GPR55 has recently attracted much attention as another member of the cannabinoid family, potentially explaining physiological effects that are non-CB1/CB2 mediated. However, the data gathered so far are conflicting with respect to its pharmacology. We review the primary literature to date on GPR55, describing its discovery, structure, pharmacology and potential physiological functions. The CB1 receptor antagonist/inverse agonist AM251 has been shown to be a GPR55 agonist in all reports in which it was evaluated, as has the lysophospholipid, lysophosphatidylinositol (LPI). Whether GPR55 responds to the endocannabinoid ligands anandamide and 2-arachidonylglycerol and the phytocannabinoids, delta-9-tetrahydrocannabidiol and cannabidiol, is cell-type and tissue-dependent. GPR55 has been shown to utilize Gq, G12, or G13 for signal transduction; RhoA and phospholipase C are activated. Experiments with mice in which GPR55 has been inactivated reveal a role for this receptor in neuropathic and inflammatory pain as well as in bone physiology. Thus delineating the pharmacology of this receptor and the discovery of selective agonists and antagonists merits further study and could lead to new therapeutics.

4.1 Pharmacology in vitro- Transfected Cells

GPR55 has been examined in transfected HEK293 cells with a number of cannabinoid ligands. The results of these studies are quite mixed (please see Table 1for a summary of compounds reported in multiple studies). Ryberg et al (2007)used a GTPγS functional assay and found that hGPR55 stably transfected in HEK293s cells was activated by nanomolar concentrations of the endocannabinoids 2-arachidonylglycerol (2-AG), virodhamine (O-arachidonoyl ethanolamine), noladin ether (2-arachidonoyl glyceryl ether), oleoylethanolamide and palmitoylethanolamide (PEA). PEA had originally been suggested to be an endogenous ligand for the CB2 receptor (Facci et al., 1995), but subsequent studies found it had little affinity for CB2 (Griffin et al., 2000; Showalter et al., 1996). PEA is a potent anti-inflammatory, anti-excitotoxic and anti-hyperalgesic compound (Jaggar et al., 1998; Skaper et al., 1996), so finding the receptor(s) involved in its activity is of interest. Ryberg et al (Ryberg et al., 2007) also reported that the phytocannabinoid compound Δ9-THC as well as the structurally related compounds CP55940, HU210, O-1602 and abnormal-cannabidiol (abn-CBD) stimulated GTPγS binding. In addition, AM251, a pyrazole CB1 antagonist/inverse agonist, but not AM281, a highly related compound, acted as an agonist at GPR55 (Ryberg et al., 2007). CBD antagonized the effects of CP55,940 and anandamide with an IC50 of 440 nM. Cannabinol had no activity at GPR55, nor did WIN 55,212-2 (Ryberg et al., 2007).

Table 1

List of agonists and antagonists reported in multiple studies on GPR55. For a complete list of compounds see text.

Ryberg et al (2007) investigated downstream signaling pathways for hGPR55. The G protein involved was not pertussis toxin sensitive nor did they find evidence of Gq coupling using a FLIPR assay. Instead, they found using peptides and/or antibodies directed against the C-terminus of Gα subunits that Gα13 was responsible for the GTPγS activation produced by 1 µM O-1602, a maximally active concentration of this compound. Co-transfection with Gα13 augmented the signal produced. Consistent with a Gα12/13-mediated response, RhoA was activated, as was rac1 and cdc42, by 1 µM O-1602 or 1 µM anandamide; this effect was blocked by 10 µM cannabidiol.

Subsequent studies have reproduced only some of these data reported by (Ryberg et al., 2007). Johns et al (2007) reported that nanomolar concentrations of abn-CBD and O-1602 but not WIN 55,212-2 stimulated GTPγS binding in HEK293T cells transiently transfected with hGPR55. No other compounds were reported in this study, but the authors generated GPR55 KO animals in which the vasodilator effects of abn-CBD were retained and antagonized by O-1918. An “abn-CBD receptor” had previously been documented in several studies (reviewed in (Godlewski et al., 2009)); thus GPR55 does not appear to be that site.

