CB2 Cannabinoid Receptors: Molecular, Signaling, and Trafficking ...

Chapter 6

CB2 Cannabinoid Receptors: Molecular, Signaling, and Trafficking Properties Paul L. Prather

Abstract Two G protein-coupled receptors, CB1 and CB2, have thus far been identified and are responsible for most of the effects produced by cannabinoids. Cannabinoids, such as ∆9-THC, produce psychoactive effects through activation of neuronal CB1 receptors, while CB2 receptors mediate the immune properties of this class of drugs. The molecular, signaling, and trafficking properties of CB2 receptors will be the focus of this review. The cloning of CB2 receptors will be described, along with evidence that individual cannabinoid ligands, differing subtly in structure, might bind to CB2 receptors in distinct fashions. In addition, potential mechanisms underlying the dramatic upregulation of CB2 receptors in response to inflammatory stimuli will be discussed. Next, the currently known signal transduction pathways associated with CB2 receptor activation will be detailed, from G protein coupling to regulation of intracellular effectors. Evidence for the ability of different CB2 receptor agonists to distinctly regulate multiple effectors, known as agonist-directed trafficking of response (ADTR), will also be presented. Furthermore, a potential relationship between CB2 receptor ADTR and immune cell function will be discussed. Lastly, two distinct aspects of CB2 receptor signaling will be described that may help to explain the well-documented interactions of cannabinoids with other receptor systems. It is hoped that this brief review will provide a basic understanding of CB2 receptor signaling necessary to appreciate the exciting future approaching for the development of potentially therapeutic selective CB2 receptor ligands.

Overview of Cannabinoid Receptors Cannabis sativa has been used both therapeutically and recreationally for centuries (see Chap. 1). ∆9-Tetrahydrocannabinol (∆9-THC) has been acknowledged to be the main psychoactive ingredient in marijuana and mediates its effects primarily through activation of two G protein-coupled receptors, CB1 and CB2 (Howlett, 1995). Identified in 1990 (Matsuda et al., 1990), the human CB1 receptor was found to be primarily localized in central and peripheral nervous tissue (Herkenham et al., 1990; Ishac et al., 1996). The CB1 receptor has been identified as a therapeutic A. Köfalvi (ed.), Cannabinoids and the Brain. © Springer 2008

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target in a variety of disease states, such as obesity (Ravinet et al., 2002), alcohol dependence (Racz et al., 2003), Parkinson’s disease (Brotchie, 2003), and pain (Iversen and Chapman, 2002) (for further details, consult with Chaps. 14, 21, 22). The second G protein-coupled cannabinoid receptor, CB2, was cloned two years later (Munro et al., 1993). These receptors are prevalently found in immune tissues, most abundantly in the spleen and leukocytes (Galiegue et al., 1995). As the localization of the CB2 receptors might indicate, selective CB2 receptor ligands have potential therapeutic use as immune modulators for tumor suppression (Klein et al., 2003) and inflammation (Conti et al., 2002). Recently, CB2 receptor agonists have also been shown to produce potent and efficacious analgesia of neuropathic pain (Ibrahim et al., 2003; Scott et al., 2004). This finding is of particular benefit due to the localization of CB2 receptors outside of the CNS; therefore, agonists that selectively activate the CB2 receptor may produce effective analgesia without the unwanted psychoactive CNS effects associated with CB1 receptor agonists (Cravatt and Lichtman, 2004).

