18), F-12 medium supplemented with G-418 and 2 mML-glutamine. and Akt serine/threonine protein kinase (8, 11). HEK293 cells were serum-starved in DMEM/F-12 medium Here, we report on the GPR55 pharmacology of arylpyrazole supplemented with 2 mML-glutamine. Cells were assayed in analogues, CB agonists, and a number of C. sativa constituents DMEM/F-12 medium containing L-glutamine and incubated (for structures see Table 3). We show that arylpyrazole ana- for 20 or 60 min at 37 °C in a humidified atmosphere. Drugs logues act both as agonists alone and as inhibitors of LPI signal- were dissolved in DMSO, and stocks at a concentration of 10 ing. The compounds significantly decrease the E value for max mM were kept at 20 °C. LPI was stored at 80 °C for up to 3 the GPR55 endogenous agonist, LPI, which is characteristic months. Drugs were tested in the absence of LPI at a final con- of a noncompetitive mode of action; this may suggest allos- centration of 0.1% DMSO or in the presence of LPI at a final tery. These results provide a possible explanation for the concentration of 0.2% DMSO, unless stated otherwise. At the controversy surrounding the pharmacology of certain end of the assay, the medium was removed, and cells were lysed ligands at GPR55, which have been reported, by different with lysis buffer supplied in the AlphaScreen SureFire ERK groups, to behave as both agonists and antagonists. Here, we kit. demonstrate for the first time that a single ligand can display AlphaScreen SureFire ERK Assay—The assay was per- both behaviors in the same assay. Furthermore, we show that formed in 384-well white Proxiplates according to the manu- certain CB receptor-selective agonists also act as antago- nists of GPR55; this may have implications for the mecha- facturer’s instructions. Briefly, 4 l samples were incubated nism of action underlying the reported antinociceptive with 7 l of mixture containing the following: 1 part donor actions of these compounds (19). beads, 1 part acceptor beads, 10 parts activation buffer, 60 parts reaction buffer. Plates were incubated at room temperature and EXPERIMENTAL PROCEDURES read with the Envision system (PerkinElmer Life Sciences) 9 9 Materials—Cannabis constituents -THC,  -THCV, using AlphaScreen settings. ()CBD, CBDA, CBDV, CBG, CBGA, and CBGV were sup- Analysis—Raw data were presented as “Envision units.” Basal plied by GW Pharmaceuticals, and SR141716A was from level was defined as zero. Results were presented as means and Sanofi-Aventis (Montpellier, France). ()CBD, AM251, variability as S.E. or 95% confidence limits of the percent stim- AM281, CP55940, WIN55212-2, HU-308, GW405833, and ulation of phosphorylated ERK1/2 above the basal level (in the JWH-133 were from Tocris Cookson (Avonmouth, UK). PLX- presence of vehicle). Data were analyzed using nonlinear anal- 4720 was from Selleck, Houston, TX. ABD824 was synthesized ysis of log agonist versus response curve using GraphPad Prism using similar methodology to that described previously for 5.0 (GraphPad, San Diego). The results of this analysis were SR141716A (Iain R. Greig, University of Aberdeen). (E)-- presented as E  S.E. and pEC  S.E. (logEC )orEC max 50 50 50 Caryophyllene was gifted by Prof. Gertsch (Institute of Bio- 95% confidence limits (CL; where appropriate). In Fig. 6A, for chemistry and Molecular Medicine, University of Bern, Swit- each kinase inhibitor, the values for percent stimulation of zerland). PD98059 and AM1241 were from Cayman. G-418 was phosphorylated ERK1/2 were normalized to the mean value from PAA (United Kingdom), DMEM/F-12, DMEM, newborn produced by 10 M LPI (in the presence of vehicle) in matched calf serum, and penicillin/streptomycin solution were obtained experiments. Curves of LPI-induced response were not differ- from Fisher. L-Glutamine, LPI, LY294002, and Y27632 and all ent between experiments; therefore, the data were pooled. Data the other chemicals were obtained from Sigma-Aldrich (Dor- were presented as “pERK” stimulation as percent of LPI. The set, U.K.). AlphaScreen SureFire phospho-ERK kit (catalogue statistical significance of E  S.E. or logEC  pEC was TGRES10K) was from PerkinElmer Life Sciences. max 50 50 determined with an unpaired Student’s t test (95% confidence Cell Culture—Untransfected HEK293 cells were maintained interval). When curves could not be fitted on a nonlinear anal- in DMEM containing 2 mML-glutamine medium supple- ysis of log agonist versus response, the statistical significance of mented with 10% fetal bovine serum. The preparation of the stimulation was determined with an unpaired Student’s t HEK293 stably expressing the tagged human GPR55 receptor test at each specific concentration. Results were considered sig- (hGPR55-HEK293) has been published previously by Hen- nificant only when the F-test comparing the variance was not stridge et al. (4). Briefly, the GPR55 receptor was tagged with a triple hemagglutinin (HA epitope) at the N terminus (3HA- significantly different. 