The Cannabinoid Receptor CB1 Modulates the Signaling Properties of the Lysophosphatidylinositol Receptor GPR55*

Background: G protein-coupled receptors (GPCR) can form heteromers and thereby alter their signaling properties. Results: GPR55 and cannabinoid 1 (CB1) receptor signaling is modulated if receptors are co-expressed. Conclusion: GPR55 signaling is inhibited in the presence of CB1 receptors; in contrast, CB1 receptor-mediated signaling is enhanced if GPR55 is co-expressed. Significance: Cross-regulation of CB1 receptor and GPR55 may affect cell function when endogenously co-expressed. The G protein-coupled receptor (GPCR) 55 (GPR55) and the cannabinoid receptor 1 (CB1R) are co-expressed in many tissues, predominantly in the central nervous system. Seven transmembrane spanning (7TM) receptors/GPCRs can form homo- and heteromers and initiate distinct signaling pathways. Recently, several synthetic CB1 receptor inverse agonists/antagonists, such as SR141716A, AM251, and AM281, were reported to activate GPR55. Of these, SR141716A was marketed as a promising anti-obesity drug, but was withdrawn from the market because of severe side effects. Here, we tested whether GPR55 and CB1 receptors are capable of (i) forming heteromers and (ii) whether such heteromers could exhibit novel signaling patterns. We show that GPR55 and CB1 receptors alter each others signaling properties in human embryonic kidney (HEK293) cells. We demonstrate that the co-expression of FLAG-CB1 receptors in cells stably expressing HA-GPR55 specifically inhibits GPR55-mediated transcription factor activation, such as nuclear factor of activated T-cells and serum response element, as well as extracellular signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can form heteromers, but the internalization of both receptors is not affected. In addition, we observe that the presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear factor of activated T-cell activation. Our data provide the first evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor has functional consequences in vitro. The GPR55-CB1R heteromer may play an important physiological and/or pathophysiological role in tissues endogenously co-expressing both receptors.

CB1Rs predominantly couple to G␣ i/o proteins and thereby inhibit adenylyl cyclase, activate mitogen-activated protein kinases (MAPKs), and further activate numerous transcription factors. In addition, CB1Rs have been described to mediate the activation of several potassium channels (16,17). Multiple GPR55-mediated signaling pathways have been described (6, 7, 18 -20), whereby the most consistent reports suggest that GPR55 couples to G␣ 13 in recombinant HEK cells transiently or stably expressing GPR55 (9,18,19,(21)(22)(23). GPR55 signaling can be mediated via small GTPases (6,21,22) and the mobilization of intracellular calcium stores (21,22,24,25). This leads to the activation of several transcription factors, such as nuclear factor of activated T-cells (NFAT), nuclear factor -light chain-enhancer of activated B cells (NF-B), cyclic AMP responseelement binding protein, and activating transcription factor 2 (21,22,26). In addition, the activation of MAP kinases, such as p38 and ERK1/2 MAPKs were described to be induced after GPR55 stimulation (22,26). Furthermore, the formation of filamentous actin (F-actin) upon GPR55 activation was reported in HEK293 cells and human neutrophils and this process is mediated by G␣ 13 and RhoA (6). The formation of F-actin is related to the induction of serum response elements (SRE) and is under control of the G␣ 13 -mediated RhoA signaling (27,28).
7TM/GPCRs can form homomers and heteromers and thereby alter the biochemical properties of the receptors involved (36,37). These interactions modulate the potential signaling pathways and have been shown to have functional relevance in vitro (38) and in vivo (39 -41). The existence of CB1R homomers was detected by using antibodies specifically recognizing CB1R dimers (42). In addition, it was reported that CB1Rs can form heteromers (3). The G protein coupling is altered in CB1R-dopamine D2R heteromers (43), CB1Rorexin-1 receptor heteromers show different trafficking and signaling properties (44) and the signaling pathways are modulated in CB1R-adenosine A2A receptor heteromers (45).
To date, it is unknown whether GPR55 can form functional heteromers with other 7TM/GPCRs. Here we show that GPR55 and CB1Rs can physically interact and modulate each others signaling properties. Our data show that GPR55 signaling is specifically inhibited in the presence of the CB1 receptor.
