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Home , Cannabinoid receptor, GPR55, Lysophosphatidylinositol, Orphan receptor

J Basic Clin Physiol Pharmacol 2016; 27(3): 297–302

Review

Hyewon Yang, Juan Zhou and Christian Lehmann* GPR55 – a putative “type 3” in

DOI 10.1515/jbcpp-2015-0080 Clinical studies using and Received July 6, 2015; accepted October 18, 2015; previously published antagonists have suggested their possible use in modu- online December 15, 2015 lating acute and/or chronic diseases, e.g. hypertension, glaucoma, emesis, anxiety, depression, gastrointestinal Abstract: -coupled receptor 55 (GPR55) shares and hepatic disorders, ulcerative colitis and neuropathic numerous cannabinoid ligands with CB1 and CB2 recep- and inflammatory [2, 3]. tors despite low homology with those classical cannabi- The evidence for homeostatic and protective functions noid receptors. The pharmacology of GPR55 is not yet of endocannabinoids in inflammation encourages studies fully elucidated; however, GPR55 utilizes a different sign- of ECS as a therapeutic target in sepsis. Further elucida- aling system and downstream cascade associated with tion of endocannabinoid signaling pathways will aid in the receptor. Therefore, GPR55 has emerged as a putative the design of compounds with site specificity and high “type 3” cannabinoid receptor, establishing a novel class affinity to various receptor targets [4, 5]. In this review, of cannabinoid receptor. Furthermore, the recent evidence current knowledge of the expression and function of the of GPR55-CB1 and GPR55-CB2 heteromerization along orphan G protein-coupled receptor 55 (GPR55), a putative with its broad distribution from “CB3” receptor, is summarized, highlighting its potential to peripheries suggests the importance of GPR55 in vari- to be a therapeutic target in inflammation. ous cellular processes and pathologies and as a potential therapeutic target in inflammation.

Keywords: CB2; ; GPR55; immune modulation; inflammation; sepsis. Endocannabinoid system

The marijuana plant, sativa, has been used for more than 4000 years for a variety of purposes from Introduction medicine to recreation owing to their psychoactive prop- erties. There are over 80 active phytocannabinoids in the The endocannabinoid system (ECS) has a tremendous marijuana plant, which exert their effects on the central potential for experimental and clinical research, based on and peripheral nervous system by binding and activat- their ubiquitary presence in vertebrates and role in funda- ing specific receptors in the [6]. The most mental physiological and pathological processes, includ- prominent and best-investigated phytocannabinoids from ing pain modulation, immune function, neuroprotection, C. sativa are Δ9-tetrahydrocannabidiol (THC) and canna- cancer, cardiovascular diseases, fertility and appetite [1]. bidiol [7]. Although these phytocannabinoids have been studied extensively for their psychoactive properties, their *Corresponding author: Christian Lehmann, Department of potential use as therapeutic agents have been under- Anesthesia, Pain Management and Perioperative Medicine, mined until the discovery of the ECS in the early 1990s [6]. Dalhousie University, QE II Health Sciences Centre, 10 West Victoria, 1276 South Park Street, Halifax, Nova Scotia B3H 2Y9, Canada, The ECS consists of three components: endogenous Phone: +19024944454, E-mail: [email protected]; -signaling molecules (endocanabinoids), G protein- Department of Microbiology and Immunology, Dalhousie University, coupled cannabinoid receptors and the for Halifax, Nova Scotia, Canada; and Department of Pharmacology, biosynthesis and degradation. Endocannabinoids are Dalhousie University, Halifax, Nova Scotia, Canada endogenous agonists of cannabinoid receptors and are Hyewon Yang and Juan Zhou: Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada; found to mimic the pharmacological actions of the phyto- and Department of Anesthesia, Pain Management and Perioperative [6, 8]. The first identified endocannabinoid Medicine, Dalhousie University, Halifax, Nova Scotia, Canada was N-arachidonyl ethanolamine (anadamide or AEA) in 298 Yang et al.: GPR55 in inflammation