92 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 1 •JANUARY 2, 2012 Pharmacology of GPR55 Ligands FIGURE 1. Detection of LPI-induced ERK1/2 phosphorylation in hGPR55-HEK293 cells using the AlphaScreen SureFire ERK assay. A, mean log concentration-response curves for the effect of LPI on ERK1/2 phosphorylation in hGPR55-HEK293 cells after 20 min (n  4 each in triplicate) or 60 min (n  3 each 2–3 repeats) of incubation at 37 °C. Increasing the incubation time significantly (p  0.05) reduced the potency of LPI-induced ERK1/2 phosphorylation but did not affect LPI-induced maximal stimulation (E ) of the GPR55 receptor. B, compared with a 20-min incubation time at 37 °C, a 60-min incubation max significantly (p  0.05) reduced the basal level of ERK1/2 phosphorylation (n  4, each in 12 repeats). Raw data of AlphaScreen SureFire ERK assay are presented as Envision units, Student’s t test, ***, p 0.05. C, no significant differences were observed in basal levels of phosphorylated ERK1/2 in untransfected HEK293 and hGPR55-HEK293 incubated for 20 min in 0.1 or 0.5% DMSO. C, effect of DMSO on LPI-induced ERK1/2 phosphorylation was assessed at 0.1 or 0.5% DMSO in either hGPR55-HEK293 cells or untransfected HEK293 cells. In untransfected HEK293 cells, no stimulation of ERK1/2 phosphorylation was detected at any concentration of LPI at any concentration of DMSO (untransfected and hGPR55-HEK293 cells were seeded on the same plate). Each symbol represents the mean percentage change in bound phosphorylated ERK1/2 protein. RESULTS centration of DMSO (Fig. 1C). These results are similar to the results published by other groups who reported the Studying the Pharmacology of GPR55 Using AlphaScreen potency of LPI in ERK1/2 phosphorylation assays (5, 13) or Surefire pERK1/2 Assay—The phosphorylation of ERK1/2 other readouts (6). protein has been reported as one of the main signaling path- LPI Induces Sustained Activation of ERK1/2 Phosphorylation ways initiated upon stimulation of the GPR55 receptor; there- in hGPR55-HEK293 Cells—Sustained activation of ERK1/2 fore, we focused our research on phosphorylated ERK1/2 pro- phosphorylation has been implicated as a measure for cancer tein and established a high throughput system using the progression, increase in cell metastasis, and invasiveness of AlphaScreen SureFire phospho-ERK assay. We validated the tumor cells (20, 21). Importantly, GPR55-induced ERK1/2 assay by time (data not shown) and vehicle responses and using phosphorylation regulates human cancer cell migration in vitro untransfected HEK293 cells (Fig. 1 and Table 1). and proliferation in vivo (3, 8, 11). It also appears to govern the LPI (0.1% DMSO) produced a maximal stimulation of maintenance of persistent inflammatory pain (22, 23). There- 129.0  9.65% (E ) (Fig. 1A), which was not significantly max different from the maximal stimulation with 0.2% DMSO fore, we investigated the ability of LPI to maintain ERK1/2 (n  15 each in triplicate; Table 1; e.g. Fig. 2A). The potency phosphorylation after 20 and 60 min of incubation (Fig. 1A). of LPI was not different either (Table 1). In untransfected The potency of LPI after 20 min of incubation, with an EC of HEK293 cells, no stimulation of phosphorylated ERK1/2 was 0.27 M (0.10–0.76), was significantly (p  0.05) reduced to detected at any concentration of LPI nor in any given con- 4.61 M (0.75–28.3) after 60 min. Prolonged incubation JANUARY 2, 2012• VOLUME 287 • NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 93 Pharmacology of GPR55 Ligands reduced both basal and LPI-induced stimulation (Fig. 1B and Structural Analogues of SR141716A Inhibit LPI-induced Table 1) but did not affect the net effect of LPI-induced Activation of ERK1/2 Phosphorylation, Implications for Allos- maximal stimulation of ERK1/2 phosphorylation (Fig. 1A), teric Inhibition—There is still a major controversy surrounding showing that percent stimulation was sustained. This is in the profile of the arylpyrazoles at GPR55. In some studies, contrast, for example, to CBD (10 M) that significantly SR141716A is reported to be an inhibitor (6), and in other stud- increased the percent stimulation after 60 min (supplemen- ies, the compound is reported to be an agonist of GPR55 (5, 13, tal Fig. 2). 14). This is of major importance as SR141716A (Rimonabant) was widely prescribed to patients as an anti-obesity agent and was withdrawn because of mental health issues. We compared TABLE 1 Cannabinoid-mediated stimulation of ERK1/2 phosphorylation in the ability of AM251, AM281, SR141716A, and ABD824, an hGPR55-HEK293 cells AM251 analog in which the iodine was substituted with bro- Cells were treated with each drug for 20 min or for the indicated time at 37 °C. CL mine (Fig. 2, Table 1, and see Table 2 for structures), to induce means confidence limits, and NA means not applicable. ERK1/2 phosphorylation in hGPR55-HEK293 cells with that of Compound EC (95% CL) % E (S.E.) 50 max LPI. The efficacy of AM251 was not significantly different from LPI (0.1% DMSO) 20 min 0.27 (0.10–0.76) 129.0  9.65 that of LPI, but the compound was significantly less potent (Fig. LPI (0.2% DMSO) 20 min 1.12 (0.59–2.16) 126.0  9.44 a 2A and Table 1). It was not possible to obtain an accurate mea- LPI (0.1% DMSO) 60 min 4.61 (0.75–28.26) 139.8  45.70 AM251 2.34 (0.99–5.56) 153.2  22.10 surement of the E for SR141716A and AM281; however, the max ABD824 0.7 (0.11–4.44) ()34.16  22.4 (up to 1 M) compounds had efficacy that was not lower than that of LPI AM281 NA 106 (10 M) SR141716A NA 160 (10 M) (Fig. 2, A and B, and Table 1). Interestingly, ABD824 had a CP55940 NA 50 (10 M) bi-phasic response in hGPR55-HEK293 cells (Fig. 2C). JWH-133 0.16 (()30.98 to ()29.8  6.40 (up to 3 M) ()4.41) Arylpyrazole-induced stimulation appears to be GPR55-me- GW405833 1.87 (0.33 to 10.7) 54.0  13.04 diated as these ligands do not stimulate ERK1/2 phosphoryla- HU-308 NA 20 (10 M) AM1241 NA 15 (10 M) tion in non-expressing HEK293 cells (4), and