Molecular Biology of CB2 Receptors Cloning of the CB2 Receptor The cDNA for the human CB2 (hCB2) receptor was initially cloned from HL-60 cells, a human promyelocytic leukaemic cell line (Munro et al., 1993). The hCB2 receptor is a protein consisting of 360 amino acids forming a structure that is predicted to span the plasma membrane seven times, characteristic of G proteincoupled receptors. Employing splenocyte cDNA libraries, CB2 receptors have also subsequently been cloned from the mouse (Shire et al., 1996) and the rat (Brown et al., 2002). In contrast to the CB1 receptor which has been cloned from a diverse array of vertebrates such as mammals (Gerard et al., 1991), birds (Soderstrom and Johnson, 2000), fish (Yamaguchi et al., 1996), and amphibians (Soderstrom et al., 2000), the CB2 receptor has only been cloned in mammals. The hCB2 receptor is also quite different from the human CB1 (hCB1) receptor on a structural basis, sharing only 44% overall homology, increasing to 68% identical amino acid identity when only the seven transmembrane domains are considered. Furthermore, the amino terminal domain of the hCB2 receptor is much shorter than, and has no significant conservation with, the hCB1 receptor. When comparing the CB2 receptor across species, a high degree of homology exists when hCB2, mCB2, and rCB2 receptors are aligned except in the carboxyl terminus. In this region, the rCB2 receptor is 50 and 63 amino acid residues longer than the hCB2 and mCB2 receptors, respectively. The genes encoding for the mCB2 and hCB2 receptors have been mapped to distal locations on their respective chromosomes [mouse #4, human #1P36, (Valk et al., 1997)] and are encoded by a single exon. However, a subsequent study reported that the rCB2 receptor was the

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first known example of an expressed cannabinoid receptor encoded by multiple exons (Brown et al., 2002).

Binding Characteristics of the CB2 Receptor Mutagenesis of cannabinoid receptors has revealed insight into the basis for CB2/CB1 receptor selectivity. As predicted by molecular modeling, mutation of a phenylalanine in transmembrane domain 5 (F5.46) of the hCB2 receptor decreased the affinity of the CB2 receptor-preferring ligand WIN55212–2 for the hCB2 receptor by 14-fold. In contrast, mutation of a valine in the analogous position (V5.46) of the hCB1 receptor to phenylalanine increased the affinity of WIN55212–2 for the hCB1 receptor by 12fold (Song et al., 1999). Furthermore, a comparison of CB2 and CB1 receptor binding sites by docking studies of WIN55212–2 complexed with both hCB1 and hCB2 receptors suggests that CB2/CB1 receptor selectivity is determined primarily by interaction with serine (S3.31) and F5.46 residues in the hCB2 receptor (Tao et al., 1999; Tuccinardi et al., 2006). Specifically, it is proposed that selectivity for the CB2 receptor may be enhanced by developing ligands with a lipophilic group able to interact with F5.46 of hCB2 and a group able to form a H bond with S3.31. In addition to selectivity, there is an increasing amount of evidence that individual ligands, differing subtly in structure, might bind the CB2 receptor in distinct fashions. For example, while substitution of F5.46 with valine in transmembrane domain 5 of the hCB2 receptor decreases the affinity of the aminoakylindole cannabinoid WIN55212–2 for the hCB2 receptor, the affinities for the classical cannabinoid HU-210, the nonclassical cannabinoid CP55940, or the eicosanoid cannabinoid anandamide are unchanged (Song et al., 1999). In studies examining the selectivity of the cannabinoid antagonist SR144528 for the CB2 receptor, mutation of amino acids adjacent to transmembrane domain 4 (serine 161, serine 165, or cysteine 175), eliminates CB2 receptor binding by SR144528, but has minimal effect on the affinity of CP55490 or WIN55212–2 (Gouldson et al., 2000). If cannabinoid ligands derived from diverse structural classes bind to CB1 and CB2 receptors in distinct manners, it is likely that individual agonists might selectively activate signal transduction pathways (i.e., agonist-directed trafficking of response, ADTR). If so, agonists might be developed that at optimal concentrations preferentially activate signal transduction pathways responsible for the therapeutic effects of cannabinoids (i.e., antinociception), while avoiding activation of other pathways potentially mediating undesirable actions (i.e., disruption of short-term memory).

Regulation of CB2 Receptor Expression Although recent studies have suggested the presence of low levels of functional CB2 receptors in the CNS (van Sickle et al., 2005; Gong et al., 2006; Onaivi et al., 2006; see Chap. 10), CB2 receptors are predominantly expressed in

