Introduction

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Marijuana (Cannabis sativa), one of the oldest and most widely abused drugs, has also been used for medicinal purposes by various cultures. The primary psychoactive constituent of marijuana is a cannabinoid compound, ⁹-tetrahydrocannabinol. During the past decade, a major investigative effort on the mechanisms of action of cannabinoids has been launched. Cannabinoids have been found to act through G protein-coupled receptors (GPCRs) on cell membranes (Devane et al., 1988). Several cDNAs and genes encoding cannabinoid receptors have been cloned, including CB₁ and CB₂ (Matsuda et al., 1990; Munro et al., 1993). The putative endogenous cannabinoid ligand anandamide has been isolated from the brain (Devane et al., 1992), and high-affinity cannabinoid mimetics with a variety of chemical structures have been synthesized (Musty et al., 1995).

Based on their chemical structures, cannabinoid agonists can be classified into at least four groups: classical cannabinoids, bicyclic or nonclassical cannabinoids, fatty acid amides and esters, and aminoalkylindoles (AAIs). AAIs are a class of cannabimimetics with structures entirely different from those of natural cannabinoids. AAIs have been shown to bind cannabinoid receptors and exhibit cannabinoid-like activities (Compton et al., 1992; Kuster et al., 1993).

Most cannabinoid agonists exhibit little or no subtype selectivity (Felder et al., 1995). Recent studies have demonstrated that WIN55212-2 (Fig. 1), a prototypic aminoalkylindole, has higher affinity for CB₂ than for CB₁, with a K_i ratio (CB₁:CB₂) of 19 (Felder et al., 1995). The molecular basis for the selectivity of WIN55212-2 for CB₂ is currently unknown. In the present study, to investigate the molecular basis for the CB₂ selectivity of WIN55212-2, models of the CB₁ and CB₂ transmembrane helix (TMH) bundles were first probed for possible AAI interaction sites.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   The chemical structure of WIN55212-2.

In the cationic neurotransmitter GPCRs, a negatively charged residue (Asp at 3.32) is commonly hypothesized to serve as a counter-ion for protonated amine ligands (Dixon et al., 1987). This hypothesized interaction site can be used as an "anchor" point for the ligand, a point from which secondary interaction sites can be identified. Unlike the cationic neurotransmitters, cannabinoid ligands are uncharged. Furthermore, the cannabinoid receptors lack the negatively charged residue at 3.32 that is found in the cationic neurotransmitter GPCRs. The cannabinoid receptors possess, instead, a positively charged amino acid (Lys) at position 3.28, which is one turn up from position 3.32 toward the extracellular side of the TMH bundle. In rhodopsin, position 3.28 is of functional importance because Glu 3.28 (113) is an interaction site for the cis-retinal chromophore (Rao et al., 1994). The Lys at 3.28 in the cannabinoid receptors should be capable of hydrogen bonding with cannabinoid ligands. Position 3.28, therefore, would appear to be a logical interaction site for cannabinoid ligands. In fact, K3.28 has been proposed to be a primary interaction site for the nonclassical cannabinoid CP-55940 at the CB₁ (Tao et al., 1999).

Song and Bonner's (1996) K3.28(192)A mutation study of the CB₁ revealed that K3.28(192) is crucial to the binding and activation produced by HU-210, a classical cannabinoid; CP-55940, a nonclassical bicyclic cannabinoid; and anandamide, an endogenous cannabinoid. However, the K3.28(192)A mutation did not affect the affinity or activity of WIN55212-2, an AAI at the CB₁. This result indicates a difference between the AAI binding site and that (those) of the other three cannabinoid ligand classes.

Recently, the carbonyl oxygen of AAIs (which could serve as a hydrogen bond acceptor) was shown to be unnecessary for cannabinoid receptor affinity and activity. Rigid AAI analogs, which lack this carbonyl group, the E naphthylidene indenes, have been reported to possess high affinity at both the CB₁ and CB₂ receptors (Reggio et al., 1998). Another possible hydrogen-bonding moiety in the AAIs is the morpholino ring. Huffman et al. (1994) have shown, however, that this morpholino ring can be replaced with an alkyl chain without loss of CB₁ affinity or efficacy. Taken together, both mutation and structure-activity relationship results suggest that hydrogen bonding may not be the primary interaction of AAIs at the CB₁.