CP55940 alone BCP NA 22 (10 M) did not induce a significant stimulation of ERK1/2 phosphor- p  0.05 versus LPI in 0.1% DMSO for 20 min (first raw). p  0.01 versus LPI in 0.1% DMSO for 20 min (first raw). ylation at any concentration (Fig. 2B). FIGURE 2. Structural analogues of SR141716A-induced ERK1/2 phosphorylation in hGPR55-HEK293 cells. A, mean log concentration-response curves for percent stimulation of ERK1/2 phosphorylation by LPI (n  4), AM251 (n  3), or AM281 (n  3) after a 20 min stimulation at 37 °C. B, mean log concentration- response curves of LPI (n 4), SR141716A (n 3), or CP55940 (n 3), a nonselective cannabinoid CB /CB agonist. C, mean log concentration-response curves 1 2 of LPI or ABD824, an AM251 analog in which iodine was substituted with a bromine (n  3, each in duplicate). The effect of SR141716A analogues on LPI induced ERK1/2 phosphorylation in hGPR55-HEK293 cells. D, LPI (n  4) in the presence or absence of 100 nM AM251 (n  3) or 1 M AM251 (n  4). E, effect of LPI in the presence or absence of 1 M SR141716A (n  4) or 1 M AM281 (n  3). F, effect of LPI in the presence or absence of 1 M and 3 M ABD824 (n 4, each in duplicate). Each symbol represents the mean percentage change in bound phosphorylated ERK1/2 S.E. over the basal level, and each independent experiment was performed in triplicate unless stated otherwise. 94 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 1 •JANUARY 2, 2012 Pharmacology of GPR55 Ligands TABLE 2 Effect of arylpyrazoles on LPI-mediated stimulation of ERK1/2 phosphorylation in hGPR55-HEK293 cells Cells were co-treated with LPI in the presence or absence of a given drug for 20 min at 37 °C. Final concentration of DMSO was 0.2%. Arylpyrazole LPI ± drug EC (µM) pEC ± S.E. % E ± S.E. % Inhibition 50 50 max Me Vehicle 1.15 5.94 ± 0.22 147.2 ± 17.76 AM251 100 nM 0.53 6.28 ± 0.07 108.6 ± 3.86 26% Cl AM251 1 µM 0.67 6.17 ± 0.29 68.70 ± 9.93 ** 53% Cl Me Vehicle 0.79 6.10 ± 0.20 161.9 ± 15.14 Cl Cl SR141716A 1 µM 0.56 6.25 ±0.29 108.3 ± 14.3* 33% Cl Me Vehicle 0.51 6.29 ± 0.24 107.4 ± 10.78 Br ABD824 1 µM 0.45 6.35 ± 0.37 70.39 ± 10.65 34% Cl ABD824 3 µM 0.42 6.38 ± 0.58 59.7 ± 11.7* 44% Cl O O Me Vehicle 0.89 6.05 ± 0.12 131.6 ± 8.9 Cl AM281 1 µM 0.25 6.59 ± 0.24 81.49 ± 8.71 * 45% Cl *, p  0.05; **, p  0.01 versus LPI in each experiment. % inhibition is percentage inhibition of E relative to LPI (100%). max In some studies, SR141716A was reported to inhibit GPR55 induced -arrestin trafficking and LPI-induced ERK1/2 phos- activity (6), and we therefore assessed if certain arylpyrazoles phorylation (5). For comparison, in this study, LPI-induced could inhibit LPI-induced GPR55 stimulation, in the same stimulation was 144.0  16.7% and CP55940 at 1 M reduced it experimental system. The arylpyrazoles AM251, AM281, by only 13% (124.9 16.0%), which was not significantly differ- SR141716A, and ABD824 reduced the maximal stimulation ent from LPI alone (supplemental Fig. 1C). (E Effect of CB ) by LPI of ERK1/2 phosphorylation but did not signifi- Agonists on LPI-induced Activation of ERK1/2 max 2 cantly alter the potency of this bioactive lipid (results are sum- Phosphorylation, Implications for Neuropathic Pain and Posi- marized in Fig. 2, E and F, and in Table 2). tive Allosteric Modulation—The antinociceptive effect of CB Importantly, these data provide the first demonstration of a receptor agonists has been extensively investigated (reviewed in dual action (both agonist and inhibitor) of these compounds in Ref. 24). Subsequently, CB receptor agonists have been devel- the same assay. The inhibition appears noncompetitive and oped by scientists and pharmaceutical companies as alternative may indicate allostery. Of the tested analogues, AM251 was the treatments aimed at alleviation of neuropathic pain. Interest- most effective as an inhibitor of LPI (Fig. 2D). The E of LPI of ingly, a comparison of the behavioral responses of wild type max M and to mice with GPR55 knock-out mice in pain models revealed the 147.2  17.8% was reduced to 108.6  3.86% by 100 n 68.7  9.93% (p  0.01) by 1 M AM251. involvement of GPR55 in the conduction of neuropathic pain We continued to explore the effects of high concentrations of (1). These results suggest that the analgesic effects of certain AM251 and SR141716A. The maximal stimulation by LPI in the CB receptor-selective ligands may be mediated by GPR55. We presence of high concentrations of AM251 or SR141716A was compared several CB agonists that have been intensively not significantly different from that of LPI only. This would be investigated in the past and thus form a core of active structures expected because the compounds are agonists alone at these for GPR55-mediated ERK1/2 phosphorylation. JWH-133 (up concentrations (supplemental Fig. 1, A and B). to 3 M) significantly reduced basal pERK levels. GW405833 Previous studies reported that CP55940, a nonselective can- behaved as a partial agonist of GPR55 (E 54  13.0% versus max nabinoid receptor agonist, is an inactive ligand at GPR55 that LPI 106.0  10.5%; p  0.05), with a similar potency to that of could not increase intracellular calcium level (6) but behaves as LPI (1.9 M (0.3–10)) (Fig. 3A). In contrast to the arylpyrazoles, a competitive antagonist in the presence of LPI (4); another GW405833 enhanced LPI-induced ERK1/2 phosphorylation at group showed it antagonized LPI, AM251, and SR141716A- a concentration that alone had no effect on pERK (Fig. 3B). JANUARY 2, 2012• VOLUME 