In contrast, Oka et al (2007) found that numerous cannabinoid compounds (including 2-AG, anandamide, PEA, oleoylethanolamide, virodhamine, CP55940, HU-210, WIN55,212-2, THC, abn-CBD and SR141716A) had no effect in HEK293 cells stably transfected with a tetracycline-inducible human construct. Instead, lysophosphatidylinositol (LPI) induced phosphorylation of ERK1/2 in hGPR55-expressing cells in a concentration-dependent manner with an EC50 of 200 nM. LPI also induced a rapid Ca2+ transient in hGPR55-expressing cells with a similar EC50; this was attenuated by siRNA treatment. LPI stimulated GTPγS binding in a concentration-dependent manner; this EC50 appears to be in the high micromolar range (Oka et al., 2007). As LPI can be a mixture of fatty acids, a subsequent study by this group identified 2-arachidonoyl-sn-glycero-3-phosphoinositol (2-AGPI) as the most potent lipid activator of hGPR55 (Oka et al., 2009c). Very recently, they reported that LPI rapidly induced p38 MAP kinase with an EC50 value of ~300 nM and also the phosphorylation of activating transcription factor-2 (Oka et al., 2009a;Oka et al., 2009b).

Lauckner et al (2008) reported that 3–5 µM concentrations of Δ9-THC, the anandamide analog methanandamide, and the CB2 agonist JWH-015, increased intracellular calcium in HEK293 cells transiently transfected with hGPR55 as well as in large dorsal root ganglion (DRG) neurons from mice. HEK293 cells transiently transfected with mouse GPR55 produced a similar profile of responses, consistent with the data obtained from the DRG. Anandamide was a partial agonist, producing a 45 nM rise in intracellular calcium, as compared with a ~100 nM increase produced by THC, methananandamide and JWH-015. 3 µM LPI increased intracellular calcium in DRG neurons to a similar extent as the cannabinoid compounds. Co-addition (co-perfusion) of LPI with the CB1 receptor antagonist/inverse agonist SR141716A reduced the responses (in both hGPR55-HEK293 cells and mouse DRG) without having an effect on its own. Several other cannabinoids were evaluated and found to have negligible agonist activity in hGPR55-expressing HEK293 cells, including 2-AG, CP55940, PEA, virodhamine, abn-CBD, CBD and WIN55,212-2. Using a combination of inhibitors and dominant negative constructs, the calcium response was found be mediated by both Gq- and G12- mediated pathways and was dependent on an intact actin cytoskeleton. Activation of hGPR55 by 5 µM THC resulted in a 38% inhibition of a (Gq and PIP2-requiring) M-type potassium current; interestingly, 5 µM THC caused a small (11%) inhibition of this current in cells that were not transfected with hGPR55. These results differ from Ryberg et al, both in terms of the ligands that activate hGPR55 as well as the Gq coupling observed; however, both report RhoA-dependent pathways (Lauckner et al., 2008; Ryberg et al., 2007).

Other studies indicate that LPI and the rimonabant-like CB1 inverse agonist AM251 induce oscillatory Ca2+ release through Gα12/13 and RhoA in HEK293 cells stably transfected with hGPR55 (Henstridge et al., 2009b). In contrast to the studies cited above (Lauckner et al., 2008; Oka et al., 2007; Oka et al., 2009c), where single Ca2+transients were measured, Henstridge et al (2009b) report a concentration-dependent induction of oscillatory Ca2+ transients by LPI which persisted for up to 45 min after agonist removal. The EC50 of LPI was calculated to be 49 nM, and 1 µM LPI increased intracellular Ca2+ levels by 929 nM above basal levels. This signaling was shown to be mediated by Gα12/13 through a RhoA-Rho kinase (ROCK)-phospholipase C-dependent release of intracellular Ca2+ from the endoplasmic reticulum, which subsequently activated NFAT and led to its nuclear translocation. 1 µM LPI also induced internalization and translocation into intracellular vesicles. While the endocannabinoids anandamide and 2-AG increased intracellular Ca2+ levels, this was a GPR55-independent effect; i.e., the response to 3–30 µM of these compounds was the same in untransfected and in hGPR55-expressing cells. However, a concentration-dependent, hGPR55-mediated increase in intracellular Ca2+ was observed with the CB1 antagonist/inverse agonist AM251 with an EC50 of 612 nM and a lower maximal Ca2+ response (586 nM) as compared with LPI. CP55,940 did not activate hGPR55, but shifted the concentration-response curve for LPI to the right, indicating that CP55,940 acts as a competitive antagonist in this assay.