Reporter Gene Assay-Transcription factor luciferase assays were carried out as previously described (21). Briefly, HEK293, HEK-GPR55, HEK-CB1, and HEK-GPR55ϩCB1 cells were seeded in 96-well plates (40,000 cells/well) and transiently transfected with the cis-reporter plasmids (PathDetect; Stratagene) for NFAT-luc (100 -200 ng) or SRE-luc (50 ng/well) (kindly provided by Silvio Gutkind, National Institutes of Health, Bethesda, MD) using Lipofectamine 2000. For genedose experiments, HEK-GPR55 cells were additionally transfected with pcDNA FLAG-CB1, pcDNA FLAG-CCR5, or pcDNA 3.1 (25-100 ng). For ␤-arrestin overexpression studies, cells were co-transfected with 100 ng of control pcDNA-EGFP or 100 ng of ␤-arrestin 2-EGFP plasmid. 24 to 48 h post-transfection, cells were incubated with the indicated ligand concentrations for 4 h in serum-free media at 37°C. The cell number was determined in a FlexStation II (Molecular Devices) and luciferase activity was visualized using the Steadylite Plus Kit (Packard Instrument Company, Meriden, CT) and was measured in a TopCounter (Top Count NXT; Packard) for 5 s. Luminescence values are given as relative light units (RLU). For reporter gene experiments, RLU were normalized to the cell number.
Cells were centrifuged at 14,000 ϫ g for 15 min at 4°C, the supernatant was aspirated and the pellet was resuspended in 500 l of 1ϫ HME buffer and snap frozen in liquid nitrogen. Thawed pellets were sonicated on ice in HME buffer and centrifuged at 14,000 ϫ g for 15 min at 4°C. The membranes were then resuspended and protein concentration was measured using a Bradford assay.
ELISA-Enzyme-linked immunosorbent assay was carried out as previously described (48). Experiments were performed in parallel to the reporter gene assays. Cells were fixed with 3.7% formaldehyde, blocked, and permeabilized in blotto (50 mM Tris-HCl, pH 7.5, 1 mM CaCl 2 , 0.1% Triton X-100, and 3% milk) for 1 h. The expression of ␤-arrestin 2 was determined by incubating cells with ␤-arrestin 2 antibody (1:100, Santa Cruz) overnight at 4°C, followed by incubation with an HRP-conjugated anti-mouse antibody (1:2500, Jackson ImmunoResearch) for 2 h at room temperature. Cells were washed with TBS (25 mM Tris base, 135 mM NaCl, 2.5 mM KCl, 1 mM CaCl 2 ⅐2H 2 O, pH 7.4) and cell numbers were determined by means of optical density using a FlexStation II device. 3,3Ј,5,5Ј-Tetramethylbenzidine (Sigma) substrate was added and the coloring reaction was stopped by the addition of 0.5 M sulfuric acid after 2 min at room temperature. Color intensity was measured at 450 nm in a Bio-Rad xMark Microplate Spectrophotometer.
Statistical Analysis-Statistical analyses were performed using analysis of variance for comparisons between multiple groups, followed by a Bonferroni's post hoc analysis and t tests using GraphPad Prism software (GraphPad Inc., San Diego, CA). A p value of Ͻ0.05 was considered statistically significant.

The CB1 Receptor Modulates GPR55 Transcription Factor
Activation-It has previously been reported that CB1 receptors can form heteromers with other 7TM/GPCRs and thereby modulate the signaling properties of the receptors involved (3). In contrast, no reports exist to date that GPR55 is able to form functional heteromers with other 7TM/GPCRs. However, we have recently described that GPR55 modulates the signaling capacities of the CB2 receptor in human neutrophils, where both receptors are naturally co-expressed (6). Likewise, GPR55 and CB1 receptors are found to be co-expressed in several cell types (11,18) and, importantly, some cannabinoid ligands have been reported to modulate both receptors in opposite ways (e.g. SR141716A is an inverse agonist/antagonist on the CB1 receptor and an agonist on GPR55 (22,24,30,31,33)). Hence, we were interested whether GPR55 and CB1 receptors heteromerize and/or modulate each others signaling properties. In addition, we set out to elucidate the properties of the "dual acting" ligand SR141716A in a cell system where both GPR55 and CB1 receptors are co-expressed.