Aromatic stacking interactions play important roles in protein stability and drug-receptor interactions (Burley and Petsko, 1985). Because WIN55212-2 is an aromatic ligand, we hypothesized that aromatic stacking interactions may be important to the interaction of WIN55212-2 with the CB₁ and CB₂ receptors. Our modeling studies identified an aromatic cluster in the TMH3-4-5 region as the most likely binding site for WIN55212-2. In this region, WIN55212-2 has greater aromatic interactions in CB₂ than in CB₁. Specifically, in CB₂, there is an aromatic interaction for WIN55212-2 with F5.46(197), a residue unique to CB₂. It is possible, therefore, that the presence of F5.46(197) in the CB₂ versus V5.46(282) in the CB₁ may result in the reported CB₂ selectivity of WIN55212-2. To test this hypothesis, a mutagenesis study at position 5.46 of the CB₁ and CB₂ receptors was undertaken.

	Materials and Methods

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Amino Acid Numbering System. The amino acid numbering system suggested by Ballesteros and Weinstein (1995) was used. Each amino acid identifier starts with the TMH number, followed by the amino acid position relative to a reference amino acid in that helix. This reference amino acid is the most highly conserved residue across GPCRs in that helix and is assigned a locant value of 0.50. The locant is followed by the sequence number of the amino acid residue in parentheses. This numbering system for the cannabinoid receptor has been described previously (Bramblett et al., 1995). As an example, the most highly conserved amino acid in TMH 2 is an aspartic acid. In the CB₁ sequence, this is residue 163. Using the proposed numbering system, this amino acid would be called D2.50(163). The amino acid preceding the aspartic acid, an Ala, would be called A2.49(162).

Model Construction and Refinement. Helix ends of the cannabinoid CB₁ and CB₂ receptors and the degrees of lipid exposure of each helix were determined using a Fourier-Transform Analysis of the periodicity in hydrophobicity and variability exhibited by the sequences of these receptors (Bramblett et al., 1995). The helices were constructed using the Chem Protein Module of Chem-X (Chemical Design Ltd., Chipping Norton, UK). A bend and twist of exposed faces of TMH 7 before and after the Pro kink produced by the NPXXY motif were introduced as described in Fu et al. (1996). Relative helix heights were checked and adjusted using the GPCR mutation literature (Rao et al., 1994; Zhou et al., 1994; Han et al., 1995; Nakanishi et al., 1995). Helix tilts were adjusted to be consistent with the 7.5Å resolution projection map of frog rhodopsin (Unger et al., 1997; Baldwin et al., 1997). Recent scanning cysteine accessibility method results for TM helices; 3, 5, 6, and 7 in the dopamine D₂ receptor (Javitch et al., 1995a,b, 1998; Fu et al., 1996) were used as a test of the cannabinoid receptor bundles. These studies have identified those residues that are accessible from within the binding site crevice of the D₂ receptor in the subject helices. The surface of this crevice is formed by residues that can contact specific ligands (agonists and/or antagonists) and by other residues that may play a structural role and affect binding indirectly . All residues identified as accessible by scanning cysteine accessibility method were found to be accessible from within the binding site crevice of the CB₁ and CB₂ receptor models.

Structure-Activity Relationship (SAR) Criteria Used to Test the TMH3-4-5 Aromatic Cluster Binding Site for WIN55212-2. Several criteria from aminoalkylindole SAR were used to test the proposed TMH 3-4-5 aromatic cluster-binding site for WIN55212-2 (see Discussion). First, Pachecho et al. (1991) have shown that the R stereoisomer of WIN55212 (called WIN55212-2), but not the S stereoisomer of WIN55212 (called WIN55212-3), produces effects at the cannabinoid receptor. Second, Reggio et al. (1998) have shown that the C-2 methyl rigid AAI analogs, the E-aminoalkylindenes (analogous to the s-trans conformer of WIN55212-2) have significantly higher affinity and efficacy at the CB₁ and CB₂ than do the Z-aminoalkylindenes (analogous to the s-cis conformer of WIN55212-2). Finally, WIN55212-2 has been shown to be 19-fold selective for the CB₂ (Felder et al., 1995).

Distance and Angle Measurements of Aromatic Stacking Interactions. Centroids for each ring were generated using the Chem-X molecular modeling suite of programs. For fused ring aromatic moieties (i.e., naphthyl and indole rings), a centroid for each ring was generated. Distances between centroids were measured using Chem-X. The distances reported here are the shortest centroid- centroid distances between the two moieties. A normal vector to the plane of each aromatic system also was generated, and the angle between the normal vectors of two interacting rings  then was evaluated using Chem-X.