These reports were all performed in HEK 293 cells, yet each documented a distinct and conflicting chemical space of agonists that recognized GPR55. To attempt to resolve these inconsistencies in classification, Kapur et al (2009) employed an alternative approach for identifying GPR55 ligands using β-arrestin recruitment. β-arrestins are intracellular proteins that bind and desensitize activated GPCRs and in the process form stable receptor/arrestin signaling complexes (Gurevich and Gurevich, 2006; Shenoy and Lefkowitz, 2005). β-arrestin-green fluorescent chimeras can make this process attractive to monitor by forming remarkably sensitive and specific probes of GPCR activation that are independent of downstream G protein mediated signaling (Barak et al., 1997; Marion et al., 2006;McGuinness et al., 2009). Kapur et al (2009) determined hGPR55 responsiveness to a representative panel of cannabinoid ligands and LPI in the presence (and absence) of a β-arrestin2-green fluorescent protein (βarr2-GFP) biosensor in U2OS cells stably transfected with hGPR55E and in HEK293 cells transiently transfected with hGPR55E or hGPR55. The hGPR55E construct used in these studies contains a serine enhanced C-terminus to increase receptor affinity for β-arrestin without changing its response profile to ligands (Kapur et al., 2009; Oakley et al., 1999). Out of numerous cannabinoid compounds tested only two unambiguously activated hGPR55 in addition to the lysophospholipid LPI (Table 1). These compounds, LPI, SR141716A and AM251 had a rank order of potency of LPI> SR141716A> AM251 (1.2, 3.9 and 9.6 µM) and comparable efficacies. SR141716A and AM251 are cannabinoid receptor inverse agonist/antagonists (Kapur et al., 2008; Lan et al., 1999) whereas the one hGPR55 receptor antagonist identified, CP55,940 (Ki ~200 nM), is a cannabinoid receptor agonist. In contrast, AM281, which is structurally related to biarylpyrazole analogs (SR141716A and AM251), failed to activate hGPR55 at concentrations up to 30 µM (in agreement with Ryberg et al, 2007). LPI did not activate CB1 receptors in a CB1-βarr2-GFP-expressing cell line, whereas CP55,940 produced the expected response (Kapur et al., 2009). In addition to measuring βarr2-GFP trafficking, agonist-induced internalization, ERK phosphorylation and activation of PKCβII-GFP, a G-protein dependent response, were assessed (Kapur et al., 2009). These studies confirmed that the endogenous compound LPI is unequivocally a GPR55 agonist at low micromolar concentrations causing β-arrestin activation, receptor internalization, activation of PKCβII and ERK1/2 phosphorylation.

JWH015 has been shown to increase intracellular Ca2+ (Lauckner et al., 2008). However, Kapur et al. (2009) found that JWH015 (CB2 receptor agonist), SR144528 (CB2 receptor antagonist), the classical CB1 agonists (HU210 and THC), the endocannabinoids (anandamide and 2-AG), and cannabidiol had no effect on their own or on LPI-induced βarr2 trafficking in hGPR55 U2OS cells. Kapur et al (2009) also demonstrated that the endocannabinoids (anandamide and 2-AG) and atypical cannabinoids (abn-CBD, O-1602 and O-1918) failed to evoke hGPR55-modulated βarr2-GFP redistribution. These compounds had previously been reported to activate GTPγS binding (Johns et al., 2007; Ryberg et al., 2007).Lauckner et al (2008) reported that SR141716A is a GPR55 antagonist at 2 µM in a calcium signaling assay. In contrast, Kapur et al (2009) showed that 10–30 µM SR141716A produces robust activation and internalization of hGPR55. This discrepancy may be a reflection of the different range of doses and efficacy of the compounds that were utilized. Henstridge et al (2009a) reported that SR14716A was an agonist at GPR55 at 1 µM. That SR141716A (rimonabant) activates GPR55 at µM concentrations may be clinically relevant, as peak plasma levels or rimonabant were typically in the ~500 nM range. Rimonabant had been marketed for the treatment of obesity and off-target effects of this and related compounds may be manifest at GPR55.