We engineered HEK293 cells stably co-expressing HAtagged GPR55 and FLAG-tagged CB1 receptor (referred to as HEK-GPR55ϩCB1) or control cells expressing either a FLAG-CB1 receptor (HEK-CB1) or a HA-GPR55 receptor (HEK-GPR55) alone. Expression of GPR55 and CB1 receptors in HEK-GPR55, HEK-CB1, and HEK-GPR55ϩCB1 cells was determined by flow cytometry with anti-HA and Alexa 488conjugated IgG1 antibodies as well as anti-FLAG and Alexa 488-conjugated IgG2b antibodies under nonpermeabilizing conditions. Expression levels of receptors were similar in single and double expressing cell lines (data not shown). In earlier studies we have shown that various transcription factors, i.e. NF-B, cyclic AMP response-element binding protein, and NFAT can be activated by GPR55 (21,22). Here we show for the first time that the SRE can be activated by GPR55 (Fig. 1, B, D, and F). Several previously described GPR55 agonists, such as LPI, SR141716A, AM251, and AM281 (9,21,22,24,33) induced SRE in a dose-dependent manner (data not shown, Table 1).
We first tested whether NFAT and SRE induction via GPR55 was modulated by co-expressing the CB1 receptor. We observed NFAT activation and SRE induction in HEK-GPR55 (white bars), but not in HEK-CB1 (gray bars) cells after stimulation with the GPR55 agonists LPI or SR141716A (Fig. 1, A-D).
In the presence of the CB1R, GPR55-mediated transcription factor activation was reduced by ϳ50% after LPI treatment when compared with HEK cells expressing the GPR55 receptor alone (Fig. 1, A and B, compare white versus black bars). Interestingly, no activation of NFAT and SRE was observed in HEK-GPR55 ϩ CB1 cells, following treatment with 1 M SR141716A (Fig. 1, C and D, compare white versus black bars). Likewise, the GPR55-specific agonist GSK319197A failed to activate NFAT and SRE-luciferase in HEK-GPR55ϩCB1 cells (Fig. 1, E and F,  ). Baseline NFAT activation and SRE induction of all three cell lines was similar after vehicle (DMSO) treatment (Fig. 1, E and  F, veh). HEK293 cells did not show NFAT activation and SRE induction following GPR55 agonist treatment (data not shown).
The binding affinities of GSK319197A were determined in heterologous competition binding experiments using [ 3 H]SR141716A as a tracer for both, GPR55 and CB1 receptors ( Fig. 2 and Table 2). No CB1R and GPR55 binding was observed in HEK293 cells in homologous and heterologous competition binding experiments ( Fig. 2A). Competition of [ 3 H]SR141716A tracer by SR141716A (K i ϭ 4.81 nM), but not by GSK319197A, was observed in HEK-CB1 cells (Fig. 2B). The binding affinity of SR141716A on HEK-GPR55 membranes was ϳ100-fold lower (K i ϭ 492.65 nM) than on CB1 membranes. This, however, is in line with previous studies, where the potency of SR141716A in activating various GPR55-mediated signaling pathways was in the 1 M range (22). GSK319197A was slightly more potent in displacing [ 3 H]SR141716A from HEK-GPR55 membranes with a K i value of 242.78 nM. On HEK-GPR55ϩCB1 membranes, the K i value for SR141716A was 2.17 nM and 6263 nM for GSK319197A (Fig. 2, C and D). These data demonstrate that GSK319197A does not bind to the CB1 receptor, but it is a ligand for GPR55 with a K i value of ϳ200 nM.
To further investigate whether activation of transcription factors by GPR55 is specifically altered in the presence of the CB1 receptor, we performed gene dose experiments. HEK-GPR55 cells were transfected with increasing concentrations of either CB1 or CCR5 receptor DNA. Cells were stimulated with either 1 M GSK319197A (Fig. 3, A and C) or 1 M SR141716A (Fig. 3, B and D). Increasing CB1R expression results in a loss of GPR55-mediated NFAT (Fig. 3, A and B) or SRE (Fig. 3, C and D) induction in a dose-dependent manner (Fig. 3, A-D, left panels). To control whether the presence of an unrelated G␣ i -coupled 7TM/GPCR would likewise modulate GPR55-mediated signaling, HEK-GPR55 cells were transfected with increasing amounts of the chemokine receptor CCR5. In the presence of CCR5 no changes in NFAT (Fig. 3, A and B, right panels) or SRE (Fig. 3, C and D, right panels) induction were observed. Gene dose-dependent expression of CB1R (Fig. 3E) and CCR5 (Fig. 3F) was confirmed by Western blot analysis. In summary, these data show that the presence of the CB1 receptor inhibits GPR55induced NFAT and SRE transcription factor activity, whereas an unrelated 7TM/GPCR, i.e. CCR5, had no effect on GPR55-mediated signaling.