287 • NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 95 Pharmacology of GPR55 Ligands FIGURE 3. Effect of CB receptor agonists on ERK1/2 phosphorylation in hGPR55-HEK293 cells. A, mean log concentration-response curves for percent stimulation of ERK1/2 phosphorylation by LPI (n 7), GW405833 (n 4), or JWH-133 (n 3) after 20 min of stimulation at 37 °C. JWH-133 significantly reduced basal pERK levels, *, p  0.05; **, p  0.01, one-sample t test. B, GW405833 at 10, 30, and 100 nM enhanced the LPI-induced ERK1/2 phosphorylation (n  4). C, JWH-133 at 1 and 3 M inhibited the LPI-induced ERK1/2 phosphorylation (n 4). D, percent stimulation of ERK1/2 phosphorylation by LPI (n 3), AM1241 (n 4), or HU-308 (n 4) and (E)--caryophyllene (BCP)(n 3) after 20 min of stimulation at 37 °C. E, (E)--caryophyllene at 1, 3, and 10 M (n 3) did not alter the LPI-induced ERK1/2 phosphorylation nor did AM1241 (F, n 3). Each symbol represents the mean percentage change in bound phosphorylated ERK1/2 S.E. over the basal level, and each independent experiment was performed in duplicate. Conversely, JWH-133 at 1 and 3 M (Fig. 3C), but not at 10 M We then assessed the modulation of LPI-induced stimula- 9 9 (results not shown), inhibited the LPI-induced response. These tion with 1 M  -THC or 1 M  -THCV (produced stimula- results further support the data obtained with the arylpyrazoles, tion of 22.2  11.1% (not significant from zero) and 31.4 indicating that cannabinoids can act as noncompetitive inhibi- 10.0% (p  0.01 versus 0), respectively). Incubation of LPI with 9 9 tors of LPI signaling. Of the tested compounds, (E)--caryo- 1 M of  -THC or  -THCV significantly inhibited LPI-in- phyllene (25), HU-308 (data not shown), and AM1241 had lit- duced stimulation (Fig. 4, B and C, respectively, and Table 3). tle/no effect up to a concentration of 10 M (Fig. 3D) and did Neither ligand affected the potency of LPI (Table 3). These results not significantly alter the LPI-induced effect on GPR55 (Fig. 3, show for the first time that the phytocannabinoids  -THC and 9 9 E and F).  -THCV are inhibitors of LPI. Moreover,  -THCV elicited a Effect of Phytocannabinoids on LPI-induced Activation of downward shift in the log concentration-response curve of LPI, ERK1/2 Phosphorylation, Comparison of  -THC and such that LPI-induced pERK level was lower in the presence of 9 9 9 -THCV—Similarly to arylpyrazoles, the effect of  -THC,  -THCV, and this resembles negative cooperativity. the main psychoactive constituent of C. sativa, on GPR55 is also Structural Analogues of Cannabidiol—We have recently controversial. It has been shown to increase calcium release (6) shown that CBD antagonizes LPI-induced stimulation of but not to mobilize -arrestin (5) in GPR55-expressing cells. In [ S]GTPS binding in breast cancer MDA-MB-231 cells, this study, we compared the structurally related cannabis con- which highly express GPR55 (3). CBD also inhibits the metastasis 9 9 stituents,  -THC and  -THCV (Fig. 4A and Table 3). Both and aggressiveness of brain and breast cancer cells (26, 27) and compounds stimulated ERK1/2 phosphorylation. However, for LPI-induced calcium mobilization in prostate cancer cells (8). neither compound could the stimulation be fitted to a concen- Therefore, we have evaluated two cannabis constituents that are tration-response curve. Compared with the maximal stimula- structurally related to CBD, alone or in combination with LPI. tion of LPI (129.0  9.65%; Fig. 1A), these results suggest that Cannabidiolic acid (CBDA) has an acid group (COOH) on the 9 9 -THC is a low affinity partial agonist, whereas  -THCV is a benzene ring, whereas cannabidivarin (CBDV) has a shorter side low affinity, high efficacy, agonist of GPR55. The levels of chain compared with that of CBD (see Table 3 for structures). ERK1/2 phosphorylation after prolonged incubation (60 min) We compared the effects of CBD from two sources (Tocris 9 9 with  -THC or  -THCV were not significantly different and GW Pharmaceuticals). The effect of CBD on ERK1/2 phos- from those obtained after 20 min. phorylation after 20 min was not significantly different between 96 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 1 •JANUARY 2, 2012 Pharmacology of GPR55 Ligands 9 9 FIGURE 4. Effect of  -THC and  -THCV on ERK1/2 phosphorylation in hGPR55-HEK293 cells. Mean log concentration-response curves for ERK1/2 9 9 phosphorylation after 20 or 60 min of stimulation at 37 °C with -THC (n  3) or  -THCV (n  4) (A). B shows the effect of LPI in the presence or absence of 1 9 9 M -THC after 20 min of stimulation at 37 °C (n 4). C shows the effect of LPI in the presence or absence of 1 M -THCV after 20 min of stimulation at 37 °C (n 3). Each symbol represents the mean percentage change in bound phosphorylated ERK1/2 S.E. over the basal level, and each independent experiment was performed in triplicate. the two sources (supplemental Fig. 2A); However, after a 60 min gest that the previously reported CBD antagonism of LPI-in- incubation, CBD significantly increased ERK1/2 phosphoryla- duced stimulation of ERK1/2 phosphorylation can be further tion (100.5 11.9% and 220.9 33.2% each at 10 M, Tocris or enhanced by shortening its alkyl side chain from pentyl to pro- GW Pharmaceuticals, respectively). Neither CBD nor CBDA pyl, although the additional acid group mainly affects the inhi- affected basal pERK after a 20 min incubation, whereas CBDV bition of ERK1/2 phosphorylation at a low concentration of produced