Reagents. Enzymes and reagents used for recombinant DNA experiments were purchased from GIBCO-BRL (Gaithersburg, MD), or Promega (Madison, WI). Oligonucleotides were synthesized by the Gene Technology Laboratory of the Texas A&M University. Tissue culture reagents were obtained from Biowhittaker (Walkersville, MD). Adenovirus-transformed 293 cells were from American Type Culture Collection (Rockville, MD). Glass tubes used for diluting cannabinoid drugs and for ligand-binding assays were silanized through exposure to dichlorodimethylsilane (Sigma Chemical Co., St. Louis, MO) vapor while under vacuum for 3 h.

Expression of Recombinant Cannabinoid Receptors. A 1.5-kb SstI/XbaI fragment of the human CB1 gene containing the entire coding region was subcloned into expression vector pRC/CMV (Invitrogen, San Diego, CA) to construct the expression plasmid pHCB₁-RC/CMV. A 1.8-kb full-length human CB₂ cDNA was subcloned into pRC/CMV to construct the expression plasmid pHCB₂-RC/CMV.

Site-Directed Mutagenesis. Mutations were introduced through the GeneEditor in vitro site-directed mutagenesis system (Promega). This system allows mutations to be made in any vector that contains ampicillin resistance as a selectable marker. Using this system, the selection oligonucleotide, which encodes the resistance to the GeneEditor antibiotic selection mix, is annealed to DNA template at the same time as a mutagenic oligonucleotide. Subsequent synthesis and ligation of the mutant strand link the two oligonucleotides. The system uses the resistance to the GeneEditor antibiotic selection mix to facilitate the selection of the mutant DNA. For CB1V5.46F, the mutagenic oligonucleotide 5'-TGGATCGGGTTC ACCAGCG-3' was used. For CB2F5.46V, the mutagenic oligonucleotide 5'-CTGGCTCCTGGTCATCGCCTTC-3' was used. The presence of the mutation and the accuracy of the DNA sequences were verified with dideoxy sequencing.

Ligand Binding. For membrane preparations, confluent cells were washed twice with cold PBS and scraped off the tissue culture plates. Cells were homogenized in membrane buffer (50 mm Tris-HCl, 200 mM sucrose, 5 mM MgCl₂, 2.5 mM EDTA, pH 7.4) with a Tekmar Tissumizer. The homogenate was centrifuged at 32,000g for 20 min at 4°C. The pellet was resuspended in binding buffer (50 mm Tris-HCl, 5 mM MgCl₂, 2.5 mM EDTA, pH 7.4) and stored at 80°C until use. Protein concentrations were determined by the use of a bicichoninic acid protein reagent kit (Pierce, Rockford, IL).

cAMP Accumulation. cAMP accumulation assays were performed using a method published previously (Song and Bonner, 1996). Briefly, using silanize test tubes, cannabinoid ligands were diluted with Eagles #2 buffer containing 50 mg/ml fatty acid-free BSA, and drugs were added to tubes in a volume of 25 µl. Forskolin or buffer also was added to the test tubes in a volume of 25 µl. Confluent cells were lifted from culture plates with Eagle's 2 buffer containing 0.5 mm EDTA, washed twice with Eagle's 2 buffer, and incubated with phosphodiesterase inhibitor RO20-1724 for 10 min. The stimulation was initiated by adding cells to test tubes containing forskolin and cannabinoids and incubated for 5 min at 37°C. The final assay volume was 250 µl with 1 × 10⁶ cells per tube. The reaction was stopped with the addition of an equal volume of 0.1 N HCl, after which 50 µl was removed for cAMP radioimmunoassay, using a kit from DuPont-NEN (Wilmington, DE).

Data Analysis. Data from ligand-binding cAMP accumulation assays were analyzed and curves generated with the use of the Prism program (GraphPad Software, San Diego, CA). IC₅₀ and EC₅₀ values were determined through nonlinear regression analysis performed with Prism. K_d and B_max values were estimated from competition binding experiments with the following equations: K_d = IC₅₀  L and B_max = (B₀IC ₅₀)/l, where l is the concentration of free radioligand, and B₀ is specifically bound radioligand (DeBlasi et al., 1989). The K_i values were calculated with the Cheng-Prusoff equation: K_i = IC₅₀/(1 + L/K_d) (Cheng and Prusoff, 1973).

	Results

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Molecular Modeling.