ERK1/2 Phosphorylation Is Altered in HEK-GPR55ϩCB1 Cells Compared with HEK-GPR55 and HEK-CB1 Cells-We and others have previously shown that ERK1/2 MAPKs are activated upon stimulation of GPR55 in a variety of cellular backgrounds (12,22,26). To test whether the presence of the CB1R interferes with GPR55-mediated signaling at a more upstream level than transcription factors, we tested MAPK activation in HEK293, HEK-GPR55, HEK-CB1, and HEK-GPR55ϩCB1 cells. As expected, ERK1/2 phosphorylation was significantly increased in HEK-GPR55 cells after stimulation with 2.5 M of the GPR55 agonists SR141716A and GSK319197A for 25 min (Fig. 4, A, upper panel, C and D, white  bars), but not after treatment with the CB1 agonist WIN55,212-2 (Fig. 4, A, upper panel, and B, white panel). ERK1/2 activation was observed in HEK-CB1 cells only after stimulation with 2.5 M WIN55,212-2 (Fig. 4, A, middle panel,  and B, gray bar). In HEK-GPR55ϩCB1 cells, stimulation with 2.5 M of the respective GPR55 agonists GSK319197A and SR141716A resulted in only marginal ERK1/2 phosphorylation (Fig. 4, A, upper and lower panels, C and D, white versus black  bars). Interestingly, the treatment of HEK-GPR55ϩCB1 with 2.5 M WIN55,212-2 resulted in a significantly higher ERK1/2 phosphorylation when compared with HEK-CB1 cells (Fig. 4, A, compare middle and lower panels, B, gray versus black bars). No ERK1/2 activation was observed in all three cell lines after vehicle (DMSO) treatment (Fig. 3A). Likewise, WIN55,212-2, GSK319197A, and SR141716A treatment did not induce ERK1/2 phosphorylation in HEK293 cells (data not shown).
These data suggest that the CB1 receptor impairs GPR55mediated signaling at the level of ERK1/2 MAP kinases. In contrast, the presence of GPR55 seems to enhance CB1R-mediated ERK phosphorylation.
To further investigate the cross-regulation of GPR55 and CB1 receptors on ERK1/2 MAP kinase phosphorylation and NFAT activity, we stimulated the respective receptors in single or co-expressing cells with the following ligand combinations:   (Figs. 4, A and B, and 5, A, D, and G). Concomitant activation of both receptors with WIN55,212-2 and GSK319197A resulted in a significant increase in both ERK1/2 phosphorylation (Fig. 5, B and E, black bar) and NFAT activity (Fig. 5H, black bar) when compared with HEK-GPR55 (white bars) or HEK-CB1 (gray bars) cells. This observation suggests that the inhibitory effect of the CB1 receptor on GPR55 signaling may be abolished when the CB1 receptor is activated. A different picture arises when HEK-GPR55ϩCB1 cells were co-treated with WIN55,212-2 and the GPR55 agonist SR141716A instead of GSK319197A. Because SR141716A is both an inverse agonist/antagonist on CB1 and an agonist on GPR55, it was not surprising to see a significant decrease in both ERK1/2 (Fig. 5, C and F, black bar) and NFAT activity (Fig.  5I, black bar) when compared with HEK-GPR55. Importantly, these results indicate that only "inactive" CB1 receptors block GPR55-mediated signaling in HEK-GPR55ϩCB1 cells.