The profile of ligands inducing agonist-induced internalization confirmed those for βarr2-GFP trafficking (Kapur et al., 2009). CP55,940 also antagonized agonist-induced internalization. Furthermore, LPI, AM251 and SR141716A were agonists in the G-protein dependent response, activation of PKCβII-GFP. Within 60 seconds of addition of agonists, membrane rearrangements began to occur followed by protrusions and blebbing. Cytoskeletal changes were also observed in the β-arrestin trafficking assay. However, CP55,940 acted as a partial agonist in the PKCβII-GFP response, inducing some recruitment of PKCβII-GFP. There was also some degree of constitutive activity in this assay, whereby HEK293 cells co-transfected with hGPR55 and PKCβII-GFP displayed some membrane fluorescence as compared to cells transfected with PKCβII-GFP alone. Thus CP55,940 acts as a partial agonist/antagonist at hGPR55 in these studies (Kapur et al., 2009).

A similar set of ligands that activate hGPR55 stably transfected in HEK293 cells to that of Kapur et al (2009) was recently reported at the 2009 International Cannabinoid Research Symposium (Balenga et al., 2009; Henstridge et al., 2009a).Henstridge et al (2009a) showed that AM251 and SR141716A stimulate Ca2+ release with EC50‘s of ~1 µM and receptor internalization; similar to what they had previously reported with LPI (Henstridge et al., 2009b). However, in these cells, AM281 produced agonist responses, albeit at concentrations 10X higher than those for AM251, SR141716A and LPI. Furthermore, they found robust ERK1/2 MAPK activation with LPI whereas AM251, SR141716A and AM281 produced much less activation of ERK1/2. Another report used the same cell line to examine transcription factor activation and showed that LPI and AM251 were efficacious activators of NFAT and NFκB at µM concentrations, whereas AM281 was most efficacious for CREB activation (Balenga et al., 2009), suggesting biased agonism for activation of downstream pathways.

Another recently published study characterizing GPR55 activation using a related technology (β-arrestin, PathHunter) demonstrated responses to AM251, SR141716A and LPI in a HEK293 cell line transiently expressing hGPR55 (Yin et al., 2009). The PathHunter technology uses enzyme (β-galactosidase) complementation of a tagged receptor with β-arrestin2 to measure β-arrestin2 recruitment (McGuinness et al., 2009). Yin et al (2009), from the GPCR Platform, Genomics Institute of the Novartis Research Foundation, also used a GPR55 reporter gene assay to evaluate potential ligands. The pGL3 basic vector was modified to have three repeats of multiple response element consensus sequence (MRE), a cAMP-response element from a vasoactive intestinal peptide gene promoter (CRE), a serum-response element promoter (SRE) before the luciferase reporter gene (Yin et al., 2009). The pGL3-CRE-MRE-SRE-luciferase reporter gene was transiently transfected into a HEK cell line stably transfected with hGPR55 and test compounds applied for 16–24 hours. The same general ligand profile for agonist activation of hGPR55 was seen with the luciferase reporter gene and β-arrestin assays. AM251 was the most potent and efficacious compound tested, with an EC50 of ~ 3 µM. LPI had similar potency (EC50 of 3.6 µM) and slightly less efficacy. SR141716A was less potent (EC50 of 10.9 µM) and also slightly less efficacious than AM251. Several cannabinoid compounds tested exhibited no agonist activity in either assay including CP55,940, WIN55,212-2, HU210, SR144528, AM630, Abn-CBD and O-1602. THC and anandamide showed a small amount of activity (12%) in the β-arrestin assay and 2-AG showed some activity in the reporter gene assay, consistent with Ryberg et al (2007). The authors state that the effects of THC and anandamide were enhanced to a more significant level when 5 µM AM251 was co-applied, so rather than acting as partial agonists they may have allosteric interactions in this system (Yin et al., 2009). β-arrestin redistribution assays can recognize agonists, antagonists, and allosteric modulators as demonstrated in a recent study with the CB1 cannabinoid receptor (van der Lee et al., 2009).