Aromatic Stacking Interactions. Two regions of the cannabinoid receptor-binding site crevice are rich in aromatic amino acids. One region is an intrahelix aromatic stack formed by F2.57, F2.61, F2.64, and F2.67. A second region rich in aromatic amino acids is formed by residues on TMH 3, 4, and 5. This second region was deemed the more likely interaction region, because there is an upper (extracellular side) aromatic stack formed by F3.25, W4.64, W5.43, and a lower aromatic stack with F3.36 when WIN55212-2 is docked in this region. In this docking position, WIN55212-2 would create a continuous aromatic stack over several turns of helices 3, 4, and 5 that is likely to be energetically favored. Binding in this region of the cannabinoid receptors is supported by chimera studies of the CB₁ and CB₂ reported by Shire et al. (1997), which indicated that the region delimited by the fourth and fifth transmembrane regions contain amino acids important for WIN55212-2 binding to the CB₂.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   WIN55212-2 in an s-trans-conformation is shown docked here in an energy minimized CB1 (A) or CB2 (B) TMH bundle. The aromatic residues with which WIN55212-2 directly interacts are shown in yellow. Residues which are part of the TMH 3-4-5 aromatic cluster, but do not directly interact with WIN55212-2 are shown in cyan. The view is inside the membrane, looking from outside the bundle toward helix 4.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Comparison of [3H]WIN55212-2 binding to human CB1 and CB2. Competition binding assays were performed on membranes prepared from 293 cells stably expressing CB1 () or CB2 (). Points, mean ± SE of three independent experiments performed in duplicate. Curves were generated as described in Materials and Methods.

Consistency of Proposed Binding Site with AAI SAR. SAR data from amino-alkylindenes indicate that only the s-trans-conformer of C2 methyl AAIs has significant affinity for CB₁ and CB₂ (Reggio et al., 1998). When an s-cis-conformer of WIN55212-2 was docked in the receptor model, the ligand was found to have steric clashes with Val 3.32, in both the CB₁ and CB₂ bundles. This steric clash resulted in no aromatic stacking interactions in the TMH 3-4-5 region of CB₁ and CB₂ for the s-cis-conformer.

Expression of the Wild-Type CB₁ and CB₂ Cannabinoid Receptors. Radioligand-binding assays were conducted to characterize CB₁ and CB₂ expression in 293 cells. Specific high-affinity binding of cannabinoid ligand [³H]WIN55212-2 was observed with membranes prepared from 293 cells stably transfected with pHCB1-RC/CMV or pHCB2-RC/CMV (Fig. 3, Table 1). For CB₁, the K_d and B_max values were determined to be 11.9 ± 1.9 nM and 1217.6 ± 221.9 fmol/mg protein, respectively. For CB₂, the K_d and B_max values were determined to be 0.76 ± 0.04 nM and 1172.7 ± 184.9 fmol/mg protein, respectively. To determine the stereospecificity of ligand binding, WIN55212-3, the inactive enantiomer WIN55212-2, was tested. WIN55212-3 was inactive in competing for [³H]WIN55212-2 binding (data not shown).

Comparison of the Mutant and Wild-Type Cannabinoid Receptors. Radioligand-binding assays were performed to compare the pharmacologic profiles of the CB1V5.46F and CB2F5.46V mutant cannabinoid receptors with those of wild-type CB₁ and CB₂. Specific high-affinity binding of [³H]WIN55212-2 was observed in membranes prepared from 293 cells stably transfected with the mutant cannabinoid receptor DNAs. The B_max values for CB1V5.46F and CB2F5.46V were 1064.0 ± 147.2 and 1265.3 ± 114.5 fmol/mg proteins, respectively. These B_max values are not significantly different from those of the wild-type CB₁ and CB₂ (1217.6 ± 221.9 and 1172.7 ± 184.8 fmol/mg protein, respectively).

	Discussion

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A fundamental hypothesis underlying the modeling studies reported here is that aromatic stacking interactions are important to the WIN55212-2/cannabinoid receptor complexes. Aromatic stacking interactions have been shown to play important roles in protein stability and drug-receptor interactions (Burley and Petsko 1985). Aromatic clusters such as the CB₁/CB₂ TMH 3-4-5 aromatic cluster discussed in this article have been proposed to form the binding site of other GPCRs. For example, Colson et al. (1998) have proposed that the thyrotropin-releasing hormone (TRH) binding site at the TRH receptor is an aromatic (hydrophobic) cluster on TMH 5 and 6. Through mutagenesis studies, these investigators showed that F5.46(199), W6.48(279), and Y6.51(282) constrain the TRH receptor in an inactive conformation. In the dopamine D₂ receptor, four highly conserved aromatic residues F6.44 (382), W6.48(386), F6.51(389), and F6.52(390), along with a histidine (H6.5(393)), are clustered together on the water-accessible surface of the D₂ binding site crevice. These residues have been suggested to define an interconnected aromatic cluster that may be involved in ligand binding and receptor activation (Javitch et al., 1998).