a maximal stimulation of 43.0 23.0% and an EC of LPI. 1.9 M (confidence intervals 0.13–27.9 M)(supplemental Fig. Structural Analogues of Cannabigerol—Following these find- 2, B and C, respectively). ings, we sought to evaluate CBG and two structurally related The effects of CBD, CBDA, and CBDV on LPI-induced cannabis constituents that either contain an acid group on the ERK1/2 phosphorylation are summarized in Table 3 and Fig. 5, benzene ring (CBGA) or a propyl side chain (CBGV). After a 20 A–C. CBD did not significantly alter LPI efficacy or potency at min incubation, CBG did not produce a significant stimulation 1or3 M (Table 3). However, the inhibition of 32% at 3 M (supplemental Fig. 2D). CBGA decreased basal pERK with an showed a trend toward antagonism. In fact, we have found sim- E of ()79.9 27.2% (20 min) and after a 60 min incubation, max ilar results using the [ S]GTPS binding assay with MDA-MB- it was ()31.4  7.34% with an EC of 5.26 M (confidence 231 breast cancer cells (3). In these endogenously expressing intervals 0.99–27.9) and 0.83 M (confidence intervals 0.01– GPR55 receptor cells, LPI stimulates [ S]GTPS binding, 4.79), respectively (supplemental Fig. 2E). whereas 1 M CBD produces a significant downward shift We then assessed the effect of each compound on LPI-in- (6.6 5.35% versus ()17.5 3.69 with CBD) but did not affect duced stimulation of GPR55 (Fig. 5, D and E, and Table 3). The the maximal stimulation (E ) of LPI-induced [ S]GTPS effect of each compound at 1 M was not significantly different max binding (LPI, 50.1  7.5%; 48.3  21.2%). However, these cells versus zero (stimulation of ()7.7% for CBG; ()7.4% for express a spectrum of receptors making them not ideal for fur- CBGA; ()6.2% for CBGV)). At 1 M, CBGA (Fig. 5E) and ther characterization of the pharmacology of GPR55. Taken CBGV (Fig. 5F) but not CBG (Fig. 5D) significantly reduced the together, these results support the hypothesis that CBD is an maximal stimulatory effect of LPI on ERK1/2 phosphorylation inhibitor of GPR55. (p  0.05), suggesting that these compounds are also inhibitors Of the tested analogues, CBDV (9.99% stimulation at 1 M; of LPI-induced activation of the GPR55 receptor. We observed not significant versus zero) significantly reduced the maximal a similar structure-activity relationship to that found for CBD stimulation of LPI by 56% (p  0.05) (Fig. 5C and Table 3). analogues, and thus the rank order of LPI inhibition was CBGV Increasing the CBDV concentration to 3 M did not inhibit or (69%) CBGA (56%) CBG (16%). Furthermore, CBGV dis- increase LPI stimulation (data not shown). CBDA did not sig- played negative cooperativity with LPI, whereby basal pERK nificantly affect the efficacy or potency of LPI but produced a was significantly lower in the presence of both compounds. significant downward shift of its response (11.15 10.1% versus GPR55 Signaling Involves Cross-talk between MAPK and Rho ()24.1  5.81% with CBDA; p  0.05) (Fig. 5B and Table 3); GTPases Signaling Pathways—We then tested if LPI-induced this may be indicative of negative cooperativity. In summary, stimulation of ERK1/2 phosphorylation can be inhibited by the the three compounds structurally related to CBD significantly MEK1/2 noncompetitive inhibitor PD98059. LPI (10 nM to 10 inhibit LPI-induced ERK1/2 phosphorylation with a rank order M) did not stimulate the phosphorylation of ERK1/2 proteins of CBDV (56%) CBDA (16%) CBD (9%). Furthermore, both in the presence of PD98059, which significantly inhibited basal CBDA and CBDV displayed negative cooperativity with LPI. pERK alone (Fig. 6, A and B). These findings were in line with Basal pERK was significantly lower when either of these com- the receptor-independent mechanism of action of PD98059 by pounds was co-incubated with LPI. These results underlie a binding the inactive form of MEK1 and inhibiting its activation new structure-activity relationship of GPR55 ligands and sug- by upstream activators such as Raf kinases. However, this could JANUARY 2, 2012• VOLUME 287 • NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 97 Pharmacology of GPR55 Ligands TABLE 3 Effect of phytocannabinoids and CB receptor compounds on LPI-mediated stimulation of ERK1/2 phosphorylation in hGPR55-HEK293 cells Cells were co-treated with LPI in the presence or absence of a given drug at 1 M for 20 min at 37 °C. Final concentration of DMSO was 0.2%. not explain previous observations showing that GPR55 is pre- Y27632 significantly inhibited the response to LPI under these dominantly coupled to G . To test our hypothesis for conditions. 12/13 cross-talk between MAPK and Rho GTPases signaling path- Enhancement of GPR55 Signaling by PLX-4720 Oncogene ways, we co-incubated LPI with Y27632, a p160ROCK (Rho- B-Raf Inhibitor—B-Raf is a member of Raf kinase family and associated kinase) inhibitor. After 20 min, 10 M Y27632 signif- plays a major role in regulating ERK1/2 phosphorylation. icantly inhibited LPI-induced stimulation of ERK1/2 Importantly, inherited and acquired mutations, such as V600E, phosphorylation (Fig. 6A; p  0.01), without inducing a signif- in B-Raf are associated with various diseases of which most icant change in LPI potency. Although 10 M Y27632 or 10 M are cancers (28). Therefore, B-Raf inhibitors have been LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), developed as anti-cancer therapeutics. PLX-4720, a potent could equally stimulate ERK1/2 phosphorylation (Fig. 6B), only and selective