In addition, we tested whether endogenous cannabinoid ligands, such as AEA, could equally regulate GPR55 signaling in HEK-GPR55ϩCB1 cells. In fact, we found a very similar profile of ERK1/2 phosphorylation and NFAT activity when HEK-GPR55ϩCB1 cells were stimulated with AEA instead of WIN55,212-2 in all combinations described above. Although stimulation with AEA had no effect on ERK1/2 phosphorylation and NFAT activity in HEK-GPR55 cells (Fig. 6, A, B, and E), a significant increase was observed in HEK-GPR55ϩCB1 cells (Fig. 6, A, B, and E), when compared with HEK-CB1 cells. Concomitant stimulation of cells with AEA and GSK319197A in the double expressing cell line led to an increase in pERK1/2 and NFAT activation (Fig. 6, A, C, and F), whereas the combination of AEA and SR141716A showed significantly reduced pERK1/2 and NFAT levels (Fig. 6, A, D, and G). Taken together, these data suggest that both, synthetic (WIN55,212-2) and endogenous (AEA) CB1R agonists are capable of restoring GPR55mediated signaling properties in cells co-expressing these receptors.
G␣ i Signaling and ␤-Arrestin 2 Are Not Involved in the Crosstalk between CB1R and GPR55-CB1 receptors have been reported to couple to G␣ i proteins (16,17). Here, we wanted to test whether G␣ i signaling is involved in the CB1R-mediated inhibition of GPR55 signaling. We tested whether treatment of cells with pertussis toxin (PTX) was able to restore GPR55mediated NFAT and ERK1/2 activation in HEK-GPR55ϩCB1 cells. PTX treatment did not alter GPR55-mediated NFAT or

HEK-GPR55؉CB1
NFAT activation (EC 50 DECEMBER 28, 2012 • VOLUME 287 • NUMBER 53 ERK activation in HEK-GPR55 cells (Fig. 7, A, compare Ⅺ versus f, and D) or HEK-CB1 cells (Fig. 7, B, compare Ⅺ versus f, and D) when stimulated with the GPR55 agonist GSK319197A. Control experiments on HEK-CB1 cells showed that PTX was active and could block ERK1/2 phosphorylation in WIN55,212-2-stimulated cells (data not shown). In contrast, we detected a decrease in both NFAT and ERK1/2 activity in GSK319197Astimulated HEK-GPR55ϩCB1 cells after PTX treatment (Fig. 7, C, compare Ⅺ versus f, and D), pointing toward an involvement of G␣ i proteins in the signaling capacity of the GPR55-CB1 heteromer when activated by a GPR55 selective ligand. It is well accepted that ␤-arrestins can also serve as signal adaptors and/or transducers for 7TM/GPCRs (50,51). In cells overexpressing two 7TM/GPCRs, it is hence possible that the endogenous ␤-arrestin levels are not high enough to serve as signaling partners for both receptors. We therefore tested whether overexpression of ␤-arrestin 2 could possibly restore GPR55-mediated transcription factor activation and ERK1/2 phosphorylation in HEK-GPR55ϩCB1 cells. However, we could observe no difference in both, NFAT (Fig. 8, A-C) or ERK phosphorylation (Fig. 8D) assays when all three cell lines were overexpressing ␤-arrestin 2. ␤-Arrestin 2 overexpression was verified by SDS-PAGE and ELISA experiments (Fig. 8, D and E).

DISCUSSION
GPR55 pharmacology and signaling properties were extensively studied in the past years, using many different cell sys-tems and signaling readouts (7,53). Nevertheless, the pharmacology of GPR55 is still rather controversial and seems to be highly cell system dependent (3,53). It is well known that the co-expression of 7TM/GPCRs in specific tissues can lead to altered binding and/or signaling properties via the respective receptors (54 -59). For instance, modulation of G protein activation was observed in the -opioid receptor (MOR)/␦-opioid receptor (DOR) (57) and the MOR/␤ 2 -AR heteromers (60), respectively. Although the MOR activity was decreased in the MOR/DOR heteromer, its G protein coupling was enhanced in the MOR/␤ 2 -AR heteromer. These data suggest that heteromers exhibit distinct and unique signaling profiles. Both GPR55 and CB1 receptors are highly expressed at similar levels in several brain regions, such as the striatum, hypothalamus, and brain stem (9). In light of these studies, we set out to investigate whether co-expression of CB1 and GPR55 in a recombinant HEK293 cell line could explain some of the controversial pharmacology reported for GPR55.