1992, followed by 2-arachidonyl (2-AG) in 1995, which are (HU-210), CP55940, and R-(+)-WIN55212 are able to acti- the best-studied endocannabinoids to date [9, 10]. vate both CB1 and CB2 receptors [6]. Biosynthesis of endocannabinoids takes place on demand either by activity-dependent or receptor­- stimulated cleavage of arachidonic acid in the cell membrane, which activates enzymes including N-acyl-­ GPR55 expression and signaling phosphatidylethanol- amine-hydrolyzing In the last decade, accumulating evidence from studies D for AEA synthesis and two isoforms of diacylglycerol using either selective CB1 and CB2 ligands or CB1 and/ lipases (DAGLα and DAGLβ) for 2-AG synthesis [11, 12]. or CB2 knockout mice has suggested the existence of one Because of the lipophilic nature of endocannabinoids, or more additional cannabinoid receptors distinct from they are not suitable for storage in vesicles and thus are CB1 and CB2 receptors [23]. Recently, the orphan GPR55 released immediately after their production. Followed by showed binding with some cannabinoids and non-­ the biosynthesis, these lipid transmitters then locally acti- cannabinoid ligands and therefore presented as the main vate cannabinoid receptors in response to different physi- candidate to be considered as the “third” cannabinoid ological and pathological stimuli [5]. receptor [24]. Endocannabinoids are degradated by enzymatic In 1999, Sawzdargo et al. first isolated and cloned hydrolysis mediated by specific intracellular enzymes to human GPR55 (hGPR55) with high expression in the limit excessive endocannabinoid signaling. The major regions of the CNS including the , caudate, degradation enzymes responsible for these reactions putamen, hypothalamus, cerebellum, thalamus and have been identified as amide hydrolase (FAAH) pons. It is also expressed in peripheral tissues such as and (MAGL), for AEA and 2-AG, the endothelial cells, adrenal glands and gastrointesti- respectively [13, 14]. To inhibit the degradation of endo- nal tract [24, 25]. More recently, high expression of GPR55 cannabinoids, inhibitors are developed. The most is also found on lymphocytes and spleen, as well as on well-characterized inhibitors of degradation enzymes are many cancer cells, which correlates with the rate of cancer URB597 and JZL184 for inhibiting FAAH and MAGL degra- cell proliferation [26, 27]. Although GPR55 shares several dation, respectively [15, 16]. cannabinoid ligands with CB1 and CB2, GPR55 exhibits a The cannabinoid receptors are members of seven- low identity to CB1 (13.5%) and CB2 (14.4%). transmembrane spanning class A G protein-coupled However, GPR55 is clearly a member of class A GPCRs, receptor (GPCR) subfamily. In the late 1980s and early based on amino acid homology with P2Y5 purinergic 1990s, two high-affinity cannabinoid receptors that bind receptor (29%), GPR23 (30%) and GPR35 (27%) [25]. and 2-AG have been identified by molecular The endocannabinoid receptor signaling pathways cloning: cannabinoid receptor type 1 and type 2 (CB1 and are not yet fully elucidated. The classical CB1 and CB2 CB2) [17–19]. CB1 receptors are predominantly expressed signaling are known to utilize primarily Gα heteromeric in the central nervous system (CNS) and mediate most i/o proteins and adenylate cyclase to activate multiple differ- psychoactive effects, whereas CB2 receptors are mainly ent downstream cascades. However, it is currently found distributed in peripheral and immune cells, mediat- that the pathway of GPR55 differs from ing immunosuppressive effects [19–21]. When activated that of CB1 and CB2. It has been found that GPR55 only by endocannabinoids, both CB1 and CB2 receptors are couples to Gα proteins, thereby activating ras homo- coupled to G heteromeric G protein and thereby inhibit 12,13 i/o logue family member A (RhoA) and Rho-associated , activate mitogen-activated protein protein kinase (ROCK). Activation of RhoA and ROCK kinases and further turn on numerous transcription elicits (PLC) pathway to increase intra- factors [5]. cellular Ca2+, activates small GTPase proteins rhoA, Rac The discovery of cannabinoid receptors is followed by and cdc42, and turns on extracellular regulated kinase development of selective CB1 and CB2 receptor agonists (ERK) phosphorylation (Figure 1) [24, 28, 29]. and antagonists [22]. The best known of CB1-selective agonists include R-(+)-, arachidonyl- 2′-chloroethylamide, arachidonyl-cyclopropylamide and O-1812, and antagonists are AM251 and AM281. As for CB2- GPR55 pharmacology selective receptors, there are JWH133, HU308 and AM1241 for agonists and SR144528 and AM630 for antagonists [6]. Despite the low homology with the classical cannabinoid Compounds such as (–) 11-hydroxy-Δ8-THC-dimethylheptyl receptors (CB1 and CB2), several cannabinoids are found Yang et al.: GPR55 in inflammation 299