4.2 Pharmacology in vitro-endogenous cell lines and tissue

Most of the studies published have tried to characterize the pharmacological pattern of GPR55 in HEK293 transfected cells. To get a better understanding of the pharmacological and functional identity of GPR55, it is important to examine its functionality in its “natural” environment, i.e., in endogenous systems.

4.3 Pharmacology in vivo

To date, the conflicting data regarding GPR55 agonists and antagonists makes it difficult to conduct in vivo pharmacological studies. However, studies from GPR55 knockout mice may shed some light on its physiological relevance in vivo.

Whyte et al (2009) found that GPR55 affects osteoclast and osteoblast differentiation to influence bone mass. In this study, receptor knockout mice were generated by deletion of the entire coding region of GPR55 by homologous recombination. The GPR55−/− mice were backcrossed for 6 generations onto the C57Bl/6 background; background differences have previously been shown to be important for bone morphological studies with the CB1 receptor (Bab and Zimmer, 2008). Male GPR55−/− mice have increased numbers of morphologically inactive osteoclasts resulting in a significant increase in the volume and thickness of trabecular bone and the presence of unresorbed cartilage. Treatment of male mice for 8 weeks with 10 mg/kg CBD (3 times per week) significantly decreased the level of serum type 1 collagen C-terminal telopeptide fragments, a biochemical marker of bone resorption, by 18%. Microtomographic analysis of the proximal tibiae also revealed a trend toward increased bone volume/tissue volume (+ 10%) and trabecular number (+ 10%), with a decrease in trabecular separation (− 7%), trabecular pattern factor (− 5%), and structure modulus index (− 8%) in the tibia of the CBD-treated mice relative to control. These findings were consistent with a decrease in bone resorption in CBD-treated mice, although these changes were not statistically significant over this treatment schedule (Whyte et al., 2009). CBD acted as a GPR55 antagonist in osteoclast cultures, although the effects of CBD on osteoclasts prepared from GPR55−/− mice were not reported. Nonetheless, these studies indicate a role for GPR55 in bone resorption, and indicate that CBD and GPR55 antagonists may be useful for the treatment of osteoporosis.

Staton et al (2008) reported that GPR55 −/− mice (also backcrossed to a C57Bl/6 background), possessed no overt phenotype, but were protected in models of inflammatory and neuropathic pain. The strategy for targeting the gene in these animals left intact the first 118 bp of GPR55 as well as the distal portion of the sequence (beyond lysine 281). As this latter sequence contains several methionines, it is possible that if it is transcribed it will be translated. Staton et al (2008) reported that no GPR55 mRNA could be detected by RT-PCR in the animals. Also the levels of CB1 and CB2 mRNA were unchanged in the brain and spleen, respectively, indicating that compensatory changes in these two receptors did not occur in the mice. Female GPR55−/− did display a reduction in withdrawal latency in an acute pain model, the hot water tail flick latency test, when tested at 50 °C, but not at higher temperatures (52.5 °C, 55 °C. A more robust difference was seen in two models of hyperalgesia. In mechanical hyperalgesia induced by intraplantar administration of Freund’s complete adjuvant (FCA), inflammatory mechanical hyperalgesia was completely absent in both male GPR55 KO mice up to 14 days post-injection. Female GPR55−/− mice were protected for one day post-FCA. In the partial nerve ligation model of neuropathic hypersensitivity, GPR55−/− mice of both sexes failed to develop mechanical hyperalgesia up to 28 days post-ligation.