Here we demonstrate that GPR55 and CB1 receptors form heteromers and influence each others signaling properties in HEK293 cells co-expressing both receptors. Specifically, we show that GPR55-mediated signaling is inhibited in the presence of the CB1 receptor. Interestingly, this effect is only apparent when the CB1 receptor is inactive. In contrast, we show that the signaling capacity of the CB1R is enhanced in the presence of GPR55.  DECEMBER 28, 2012 • VOLUME 287 • NUMBER 53

JOURNAL OF BIOLOGICAL CHEMISTRY 44241
GPR55-mediated signaling is lowered or inhibited in the presence of unstimulated CB1 receptor at the level of MAP kinases (Fig. 4) and transcription factors, such as NFAT and SRE (Figs. 1 and 3). The signaling capacity of the CB1 receptor is enhanced in the presence of GPR55 (Figs. 4 -6). Upon stimulation of the CB1 receptor with the synthetic ligand WIN55,212-2 (Figs. 4 and 5) or the endogenous ligand anandamide (Fig. 6), we observed elevated ERK1/2 signals and NFAT activation in HEK-GPR55ϩCB1 cells compared with HEK-CB1 cells. Interestingly, signaling via GPR55 is restored in the presence of activated CB1 receptors (Figs. 5 and 6), because costimulation of HEK-GPR55ϩCB1 cells with both GSK319197A and WIN55,212-2 or AEA resulted in a dramatic increase of pERK and NFAT activity (Figs. 5, B, E, and H, and 6, A, C, and F). In line with these data, the GPR55 signal was effectively blocked when HEK-GPR55ϩCB1 cells were co-stimulated with WIN55,212-2 or AEA and SR141716A (Figs. 5, C, F, and I, and  6, A, D, and G). SR141716A is both an inverse agonist/antago-  A, B, and C). pERK1/2 was normalized to total ERK1/2 and data are means of three independent experiments Ϯ S.E. Reporter gene assay data are mean Ϯ S.E. from one of three independent experiments performed in triplicates. Data were normalized and expressed as percent of maximum activation, which was set as 100%, *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001. nist on CB1R and an agonist on GPR55 (Figs. 4, A and C; 5, C, F, and I; and 6, A, D, and G). Hence, when the CB1 receptor is inactivated in the presence of SR141716A, GPR55 is incapable of inducing ERK1/2 and/or NFAT activation (Figs. 5, C, F, and I,  and 6, A, D, and G), despite the fact that SR141716A is a potent agonist on GPR55. These data further substantiate the hypothesis that only inactive CB1 receptors are able to inhibit GPR55mediated signaling.
We next observed that the blockade of CB1 receptor-coupling to G␣ i proteins with PTX does not restore GPR55-mediated transcription factor activation or pERK1/2 activation in HEK-GPR55ϩCB1 cells. Interestingly, we noticed a further decrease in GPR55-mediated NFAT and ERK1/2 activation after PTX treatment in HEK-GPR55ϩCB1 cells (Fig. 7). It has been reported that CB1 receptors are able to constitutively activate G␣ i proteins (16,17,61). PTX irreversibly interacts with the G␣ i subunits and thereby inhibits the interaction with 7TM/GPCRs. We hypothesize that the inhibition of the CB1 receptor-G␣ i -subunit interaction keeps the CB1 receptor in its inactive state and thereby further renders GPR55 inactive when both receptors are co-expressed.
Most 7TM/GPCRs recruit ␤-arrestins following agonist stimulation (62,63). Not only do ␤-arrestins play an important role in 7TM/GPCR internalization and desensitization, but also  D) and NFAT (G) activation is inhibited by co-stimulation of HEK-CB1 and HEK-GPR55ϩCB1 cells with 2.5 M AEA and 2.5 M SR141716A, but induced in HEK-GPR55 cells. pERK1/2 was normalized to total ERK1/2 and data are means of three independent experiments Ϯ S.E. Reporter gene assay data are mean Ϯ S.E. from one of four independent experiments performed in triplicates. Data were normalized and expressed as percent of maximum activation, which was set as 100%, *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001. DECEMBER 28, 2012 • VOLUME 287 • NUMBER 53 JOURNAL OF BIOLOGICAL CHEMISTRY 44243 often serve as signaling partners (50,51). One possible explanation for the lack of signaling response in cells overexpressing both CB1 and GPR55 receptors could be a lack of sufficient ␤-arrestin levels in our heterologous cell system. However, overexpression of ␤-arrestin 2 did not restore GPR55-mediated transcription factor activation or ERK1/2 phosphorylation in HEK-GPR55ϩCB1 cells (Fig. 8).