B-Raf inhibitor (29), induced a concentration- 98 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 1 •JANUARY 2, 2012 Pharmacology of GPR55 Ligands TABLE 3—continued *, p  0.05 versus LPI in each experiment. % inhibition is percentage inhibition of E relative to LPI (100%). **, p  0.01 versus LPI in each experiment. max FIGURE 5. Effect of CBD and CBDA and CBDV on ERK1/2 phosphorylation in hGPR55-HEK293 cells. Mean log concentration-response curves for ERK1/2 phosphorylation after 20 min of stimulation at 37 °C. The effect of LPI in the presence or absence is shown as follows: 1 M CBD (n  5) (A); 1 M CBDA (n  3) (B); 1 M CBDV (n  3) (C); 1 M CBG (n  4) (D); 1 M CBGA (n  4) (E); 1 M CBGV (n  3) (F). CBDV and CGV pronouncedly inhibited LPI compared with their analogues. Each symbol represents the mean percentage change in bound phosphorylated ERK1/2  S.E. over the basal level (n 3). CBDA, CBDV, and CBGV significantly reduced basal pERK levels, *, p  0.05; **, p  0.01; ***, p  0.001 one-sample t test. dependent stimulation of ERK1/2 phosphorylation at a mag- gests an additive relationship between intracellular inhibi- nitude similar to that induced by LPI (Fig. 6C), opposing the tion of B-Raf and extracellular stimulation of LPI-induced of inhibitory effects of the MEK signaling pathway in ERK1/2 phosphorylation, supporting a cross-talk between BRAF(V600E)-expressing cells. In the presence of LPI, MAPK and Rho GTPases signaling pathways. increasing concentrations of PLX-4720 (100, 300, 1000 nM) DISCUSSION significantly increased the E of LPI (119.2  10.53%) max reaching 220.6  13.97% at 1000 nM (p  0.01, one-way The lack of appropriate radiolabeled ligands for GPR55 pre- analysis of variance) but did not alter its potency; this sug- cludes the characterization of the binding of novel small mole- JANUARY 2, 2012• VOLUME 287 • NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 99 Pharmacology of GPR55 Ligands FIGURE 6. Effect of kinase inhibitors. A, LPI-induced stimulation of ERK1/2 phosphorylation was attenuated by 10M PD98059, a MEK1 inhibitor, and inhibited by 10 M Y27632, a Rho-associated protein kinase inhibitor (n 5 each in duplicate), but not by 10 M LY294002, a phosphatidylinositol 3-kinase inhibitor (n 3 each in duplicate). B, effect of each inhibitor alone on phosphorylated ERK1/2 level (n  4 for PD98059; n  7 for Y27632; and n  3 for LY294002, each in duplicate). PD98059 significantly reduced basal pERK levels; *, p  0.05; **, p  0.01, one-sample t test. C, diagram representing the putative GPR55/LPI signaling cascade. D, selective B-Raf inhibitor, PLX-4720, stimulated hGPR55-HEK293-expressing cells in a similar manner to LPI (n  3 each in duplicate). E, increasing concentrations of PLX-4720 significantly increased the efficacy of LPI-induced response (100 nM, p 0.05; 300 nM, p 0.01; 1000 nM, p 0.01) and the bottom of the curve (300 nM, p  0.05), (n  3 each in duplicate). cules to this receptor. In this study, we established a new rapid phosphorylation is also associated with a variety of human and sensitive AlphaScreen SureFire assay as a strategy for pathologies (38). In addition, many mutations of components studying the pharmacology of GPR55. This method has been upstream to ERK1/2 alter this signaling pathway and have been used to explore ligands of the cytokine receptors (30), LDL associated with increased cancer metastasis and invasiveness endothelial receptor (31), and potassium channel TREK-1 (20, 38). The combination of strength and duration of ERK1/2 receptors (32) and to develop antagonists for other intracellular signaling determines the distinct outcomes, ranging from sus- targets (33–35). tained high activation that can lead to apoptosis or differentia- LPI and Certain Cannabinoids Induce Sustained GPR55-me- tion to sustained lower levels of activation that are correlated diated ERK1/2 Phosphorylation—Studies have shown that with cell proliferation (39). In our case, it appears that LPI- GPR55 induces maximal ERK1/2 phosphorylation response induced activation parallels the latter scenario, supporting the after 10–20 min (this study and see Refs. 4, 5, 7); here we show role of GPR55 in cancers. Therefore, it will be interesting to that although both basal and LPI-induced stimulation levels are determine whether this mechanism involves the endogenous reduced at 60 min (Fig. 1 compared with 20 min) the percent mitogen-activated protein kinase phosphatase 1 (MKP-1) that stimulation is sustained, but the potency of LPI is significantly controls the constitutive activation of ERK1/2 (21). decreased after 60 min. GPR55 Pharmacology of Arylpyrazoles—A key finding of this Sustained ERK1/2 activation in injured neurons has been study is the demonstration that arylpyrazoles, e.g. SR141716A, suggested to reflect alterations in the intracellular feedback reg- can act both as agonists of GPR55 and inhibitors of LPI-induced ulators that normally function to terminate signaling responses activation of pERK. Thus, we demonstrate here for the first (36) and has been associated with brain ischemia (37). ERK1/2 time that both types of behavior (agonism and inhibition) as 100 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 1 •JANUARY 2, 2012 Pharmacology of GPR55 Ligands FIGURE 7. Pharmacological mechanisms for the modulation of GPR55. LPI is suggested to primarily bind the GPR55 orthosteric binding site. GPR55 may also contain an allosteric binding site. These observations raise at least two