Cytokine profiling experiments showed increased levels of IL-4, IL-10, IFN γ and GM-CSF in paws from the FCA-injected GPR55−/− mice when compared with the FCA-injected GPR55 wild-type mice (at 1 and 14 days post-injection for the female and male mice, respectively). This suggests that GPR55 signaling can influence the regulation of certain cytokines and this may contribute to the lack of inflammatory mechanical hyperalgesia in the GPR55 −/− mice. These data suggest that GPR55 antagonists may have therapeutic potential in the treatment of both inflammatory and neuropathic pain.

Allostery/Biased Agonism/Functional Selectivity

The most parsimonious explanation of this apparent disagreement between reports (Johns et al., 2007; Kapur et al., 2009; Lauckner et al., 2008; Oka et al., 2007;Ryberg et al., 2007; Yin et al., 2009) is that distinct conformations of the receptor resulting from the binding of different ligands might couple differentially and/or to multiple downstream effectors (Hudson et al., 2009; Kenakin, 2009; Ross, 2009;Violin and Lefkowitz, 2007). Indeed, the primary action of drugs on GPCRs is to promote a conformational change in tertiary conformation, an obviously complex outcome (Kenakin, 2009). Although functional selectivity (biased agonism) is treated separately from allosterism, it can be seen as a vectorial manisfestation of the general allosteric nature of GPCRs (Kenakin, 2009).

For GPR55, a common final pathway that has been reported in most but not all papers is the activation of MAPK (ERK1/2). GPCR activation can result in ERK1/2 phosphorylation by G-protein and G-protein-independent pathways (Lefkowitz and Shenoy, 2005). The observation that LPI alone induces a significant activation of ERK1/2 in cells expressing hGPR55 while cannabinoids (AM251, SR141716A, anandamide and 2-AG) either produced no (Kapur et al., 2009) or little (Henstridge et al., 2009a) response are mostly in agreement with Oka et al (2007). However, this divergence in efficacy between signaling pathways (βarrestin trafficking, Ca2+ signaling, etc., vs. ERK activation) suggests that LPI and cannabinoids display functional selectivity (Gonzalez-Maeso and Sealfon, 2009;Hudson et al., 2009; Kenakin, 2009; Violin and Lefkowitz, 2007).

Biased agonism is also suggested by the studies assessing transcription factor activation, where AM281 was the most efficacious agent activating CREB, whereas LPI and AM251 were the most efficacious at activating NFAT (and NFκB) (Balenga et al., 2009). Biased agonism may also explain the ability of CP55940 to act as a partial agonist in PKCβII-GFP activation and an antagonist in the βarr2-GFP trafficking assays (Kapur et al., 2009), although partial agonists can act as antagonists depending on the signal strength (Kenakin, 2007).

Cell line and tissue heterogeneity

It is well known that cell lines present inconsistent phenotype over time. For example, Dubi et al. (2008) have recently demonstrated that the androgen insensitive PC-3 cell line exhibited two sublines that showed distinct receptor activation. Moreover, the clonal background of HEK293 cells can differ markedly between laboratories (Henstridge et al., 2009b). In addition, Ryberg et al (2007)used a HEK293s cell line, Johns et al (2007) used a HEK293T cell line, and the HEK293 cells in other papers were from different laboratories (Henstridge et al., 2009b; Kapur et al., 2009; Lauckner et al., 2008; Oka et al., 2007). Indeed, although HEK293 cells are referred to as “human embryonic kidney” cells, a study on the origin of the line suggests they may instead be derived by adenoviral transformation of a neuronal precursor present in the human embryonic kidney cell cultures (Shaw et al., 2002).