Cross-talk of GPR55 and CB1 Receptors
We next show that GPR55 and CB1 receptors physically interact in HEK-GPR55ϩCB1 cells in the absence of any ligand (Fig. 9A). However, we did not observe any altered internalization patterns following agonist activation of both receptors in double-expressing cells when compared with single-expressing cells (Fig. 9B).
Hence, when co-expressed, the GPR55-CB1 receptors alter each others signaling properties at the level of MAPKs and ensue transcription factor activation, i.e. when unstimulated, the CB1 receptor prevents the activation/signaling of GPR55. A similar mechanism has been reported for the opioid receptor system. The presence of the DOR decreases the activity of the MOR upon stimulation with selective MOR agonists (57). However, once activated, the CB1 receptor internalizes (Fig. 9B,  WIN), thereby restoring GPR55-mediated signaling when costimulated with the specific GPR55 agonist GSK319197A (Fig.  5, B, E, and H). Another interesting finding of our study is that CB1 receptor-mediated signaling is greatly amplified in the presence of GPR55.
A recent report on the CRTH2 and DP receptor heteromers (64) describes a similar cross-talk mechanism, i.e. the DP receptor is able to amplify a CRTH2-induced Ca 2ϩ release from intracellular stores and, coincidentally, loses its own signaling capacity. In addition, a recent study by Rozenfeld et al. (65) showed that heteromerization of CB1Rs and DOR affects receptor signaling. In fact, activation of the CB1 receptor results in a decrease in receptor signaling in the presence of DOR. Moreover, this decrease in signaling is associated with increased phospholipase C-dependent recruitment of ␤-arres- . For ERK1/2 phosphorylation (D) determination in the presence or absence of PTX, HEK-GPR55, HEK-CB1, and HEK-GPR55ϩCB1 cells were serum starved overnight, preincubated with vehicle or 100 ng/ml of PTX for 4 h, and stimulated with vehicle or 2.5 M GSK319197A for 25 min. ERK1/2 phosphorylation was not altered by PTX in HEK-GPR55 cells. No ERK1/2 activity was observed after vehicle treatment in all cell lines and stimulation with the GPR55 agonist GSK319197A in HEK-CB1 cells. HEK-GPR55ϩCB1 cells were preincubated with PTX showed decreased pERK1/2 when compared with vehicle preincubated double expressing cell line. Reporter gene assay data are mean Ϯ S.E. from one of three independent experiments performed in duplicates. Data were normalized and expressed as percent of maximum activation, which was set as 100% (A-C). Representative ERK1/2 blot from three independent experiments is shown. tin 2 to the heteromer (65). Another study describes a cross-talk of the CB2 receptor and GPR55 at the level of small GTPases in neutrophils (6). Here, co-stimulation of both, CB2R and GPR55, leads to a synergistic effect of neutrophil migration and polarization via the small GTPase cdc42. In contrast, the GPR55-mediated inhibition of Rac2 activation results in reduced CB2R-mediated reactive oxygen species production and myeloperoxidase release. GPR55 activation in neutrophils enhances migration to the site of inflammation, but prevents exaggerated tissue injury mediated by CB2 receptor activation (6). Another study in a hybridoma endothelial cell line suggested that integrin clustering was a prerequisite for the inhibition of GPR55-mediated signaling in the presence of the CB1 receptor (11).
In summary, GPR55 plays an important role in several physiological and pathophysiological processes (7), such as the Reporter gene assay data are mean Ϯ S.E. from one of three independent experiments performed in triplicates. Data were normalized and expressed as percent of maximum activation, which was set as 100% (A-C). ERK1/2 phosphorylation was not altered by overexpression of ␤-arrestin 2 (D). ERK1/2 phosphorylation in the presence or absence of ␤-arrestin 2 was determined in HEK-GPR55, HEK-CB1, and HEK-GPR55ϩCB1 cells that were serum starved overnight. Cells were then stimulated with vehicle or 2.5 M GSK319197A for 25 min. A representative ERK1/2 blot out of three independent experiments is shown. In parallel to reporter gene assays, overexpression of ␤-arrestin 2 was controlled by ELISA (E). ELISA data were normalized and are mean Ϯ S.E.