Figure 1: Overview of the GPR55 signaling pathway by LPI.

Extracellular LPI can bind and activate GPR55. Activation of GPR55 is coupled to Gα12,13 proteins, thereby activating ras homologue gene family member A (RhoA) and ROCK. ROCK then turns on PLC pathway, which allows cytosolic concentration of Ca2+ to increase. Increased intracellular Ca2+ in turn leads to ERK phosphorylation and [24, 28, 29].

to interact with GPR55, including the cannabinoid , heteromers with CB2 receptor in cancer cells is Δ9-THC, CB1-selective antagonist/inverse agonist rimona- concentration dependent [37]. Furthermore, GPR55-CB2 bant and CB1 and CB2 agonist CP55, 940 [30]. It has been heteromers are shown to elicit different pathways via shown that 1- (LPI), O-1602 and ligand- and concentration-specific crosstalk (Figure 2) AM251 have agonist effects for GPR55, whereas SR141716A [38]. Overall, GPR55 heteromers with either CB1 or CB2 (), O-1918 and (CBD) have antago- exhibit function as novel signaling entities [36, 37]. nist effects [31–33]. However, there is controversial evi- dence regarding the opposite or divergent pharmacological effects through GPR55 compared to CB receptors [34]. For instance, anandamide and 2-AG are reported to activate GPR55, while other groups failed to detect the agonist effect by these cannabinoids [29, 34, 35]. Most recent studies on receptor coupling and heter- omer formation present an explanation of the aforemen- tioned controversial findings on GPR55 ligands [33, 36, 37]. Inconsistent activities of GPR55 is observed in response to cannabinoid ligands, influenced by the assay used to assess receptor function [33]. With that regard, a novel label-free assay is evaluated and successfully detected agonist activity for all GPR55 ligands tested [33]. Further- more, it is reported that CB1 and other class A GPCRs could form homomers and heteromers, which alter the biochem- ical property of the receptors. Naturally co-expressed receptors are present in several cell types. For example, GPR55 and CB2 are co-expressed on neutrophils and Figure 2: Schematic of signaling modulation of GPR55-CB2 cancer cells [37], and GPR55 and CB1 are co-expressed in heteromer. several brain regions [36]. In these co-expression assays, (A) CB2 signaling pathway is inhibited in the heteromer when both GPR55 and CB2 are activated by the agonists (negative crosstalk). GPR55-mediated signaling is inhibited with inactive CB1; (B) GPR55 antagonist inhibits CB2 agonist-induced heteromer in contrast, CB1-mediated signaling is enhanced with activation (left) or CB2 antagonist inhibits GPR55 agonist-induced GPR55 [36]. On the other hand, the activation of GPR55 heteromer activation (right, cross antagonism) [37, 38]. 300 Yang et al.: GPR55 in inflammation