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immune cells (Herkenham et al., 1990; Ishac et al., 1996). However, during chronic inflammation associated with several diseases affecting the CNS, CB2 receptor levels are dramatically upregulated in inflamed neural tissues (Benito et al., 2003; Ramirez et al., 2005; Shoemaker et al., 2007). The increase in the density of CB 2 receptors appears to occur primarily in activated microglia, the resident immune cells of the CNS. Few studies have attempted to investigate the mechanisms underlying CB2 receptor upregulation in response to inflammation. There is evidence, however, indicating a role for specific cytokines (Maresz et al., 2005) and the cyclic AMP-protein kinase A signaling pathway (Mukhopadhyay et al., 2006). For example, Maresz and colleagues (2005) demonstrated that microglial cells cultured with combinations of gamma-interferon and granulocyte macrophage-colony stimulating factor, which both promote microglial cell activation and are expressed in the CNS during many neuroinflammatory diseases, produce a synergistic eightfold to tenfold increase in the levels of CB2 receptors within these cells. In another recent study, CB2 receptors in cultured RAW 264.7 macrophages increase following exposure to the bacterial cell wall component lipopolysaccharide (Mukhopadhyay et al., 2006). CB2 receptor upregulation was partially blocked by cyclohexamide or the protein kinase A and C inhibitors H8 and bis-indolylmaleimide. Furthermore, application of dibutyryl cyclic AMP or activation of adenylyl cyclase by forskolin increased CB2 receptor levels. This data suggest that the regulation of CB2 receptor expression in macrophages following exposure to inflammatory stimuli, such as lipopolysaccharide, involves the cyclic AMP-protein kinase A-cyclic AMP response element pathway.

CB2 Receptor Signal Transduction G Protein Coupling Both CB1 and CB2 receptors are G protein-coupled receptors that traverse the plasma membrane seven times and regulate the activity of intracellular effectors through activation of intracellular G proteins. Heterotrimeric G proteins are composed of three distinct subunits, α (39–50 kDa), β (35–36 kDa), and γ (6–10 kDa) and their activation by G protein-coupled receptors produces an exchange of GTP for GDP on the subunits. This results in the dissociation of the G protein from the receptor and the separation of the α GTP from the βγ subunits. Both the free αGTP and βγ subunits then proceed to regulate various downstream effectors (Gudermann et al., 1997). Pertussis toxin (PTX)-sensitive G proteins (i.e., Giα and Goα-subtypes) appear to mediate the physiological effects of cannabinoids acting on CB1 and CB2 receptors (Howlett, 1995). However, other studies also suggest that CB1 receptors may regulate intracellular signaling via


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PTX-insensitive Gsα as well (Glass and Felder, 1997; Maneuf and Brotchie, 1997; Felder et al., 1998; see Chap. 9).

Effector Regulation CB1 and CB2 receptors couple to multiple intracellular effectors. Both CB1 and CB2 receptors regulate the activity of adenylyl cyclase (Howlett, 1985) and the extracellular regulated kinase subgroup of the mitogen-activated protein kinases (ERKMAPK) (Bouaboula et al., 1995). Activation of CB1 (Sugiura et al., 1997) and CB2 (Sugiura et al., 2000) receptors also evokes a rapid, transient increase in intracellular free Ca2+ in neuronal and immune cells. Chronic CB1 and CB2 receptor stimulation results in elevation of intracellular levels of ceramide, associated with decreased proliferation and apoptosis in glioma cells (Guzman et al., 2001). More recently, it has been shown that cannabinoids, acting at both CB1 and CB2 receptors, also promote survival of cortical neurons and oligodendrocyte progenitors through stimulation of the phosphoinositide 3-kinase/protein kinase B (PI3K /Akt) signaling pathway (Molina-Holgado et al., 2002; Molina-Holgado et al., 2005). Interestingly, only CB1, but not CB2 (Felder et al., 1995; McAllister et al., 1999), additionally couples to certain ion channels, producing inhibition of voltage-gated Ca2+ channels (Mackie and Hille, 1992) and activation of inwardly rectifying K+ channels (Mackie et al., 1995). The specific regulation of each of these intracellular effectors by the CB2 receptor will be briefly discussed below.