possibilities as follows. Left, one possibility is that certain arylpyrazole ligands actually represent bitopic ligands of GPR55. These ligands may have the capacity to modulate both the orthosteric (agonists) and the allosteric site through different pharmacophores. The second possibility is that AM251 and certain arylpyrazole analogues are only allosteric ligands that are ago-allosteric alone. Right,inthe presence of LPI, arylpyrazoles can also behave as allosteric inhibitors and GW405833 as an allosteric enhancer. In addition, a number of Cannabis sativa constituents appear to inhibit ERK1/2 phosphorylation in an allosteric manner. reported by previous studies from different laboratories can distinct binding sites at GPR55. Nevertheless, this study pro- exist in the same model system. Our data suggest that there may vides the first potential explanation for the considerable con- be two topographically distinct binding sites on GPR55 (Fig. 7). troversy in the pharmacology of cannabinoid ligands at GPR55 In the first scenario, certain ligands (e.g. AM251 and and has important therapeutic implications. GW405833) could act as agonists alone (either via the The arylpyrazoles belong to a subgroup of ligands that inter- orthosteric (LPI) binding site or a distinct (allosteric) binding act with both GPR55 and CB receptors. In this study, arylpyra- site (40). In the second scenario, the same ligands could act as zole analogues induced activation of pERK with a rank order of modulators of LPI-induced pERK activation. This suggests that potency LPI AM251 SR141716A AM281. This rank the compounds may bind to an allosteric site to induce a con- order of potency is in agreement with the study by Henstridge et formational change in the orthosteric (LPI) binding site such al. (4) using the same hGPR55-expressing HEK293 cells and is that the efficacy of LPI is reduced (e.g. AM251) or enhanced (e.g. similar to the study by Kapur et al. (5) that used hGPR55-ex- GW405833). These observations raise the possibility that cer- pressing U2OS cells and a -arrestin mobilization assay. tain arylpyrazole ligands may represent bitopic ligands of Although in the latter study arylpyrazoles could not induce the GPR55. Bitopic ligands have the capacity to interact with both phosphorylation of ERK1/2, the difference between this and the orthosteric site (as agonists) and the allosteric site (as mod- our study may be due to differences in method sensitivity ulators) through distinct chemical structures (41). (Western blot versus AlphaScreen) or may reflect cell type Allosteric binding sites have been described for other lipid differences. receptors, including allosteric modulators of anandamide at the Collectively, these results suggest that AM251, SR141716A, cannabinoid CB receptor (42) and ago-allosteric ligands for and AM281 are weak agonists of GPR55 as measured by their ability to induce ERK1/2 activation. Interestingly, ABD824, an short chain free fatty acids at FFA2 and FFA3 receptors (43). This study demonstrates the modulation of GPR55 by certain AM251 analog, decreased basal pERK at concentrations below ligands, possibly by an allosteric mechanism. The first evidence 3 M. This suggests a role for the chemical group on the aryl is the reduction in E of LPI observed in the presence of ring in shifting the GPR55 receptor between inactive and active max receptor conformations. As for the efficacy for the observed various compounds. However, it is important to note that this study does not provide direct evidence of allostery (e.g. dissoci- antagonism of LPI by arylpyrazoles, the rank order is AM251 ation kinetics), and there is a possibility that the compounds AM281 ABD824  SR141716A, which does not correspond may inhibit LPI signaling downstream of the receptor to disrupt to the relative potencies of these ligands as GPR55 agonists (as the pERK signaling cascade. The second evidence is the appar- detailed above). This suggests that the pharmacophore for inhi- ent negative cooperativity between various ligands and LPI bition of GPR55 by these pyrazoles may be different from that such that LPI-induced pERK level is significantly decreased in responsible for activation of GPR55. the presence of both ligands, an effect not observed with either SR141716A was developed as a cannabinoid CB receptor ligand alone. antagonist and progressed to clinical studies as a drug treat- Clearly, this evidence is circumstantial, and dissociation ment for obesity-related disorders (44). Interestingly, kinetic analysis (which is not currently feasible because of the SR141716A has been shown to reduce neuropathy associated lack of appropriate radioligand) or extensive mutation studies, with type-2 diabetes in patients (45). This has also been sup- will be required to confirm the existence of topographically ported by in vivo studies in which SR141716A reduced neuro- JANUARY 2, 2012• VOLUME 287 • NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 101 Pharmacology of GPR55 Ligands pathic pain in a murine model for obesity (46) and in a rodent  -THC have so far been considered to be mediated by activa- model for nerve injury (47). This is unexpected for an inverse tion of cannabinoid receptors. Recently, GPR55 has been linked agonist of CB because CB receptor agonists are effective in to inhibition of the migration and proliferation of cancer cells 1 1 neuropathic pain. Collectively, these studies suggest that by CBD (3, 8). The results warrant further