The response and signaling pathways, initiated by the same agonist, might differ among different tissues. One example comes from GPR39, which also belongs to the Class A GPCRs, recently identified as an extracellular zinc-sensing receptor (Yasuda et al., 2007). Activation of the zinc-sensing receptor leads to intracellular Ca2+release with Km values of 55 µM, 146 µM and 200 µM in salivary gland, brain and prostate cells, respectively (Besser et al., 2009; Dubi et al., 2008; Sharir and Hershfinkel, 2005). Moreover, extracellular Ca2+ had been demonstrated to allosterically modulate the receptor’s affinity (Sharir and Hershfinkel, 2005). Activation of the zinc-sensing receptor also results in phosphorylation of ERK1/2. In PC-3 cells activation of the receptor resulted in sustained ERK1/2 phosphorylation, for at least 3 hr (Dubi et al., 2008). In HT-29 cells, however, ERK1/2 phosphorylation peaked at 30 min following ligand application, and approached resting levels after 2 hr (Azriel-Tamir et al., 2004).

Receptor over-expression

Many of the in vitro studies using transfected cells were over-expressing GPR55 (Henstridge et al., 2009b; Johns et al., 2007; Kapur et al., 2009; Lauckner et al., 2008). If overexpression of the receptor induces constitutive activity, this can lead to altered behavior of ligands (Kenakin, 2001). An additional difference that arises when considering transient versus stable expression systems is that in a cell stably expressing a protein it is necessary that the protein be expressed at levels where it is not detrimental to the health and division of the cell (otherwise the cell does not survive the selection process). This might involve lower levels of expression, or a matching of expression of proteins that might ameliorate some of the toxicity of the expressed proteins. Additionally, there may also be investigator-initiated selection bias in stable lines where a line exhibiting a more reproducible response is chosen for future studies. These factors may also explain differences between the results of studies conducted using stable versus transient expression.

Modulation of the GPR55 response by other proteins or factors in the cell/cell media

Because of cell line and tissue heterogeneity, there may be proteins in the various cell lines that modify the response of GPR55. The differential signaling of anandamide due to integrin clustering in endothelial cells and CB1 activation is one published example (Waldeck-Weiermair et al., 2008). Another example is that interaction with other GPCRs alters the pharmacology of CB1 cannabinoid receptors (recently reviewed by (Hudson et al., 2009)).

The differential response of GPR55 in different laboratories may also be dependent on cell culture conditions. The presence of endocannabinoids in serum has been documented (Valk et al., 1997) and other growth factors are present as well. In addition, HEK293 and other cells can synthesize endocannabinoids and this may alter the measured response (Turu et al., 2009).

Phospholipase C Activation

LPI is synthesized by phospholipase A-mediated removal of acyl moieties of phosphatidylinostol. In this regard, it is similar to 2-AG which can be generated by phospholipase C or diacylglycerol lipase (PLC & DAG). One hypothesis that has been put forward is that cannabinoids acting at CB1 or CB2 receptors or a “receptor X” generate LPI that then acts at GPR55 (De Petrocellis and Di Marzo, 2009). One would have to invoke the “other receptor” to explain the CB1 receptor antagonists/inverse agonists SR141716A and AM251’s agonist activity at GPR55 and the antagonism of LPI’s effects by CP55,940. Thus, it seems more likely that these are direct actions at GPR55. However, since near micromolar concentrations of these compounds are generally reported for these compounds it is not possible to conduct radioligand binding assays. Thus, this possibility cannot be eliminated at this time.

This work was supported from NIH/NIDA – DA023204 and DA05274. The authors would like to acknowledge Dr. Patricia H. Reggio, Dow P. Hurst and Evangelia Kotsikorou from UNC-Greensboro for their input into structural features of GPR55 and its ligands. We wish to thank Jahan Marcu and Pingwei Zhao, members of the Abood laboratory, for their critical reading during the preparation of this manuscript.

2-AG

2-arachidonylglycerol

2-AGPI

2-arachidonoyl-sn-glycero-3-phosphoinositol

e-aR

anandamide endothelial receptor

abn-CBD

abnormal cannabidiol

βarr2-GFP

β-arrestin2-green fluorescent protein

CBD

cannabidiol

Δ9-THC

delta-9-tetrahydrocannabidiol

DRG

dorsal root ganglion

ERK

extracellular signal-regulated kinase

LPI

lysophosphatidylinositol

PEA

palmitoylethanolamide

PKCβII-GFP

Protein kinase C betaII-green fluorescent protein

ROCK

Rho A-associated kinase

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