Adenylyl Cyclase Initial studies demonstrated that cannabinoids produce concentration-dependent inhibition of adenylyl cyclase activity in CHO (Bayewitch et al., 1995) or COS (Slipetz et al., 1995) cells, transfected with the CB2 receptor. Cannabinoids also reduce intracellular cAMP levels in human lymphocytes and mouse spleen cells expressing endogenous CB2 receptors (Howlett and Mukhopadhyay, 2000). In all studies, CB2 receptor-dependent adenylyl cyclase inhibition is PTX-sensitive, indicating the requirement for Gi/oα subtypes of G proteins in the signaling cascade. It has been suggested that the regulation of immune function by the CB2 receptor is mediated, in part, by a reduction in adenylyl cyclase activity (Kaminski et al., 1994). Surprisingly, in cells pretreated with PTX to eliminate Gi/oα -coupling, CB1 (but not CB2) receptor agonists are still able to couple to Gsα to produce stimulation of adenylyl cyclase activity (Glass and Felder, 1997; Maneuf and Brotchie, 1997; Felder et al., 1998). These data demonstrate that, in addition to being unable to regulate ion channels (Felder et al., 1995; McAllister et al., 1999), CB2 receptors also cannot couple to Gsα. Collectively, these studies importantly indicate that CB1 and CB2 receptors transduce intracellular signals in significantly different manners.

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ERK-MAPK Activation of CB2 receptors by cannabinoids also stimulates the activity of p42/p44 ERK-MAPK in HL-60 cells endogenously expressing CB2 receptors (Kobayashi et al., 2001) and in CB2 receptor-transfected CHO cells (Bouaboula et al., 1996). In both studies, the cannabinoid-mediated effect on ERK-MAPK was time- and concentration-dependent and blocked by pretreatment with either PTX or the selective CB2 receptor antagonist SR144528. In PC-3 cells, a human prostate epithelial cell line, the activation of ERK-MAPK by cannabinoids appears to be mediated via a PI3K/Akt pathway that produces membrane translocation of Raf-1 with subsequent phosphorylation of p42/p44 ERK-MAPK (Sanchez et al., 2003). This response was blocked by pretreatment of cells with SR144528, indicating the involvement of CB2 receptors. CB2 receptor-mediated activation of ERK-MAPK by endogenous cannabinoids in immune cells appears to be associated with their migration. For example, in HL-60 cells differentiated into a macrophage-like state, the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) produces marked migration through a CB2 receptor- and ERK-MAPK-dependent pathway (Kishimoto et al., 2003). 2-AG also results in pronounced ERK-MAPK-dependent migration of myeloid precursor cells via overexpressed CB2 receptors (Jorda et al., 2002). Microglial cell migration, a neuroinflammatory response to dying neurons, is initiated in response to CB2 receptor activation by 2-AG and is dependent on ERK-MAPK activation (Walter et al., 2003). Lastly, cannabinoids can also inhibit ERK-MAPK in stimulated mouse splenocytes, presumably via CB2 receptor (although not directly demonstrated) (Kaplan et al., 2003). By use of the mitogen-activated kinase (MEK) inhibitor PD098059, the authors suggest that cannabinoid-mediated reduction in ERKMAPK may inhibit IL-2 production in these cells, contributing to the mechanism for immunosuppression commonly observed with cannabinoids.

Ca2+ Transients Stimulation of CB2 receptors produces transient increases in intracellular free Ca2+ concentration via a phospholipase-Cβ (PLCβ-mediated mechanism in HL-60 cells expressing endogenous CB2 cannabinoid receptors (Sugiura et al., 2000) and in CHO cells stably transfected with CB2 (Shoemaker et al., 2005b). In both studies, the Ca2+ transients produced were concentration-dependent and blocked by pretreatment with either PTX or selective CB2 antagonists. In CHO-CB2 cells, the cannabinoid-elicited rise in intracellular free Ca2+ concentration was blocked by preincubation with the active (U73122), but not the inactive (U73343), inhibitor of PLCβ. This provides rather strong evidence that activation of PLCβ is involved in the observed CB2 receptor-mediated production of Ca2+transients in transfected CHO cells. Interestingly, a previous study reported that activation of transfected CB2 receptors in CHO cells is unable to elevate intracellular free Ca2+


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concentration (Felder et al., 1995). While the exact reasons for the differences between these studies are not known, one potential explanation might be due to the choice of agonists employed. While Felder and colleagues (1995) observed no effect on intracellular calcium concentrations in response to WIN55212–2, anandamide, and HU-210, the agonist evaluated by Shoemaker and colleagues (2005a,b) (2-AG) was not examined.