investigation into the SR141716A may act on a target other than the CB receptor to potential therapeutic use of various phytocannabinoid. CBDA inhibit pain. As GPR55 knock-out mice are less sensitive to the and CBG have been shown to activate TRPA1- and TRPV1- development of chronic pain (1), our findings that LPI-induced expressing cells and to antagonize TRPM8, implying a role in activation of GPR55 is inhibited by SR141716A suggests a novel analgesia and prostate cancer for these compounds (58, 59). mechanism of action for the regulation of pain and possibly for Our study suggests that GPR55 is an additional target for these metabolic syndrome by this compound. This effect may be compounds; inhibition of GPR55 would therefore further sup- mediated by a putative allosteric binding site on GPR55. port their potential role in the treatment of carcinoma and pain GPR55 Pharmacology of CB Agonists—This study has iden- (51). tified GW405833 as a GPR55 partial agonist. JWH-133 signifi- GPR55-mediated MAPK Signaling—Importantly, our find- cantly reduced basal pERK and as such may be an inverse ago- ings demonstrate that LPI-induced ERK1/2 phosphorylation is nist of GPR55. In line with our results, JWH-133 has been controlled, at least in part, by ROCK, supporting the role for previously studied for its analgesic effects and has been shown G in LPI signaling and indicating cross-talk between 12/13 to produce antinociception via nicotinic-dependent pathways MAPK and Rho GTPases signaling, which is in line with (48). However, its effect could not always be reversed Andradas et al. (11). Such cross-talk may be communicated via by SR144528, a CB receptor antagonist (49). Similarly, the serine/threonine kinase Raf family, of which the expression GW405833 was found to promote a pro-antinociceptive level of the oncogene B-Raf in turn controls the stimulation of response in a rat model of osteoarthritis in contrast to its behav- ROCK (60). In line with Hatzivassiliou et al. (16), the selective ior as a CB receptor partial agonist (50). These studies suggest B-Raf inhibitor PLX-4720 unregulated ERK1/2 phosphoryla- that JWH-133 and GW405833 have an additional target(s), and tion in hGPR55-HEK293 cells that do not carry the our results suggest that GPR55 signaling may be one of their BRAF(V600E) mutation; interestingly, the magnitude of the targets. response closely resembled that of LPI. Furthermore, ERK1/2 Phytocannabinoids That Inhibit LPI-mediated Activation of phosphorylation was unregulated following the co-incubation GPR55—The pharmacological actions of several C. sativa con- of PLX-4270 with LPI in an additive manner, supporting a stituents have been recently reviewed; both the psychoactive cross-talk between B-Raf and ROCK signaling pathways in LPI- and nonpsychoactive compounds have multiple targets and mediated GPR55 signaling. Further support for this interaction convey a range of pharmacological actions (51). In this study, comes from B-Raf knock-out mice in which ROCKII expression we have evaluated a range of C. sativa constituents for their is reduced (61). ability to modulate GPR55. Although  -THC activates CB Conclusions—We have established a rapid and sensitive and CB receptors and GPR55 and behaves as a partial agonist method to study the pharmacology of the GPCR GPR55 using at these receptors,  -THCV is a CB antagonist and a CB the AlphaScreen SureFire ERK assay. It is important to note 1 2 partial agonist. In this study, we found that  -THCV is a weak that this study exclusively measures ERK1/2 phosphorylation agonist of GPR55 that can significantly inhibit LPI-induced as a readout of GPR55. This readout may not allow the distinc- stimulation of pERK in GPR55-expressing cells. Thus, the anal- tion between various signaling pathways; thus, this work might gesic actions of -THCV in vivo (52) might, at least in part, be be limited by the fact that the signaling cascade leading to mediated by modulation of GPR55. Expression of GPR55 ERK1/2 activation may vary with the compound used. That mRNA has been detected in adipose tissue (15), and a polymor- said, to our knowledge this study provides the first evidence phism in the GPR55 gene has been found in females with ano- that certain cannabinoids can display both activation of GPR55 rexia nervosa (53). Although the precise role of GPR55 in food and inhibition of LPI-mediated pERK stimulation in the same intake and metabolism remains to be elucidated, it is possible assay. Further studies should lead to a resolution of the contro- that the inhibition of food intake reported for  -THCV (54) versy regarding the pharmacology of cannabinoids at GPR55. and other cannabinoids may be mediated, at least in part, by Furthermore, we show that certain ligands previously thought modulation of GPR55. to be selective for the CB receptor also modulate GPR55. Our Here, we report that the little investigated cannabis constit- findings also suggest that GPR55 may be a new pharmacologi- uents CBDV, CBGA, and CBGV are potent inhibitors of LPI- cal target for the following C. sativa constituents:  -THCV, induced GPR55 signaling. CBD is the most investigated non- CBDV, CBGA, and CBGV. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Jan 2, 2012

Keywords: Allosteric Regulation; Cannabinoid Receptors; Cannabinoids; G Protein-coupled Receptors (GPCR); Pharmacology; Acomplia; AlphaScreen; Bitopic Ligand; GPR55; PLX-4720 BRAF Inhibitor

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