GPR55 in inflammation remark on the potential role of GPR55 in innate immu- nity modulation and inflammation. Therefore, GPR55 is a novel candidate for treatment of inflammatory diseases. GPR55 is widely distributed throughout the body, and its role has been suggested to be involved in many physiolog- ical and pathophysiological processes, including the role in pathophysiology of the gut [39–41], inflammatory and Conclusions [42] and modulation of innate and adap- tive [43–45]. The ECS is under investigation as an attractive target of As GPR55 is expressed throughout the rodent gastroin- drug development for inflammatory diseases. Cannabi- testinal tract, many gastrointestinal disease models were noid receptor expression in different tissues and cells, adopted to reveal the role of GRP55 in intestinal inflam- along with their high affinity to bind cannabinoid ligands, mation. For example, increased expression of GPR55 is contributes to their ability to modulate numerous cellular observed in lipopolysaccharide (LPS)-treated intestine processes and pathologies. [41] in vivo suggesting GPR55 involvement in intestinal GPR55, a putative “type 3” cannabinoid receptor, trig- inflammation. In addition, GPR55 knockout mice showed gers distinct signaling pathways in response to inflam- less severe colitis compared to CB1 or CB2 knockout mice, matory mediators. Although recent studies have rapidly suggesting pro-inflammatory role of GPR55 in experi- expanded G protein-coupled receptor pharmacology and mental colitis induced by dextran sulfate sodium [39]. pathophysiology, more research for selective agonists and In another experimental colitis model, an endogenous antagonists is required to resolve the controversial phar- anandamide-related lipid, (PEA), macology of GPR55. In addition, biochemical insights in is shown to reduce neuro-inflammation and chronic pain the receptor coupling and in vivo studies in knock-out in the colitis induced by intracolonic administration of mice will aid to further elucidate GPR55 mechanism of dinitrobenzenesulfonic acid [46]. In this study, mRNA action and to develop specific GPR55 agonists and antago- expression of GPR55 is significantly increased compared nists for the use in inflammation. to CB2 when PEA is administered [46]. Furthermore, in a mechanical hyperalgesia model using Freund’s complete Author contributions: All the authors have accepted adjuvant, inflammation and neuropathic hypersensitiv- responsibility for the entire content of this submitted ity are absent in GPR55 knockout mice, suggesting a pro- manuscript and approved submission. inflammatory role of GPR55 [42]. Research funding: None declared. While the role of GPR55 was not fully elucidated, Employment or leadership: None declared. CB2R has been thought to be responsible for activation Honorarium: None declared. and recruitment of neutrophils as well as regulation of Competing interests: The funding organization(s) played other immune cells. Recently, Balenga and colleagues no role in the study design; in the collection, analysis, and found that GPR55 is involved in neutrophil chemotaxis interpretation of data; in the writing of the report; or in the and recruitment via crosstalk with CB2R orchestrated decision to submit the report for publication. by various chemoattractants in downstream signaling pathway [43]. The interplay between GPR55 and CB2R during inflammation enhances neutrophil migration effi- References ciency and revokes degranulation and ROS formation in neutrophils [43]. Moreover, GPR55 found on mast cells 1. Ligresti A, Petrosino S, Di Marzo V. From endocannabinoid exerts its anti-inflammatory effect by inhibiting mast cell- profiling to endocannabinoid therapeutics. Curr Opin Chem Biol 2009;13:321–31. mediated release of growth factor and attenuating 2. Di Marzo V. The endocannabinoid system: its general strategy of angiogenesis [44]. Lastly, it has been reported that GPR55 action, tools for its pharmacological manipulation and potential is highly expressed on monocytes and natural killer (NK) therapeutic exploitation. Pharmacol Res 2009;60:77–84. cells, compared to several innate and adaptive immune 3. McPartland JM, Guy GW, Di Marzo V. Care and feeding of the cells from human peripheral blood mononuclear cells endocannabinoid system: a systematic review of potential clini- [45]. Activation of LPS-activated monocytes and NK cells cal interventions that upregulate the endocannabinoid system. PLoS One 2014;9:e89566. by GPR55 shows increase in pro-inflammatory cytokines, 4. Pacher P, Batkai S, Kunos G. The endocannabinoid system cytolytic activity of NK cells and decrease in endocytic as an emerging target of pharmacotherapy. Pharmacol Rev activity of monocytes [45]. Altogether, these evidences 2006;58:389–462. Yang et al.: GPR55 in inflammation 301

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