Ceramide Synthesis Several early reports demonstrated that ceramide accumulation participates in cannabinoid-induced apoptosis of glioma cells (Galve-Roperh et al., 2000; Gomez del Pulgar et al., 2002), a mechanism that appears to rely on the activation of the CB2 receptor (Sanchez et al., 2001). Recent studies employing Jurkat cells, a human leukemia cell line expressing endogenous CB2 receptors, further showed that CB2 receptor activation signals apoptosis via a ceramide-dependent stimulation of the mitochondrial intrinsic pathway (Herrera et al., 2006). Specifically, cannabinoid treatment resulted in a CB2 receptor-dependent stimulation of ceramide biosynthesis, and inhibition of this pathway prevented cannabinoid-induced mitochondrial hypopolarization and cytochrome-c release. These results indicate that ceramide acts at a premitochondrial level. Ceramide synthesis inhibition in this study also prevented caspase activation and apoptosis. Collectively, these reports demonstrate that CB2 receptor signaling plays a major role in the proapoptotic effect of cannabinoids and suggest that selective CB2 cannabinoids might be developed as useful agents to slow tumor growth in various forms of cancer.

PI3K /Akt Pathway Survival signaling of many cell types, including neurons, has been clearly demonstrated to be associated with the PI3K/Akt pathway (Brunet et al., 2001). Cannabinoids, acting at both CB1 and CB2 receptors, also promote survival of cortical neurons and oligodendrocyte progenitors through stimulation of the PI3K/Akt signaling pathway (Molina-Holgado et al., 2002, 2005). Specifically, the nonselective cannabinoid agonist HU-210 inhibits the death of rat primary cortical neurons induced by the neurotoxin (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (S-AMPA). The neuroprotective effect of HU-210 is reversed by antagonists selective for either CB1 or CB2 receptors. HU-210 triggers activation of Akt, but not activation of the ERK-MAPK, JNK-MAPK, or p38-MAPK signaling pathways. Furthermore, the PI3K inhibitors LY294002 and wortmannin prevent phosphorylation of Akt in response to HU-210, and reversed the neuroprotective effect of HU-210. As such, the authors suggest that the PI3K/Akt signaling pathway mediates the neuroprotective effect of the exogenous cannabinoid

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HU-210 acting at CB1 and CB2 receptors in primary cultured CNS neurons (MolinaHolgado et al., 2005).

CB2 Receptor Internalization and Trafficking in Response to Acute and Chronic Ligand Exposure Acute and chronic ligand exposure significantly regulates both CB1 and CB2 receptor signaling. A great deal of research has been conducted on this topic concerning CB1 receptor signaling and has recently been reviewed extensively by Sim-Selley (2003) (see Chap. 5). In contrast, much less has been reported about the effect of acute and chronic ligand exposure on signaling by CB2 receptors. As observed with other G protein-coupled receptors, studies with CB2 transfected CHO cells demonstrate that upon initial exposure to the full agonist CP55940, serine 353 is extensively phosphorylated and phosphorylation is maintained for up to 8 h (Bouaboula et al., 1999b). CB2 receptor phosphorylation by CP55940 can be reversed by preincubation with the CB2 receptor-selective antagonist/ inverse agonist SR144528. Furthermore, CB2 receptors desensitize in a time-and concentration-dependent manner following prolonged agonist exposure, such that cellular responses are abolished in response to challenge with CB2 receptor agonists following chronic exposure to either CP55940 (Bouaboula et al., 1999b) or to the putative endogenous cannabinoid noladin ether (Shoemaker et al., 2005a). If exposure to CP55940 is extended to 24 hours, CB2 receptors are also downregulated, reflected by over a 90% loss of receptors as measured by receptor binding (Shoemaker et al., 2005a). Interestingly, similar chronic exposure to noladin ether produces significantly less CB2 receptor down-regulation, resulting in only approximately a 50% loss of receptor binding (Shoemaker et al., 2005a). Several recent studies have revealed some very interesting findings concerning CB2 receptor localization in immune cells and the effect of acute cannabinoid exposure on CB2 receptor trafficking within these cells (Walter et al., 2003; Carrier et al., 2004; Rayman et al., 2004). Microglial cell lines and primary cultures of microglia exist in an activated state when maintained in culture (Becher and Antel, 1996). In cultured (activated) mouse microglial BV-2 cells, rat microglial RTMGL1 cells, and mouse microglial primary cultured cells, CB1 receptors appear to be localized in the intracellular compartment (Walter et al., 2003; Carrier et al., 2004). In marked contrast, CB2 receptors are expressed heterogeneously throughout the activated microglial cells, both at the cell surface and internally. Even more interesting is the observation that CB2 receptors are expressed in relatively high density at the leading edge of the lamellipodia of activated microglial cells (Walter et al., 2003). This critical positioning suggests that CB2 receptors might participate in the migration of microglial cells occurring in response to inflammatory stimuli. Indeed, microglial cell migration is initiated following exposure to the endogenous cannabinoid agonist 2-AG, an effect mediated by both CB2 and abnormal cannabidiol-sensitive receptors (Walter et al.,