In the energy-minimized model of the WIN55212-2-CB₁ complex (Fig. 1a), the indole ring of WIN55212-2 interacts with F3.36(200) on helix 3. The naphthyl ring of WIN55212-2 interacts with helix 3 [(F3.25(189)] and helix 5 [W5.43(279)]. In the energy-minimized model of the WIN55212-2/CB₂ complex (Fig. 1b), the indole ring of WIN55212-2 is positioned to have a strong aromatic interaction with F5.46(197), an aromatic amino acid unique to CB₂. In addition, aromatic interactions for the indole ring with two other aromatic amino acids occur [F3.36(117), W5.43(194)]. For the naphthyl moiety of WIN55212-2, the energy-minimized WIN55212-2-CB₂ complex reveals aromatic stacking interactions with F3.25(106) and W5.43(194).

If the selectivity of a ligand for a particular receptor subtype is attributable to a favorable interaction with a single amino acid, then introduction of this functional group via mutagenesis into the receptor subtype lacking this amino acid in an analogous position would be predicted to increase the affinity of the ligand for the latter receptor. Furthermore, the removal of this amino acid (by replacement with some other amino acid) from the wild-type receptor for which the ligand is selective should result in decreased affinity. In the experimental part of this study, the selectivity of WIN55212-2 for CB₂ was first confirmed in our system. In ligand-binding assays, WIN55212-2 exhibited 15- to 16- fold higher affinity for CB₂ (Fig. 3; Tables 1 and 2). Most important, mutagenesis studies showed that the CB1V5.46F mutation increased the affinity of WIN55212-2 for CB₁ by 12- to 13-fold, and the CB2F5.46V mutation decreased the affinity of WIN55212-2 for CB₂ by 14-fold. In contrast, these mutations did not affect the affinity of three other cannabinoid agonists for either CB₁ or CB₂. In cAMP accumulation assays, the maximum inhibition (40-60%) of forskolin-stimulated cAMP accumulation by cannabinoid agonists was unchanged on these mutant receptors. The changes in EC₅₀ values of WIN55212-2 were consistent with the changes of its binding affinity caused by the mutations. These data strongly support the hypothesis that the selectivity of WIN55212-2 for CB₂ over CB₁ is caused by the change from Val in CB1 at position 5.46 to Phe.

Residue 5.46 has been shown to be important for ligand-binding and subtype selectivity in other GPCRs as well. In the -adrenergic receptor, S5.46 (along with S5.42) has been proposed to interact with the catechol hydroxyl groups of -adrenergic ligands (Strader et al., 1989). Position 5.46 has been proposed to be the major contributor to the pharmacologic differences between human (S5.46) and rat (A5.46) 5-Hydroxytryptamine (5-HT)₂ receptors (Kao et al., 1992). The ergolines, mesulergine (5-HT_2C selective), and d-lysergic acid diethylamide (5-HT_2A selective) have been shown to reverse their relative affinities with the mutations 5-HT_2A [S5.46(242)A] and 5-HT_2C [A5.46(222)S], supporting a direct role of this locus in the selectivity of these ligands (Almaula et al., 1996).

Marijuana and its active component cannabinoids have a number of potential therapeutic properties, such as for the treatment of inflammation and autoimmune diseases (Lyman et al., 1989; Mazzari et al., 1996). However, one of the major problems for cannabinoids as therapeutic agents is their severe psychoactive side effect. The recent cloning of CB₂, a subtype of cannabinoid receptors, brought us new hope for a better medical use of cannabinoids (Munro et al., 1993). Because CB₂ is predominantly located in the immune system, and rarely in the brain, CB₂ selective compounds may prove to be potential therapeutic agents for inflammation and autoimmune diseases. CB₂ selective drugs should retain immunomodulatory effects, but be devoid of the psychoactive effects of marijuana.

By combining the approaches of molecular modeling and site-directed mutagenesis, this study has identified an amino acid residue that is important for the selectivity of WIN55212-2 for CB₂. Results described in this article suggest that cannabinoid agonists designed to have direct interactions with F5.46, in the CB₂ receptor, as well as with other aromatic amino acids in the TMH 3-4-5 cluster, may prove to be useful lead compounds for the design of more potent CB₂-selective agonists.