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2003). Furthermore, exposure of microglial cells to 2-AG significantly increases CB2 receptor internalization, but not degradation (Carrier et al., 2004). In lymphoid tissues, CB2 receptors are also expressed in distinct patterns, depending on receptor activation status (Rayman et al., 2004). For example, active CB2 receptors are present mainly in the germinal centers, while inactive CB2 receptors are confined to the mantle and marginal zones of the secondary follicles where resting cells reside. Collectively, these studies suggest that activated CB2 receptors are selectively trafficked within immune cells to specific regions, critically posed to participate in important immune cell functions such as proliferation and migration.

CB2 Receptor ADTR Definition and Observation of ADTR at CB2 Receptors Evidence suggests that G protein-coupled receptors exist in multiple active receptor conformations (Kenakin, 2002). It has been predicted that binding of a particular agonist to a GPCR results in enrichment of a unique set of receptor conformations based on the microaffinity of the agonist for each conformation. Because distinct conformations could presumably couple receptors differently to specific G proteins and intracellular effectors, individual agonists could ultimately produce distinct effects. Numerous studies provide support that individual agonists acting at several different classes of G protein-coupled receptors (Figini et al., 1997; Berg et al., 1998; Wiens et al., 1998), including CB1 receptors (Bonhaus et al., 1998), are able to traffic intracellular responses in a ligand-dependent manner. Furthermore, utilizing plasmon waveguide resonance spectroscopy, Alves and colleagues have recently provided direct evidence for the existence of distinct topographical configurations of human delta opioid receptors with discrete affinities between individual G protein subclasses and different ligand-induced states (Alves et al., 2003). Very recently, evidence for ADTR by endocannabinoids acting at CB2 receptors has been provided (Shoemaker et al., 2005b). Specifically, in CHO-CB2 cells it was shown that 2-AG, acting through CB2 receptors, most potently activates ERKMAPK, requiring greater concentrations to inhibit adenylyl cyclase, and even higher amounts to stimulate Ca2+transients. In contrast, two other cannabinoids tested (noladin ether and CP55940) most potently inhibit adenylyl cyclase, necessitating higher concentrations to stimulate ERK-MAPK and Ca2+transients.

Potential Relationship of CB2 ADTR to Function If ADTR occurs at CB2 receptors, the preferential activation of the ERK-MAPK pathway by 2-AG, relative to noladin ether and CP55940 demonstrated by Shoemaker

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and coworkers (2005a,b), might provide insight into the cellular basis for welldocumented agonist selective actions reported for cannabinoids in immune cells. For example, in HL-60 cells differentiated into a macrophage-like state, 2-AG produces marked migration through a CB2 receptor- and ERK-MAPK-dependent pathway (Kishimoto et al., 2003). In contrast, noladin ether only weakly stimulates migration, while anandamide, CP55940, WIN55212–2, and several other cannabinoids have no effect. 2-AG also results in pronounced ERK-MAPK-dependent migration of myeloid precursor cells via overexpressed CB2 receptors, whereas anandamide produces near negligible effects and other cannabinoids are devoid of activity (Jorda et al., 2002). Microglial cell migration, a neuroinflammatory response to dying neurons, is initiated in response to CB2 receptor activation by 2AG, but not by two other putative endocannabinoids and is dependent on ERKMAPK activation (Walter et al., 2003). Although involvement of ERK-MAPK was not tested, activation of CB2 receptors by 2-AG induces the migration of EoL-1 human eosinophilic leukemia cells, noladin ether is only weakly effective, and anandamide does not induce migration (Oka et al., 2004). In all the cited studies, 2-AG induces pronounced migration of cells while other endogenously occurring or synthetically derived cannabinoids produce only modest or no effects at all. In addition, migration induced by 2-AG was shown to occur through activation of CB2 receptors and ERK-MAPK. As such, it is tempting to speculate that this rather selective, robust ability of 2-AG to induce migration of variety of cell types might be due to the ability of 2-AG to preferentially regulate ERK-MAPK via CB2 receptors relative to other cannabinoids.

CB2 Receptor Interactions Inactivation of Other Gi /Go-Coupled Receptor Signaling by CB2 Receptors Many G protein-coupled receptors exhibit constitutive activity, producing spontaneous regulation of effectors in the absence of activation by agonists (Kenakin, 2001). Ligands that can reduce or abolish this spontaneous, agonist-independent activity are termed inverse agonists (Strange, 2002; Prather, 2004). CB2 receptors are constitutively active (Bouaboula et al., 1999b). The CB2 inverse agonist JTE907 demonstrates anti-inflammatory actions in several animal models (Maekawa et al., 2006; Ueda et al., 2007). Furthermore, a novel CB2 inverse agonist has recently been shown to inhibit leukocyte recruitment induced by several different chemokines (Lunn et al., 2006). While the mechanism for the blockade of leukocyte recruitment was not examined, constitutively active CB2 and CB1 receptors appear to be able to sequester Gi/o type G proteins away from other G proteincoupled receptors, interfering with their function (Bouaboula et al., 1999a; Vasquez


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and Lewis, 1999). Since chemokine receptors produce immune cell migration via activation of Gi/o type G proteins, it is possible that CB2 inverse agonists (such as JTE-907) might reduce inflammation by interfering with this critical step in the immune response mediated by chemokines. This indicates that CB2 inverse agonists might be potentially developed as drugs to treat a variety of inflammatory disorders.

Transcriptional Regulation of Other Receptors by CB2 Receptors Very recently, CB2 receptor activation has been shown to be regulating the expression of CB1, µ-, and δ-opioid receptors in the CD4+ T cell line Jurkat (Borner et al., 2006, 2007). Specifically, the upregulation of all three receptors involves activation of CB2 receptors followed by phosphorylation of signal transducer and activator of transcription 5 (STAT5) with subsequent transactivation of the gene encoding for interleukin-4 (IL-4). Transactivation of CB1, µ-, and δ-opioid receptor genes in response to IL-4 is then mediated by phosphorylation of the signal transducer and activator of transcription 6 (STAT6). Increasing the levels of CB1 receptors in T lymphocytes, and possibly other immune cells, in response to CB2 receptor stimulation would be expected to enhance the immunomodulatory effects mediated by cannabinoids in these cells. Furthermore, if CB2-mediated upregulation of µ- or δ-opioid receptors also occurs in neurons, it might help explain the well-documented synergistic analgesic effects between cannabinoids and opioids (Cichewicz, 2004).

Concluding Remarks Cannabinoids produce the majority of their effects through interaction with CB1 and CB2 receptors. CB2 receptors (the subject of this review) are expressed predominantly in immune tissues and transduce intracellular signals through coupling to the Gi/Go subtype of G proteins. Upon receptor activation by agonists, CB2 receptors regulate the activity of multiple intracellular effectors, including adenylyl cyclase, ERK-MAPK, Ca2+ transients, ceramide synthesis, and PI3K / Akt. Interestingly, different CB2 agonists bind uniquely to CB2 receptors and distinctly regulate multiple effectors. This type of intracellular signaling has been described as agonist-directed trafficking of response (ADTR). The ability of CB2 ligands to selectively traffic intracellular responses, coupled with their selective expression profile in inflamed tissues, and pronounced anti-inflammatory and neuroprotective properties, suggest an exciting future is approaching for the development this novel class of drugs for the treatment of a variety of inflammatory disorders.

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Acknowledgments This work was supported in part by National Institute on Drug Abuse (grant RO1-DA13660), Amyotrophic Lateral Sclerosis Association (ALSA) (grant 1311), and University of Arkansas for Medical Sciences (Tobacco Award).

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