Chemicals and reagents
[3H] CP-55,940 was purchased from Perkin-Elmer. The fluorescent dyes Texas Red (1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt), DilC18 (1,1′-dioctadecyl-3,3,3′, 3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt), Alexa Fluor 532 carboxylic acid, and succinimidyl ester (1H-Pyrano[3,2-f:5,5-f′] diindole-10, 12-disulfonic acid, 5-[4-[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]phenyl-2,3,7,8-tetrahydro-2,3,3,7,7,8-hexamethyl) were purchased from Molecular Probes. Restriction enzymes and DNA-modifying enzymes were obtained from New England Biolabs. The INDIA HisProbe-HRP kit for detection of polyhistidine-tagged fusion proteins was obtained from Pierce.
The cannabinoid ligands anandamide (N-(2-hydroxyethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide), CP-55,940 ((−)-cis-3[2-hy-droxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol), ACEA (arachidonyl-2′-chloroethylamide/N-(2-chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide), WIN 55,212-2 mesylate ((R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo [1,2,3-de]-1,4-bezoxazin-6-yl]-1-naphthalenyl-methanone mesylate), JWH015 ((2-methyl-1-propyl-1H-indol-3-yl)-1-napthalenylmethanone), AM281 (1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide), AM630 (6-iodo-2-methyl-1-(2-morpholin-4-ylethyl)-1H-indol-3-yl](4-methoxyphenyl)methanone, (−)-cannabidiol (2-[(1R,6R)3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol), abn-CBD (4-[(1R,6R)-3-methyl-6-(1-methylethe-nyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol), and noladin ether (2-[(5Z,8Z,11 Z,14Z)-eicosatetraenyloxy]-1,3-propanediol) were purchased from Tocris. HU210 ((6aR)-trans-3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo [b,d] pyran-9-methanol), a specific central-type cannabinoid receptor synthetic agonist SR 141716A, and Δ9-tetrahydrocannabidiol were kindly provided by Dr. G. Kunos (NIAAA, NIH).
The detergents 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), cholesteryl hemisuccinate Tris salt (CHS), and n-dodecyl-β-D-maltoside (DM) were obtained from Anatrace. N-Octyl-β-D-glucopyranoside (OG) was purchased from Calbiochem.
The lipids 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were obtained from Avanti Polar Lipids, Inc.
The deuterated chemicals D2O (99%), imidazole-d4 (98%), and dodecylphosphocholine-d38 were purchased from Cambridge Isotope Laboratories.
Construction of plasmids for CB2 expression
Plasmid pAY105 for expression of human CB2 protein (fused to the E. coli maltose-binding protein at the N-terminus, and to the E. coli thioredoxin followed by deca-histidine tag at the C-terminus) was constructed by ligating the 1.1-kb BamHI–NotI CB2-gene-encoding fragment of pRG611 into the 7.3-kb BamHI–NotI fragment of vector pRG624 (Fig. 1). The corresponding fusion protein CB2-105 consists of E. coli MBP (Lys1 to Thr391) followed by Gly-Ser, CB2 (Glu1 to Cys359), E. coli thioredoxin (TrxA, Ser2 to Ala109), Gly-Thr, and a deca-histidine tag (H10).
Plasmid pAY107 was constructed by ligating the 1.1-kb BamHI–NotI fragment of pRG 611 into the 7.5-kb BamHI–NotI fragment of vector pRG 1023. The resulting plasmid (Fig. 1) encodes a CB2-107 that contains two TEV protease-recognition sites flanking CB2 with residues Gly-Ser-Asn10-Glu-Asn-Leu-Tyr-Phe-Gln-Ser-Gly-Ser located upstream of CB2 and residues Ala3-Asn5-Gly3-Ser-Glu-Asn-Leu-Tyr-Phe-Gln-Ser-Gly3-Ser-Glu-Phe located downstream of CB2.
The expression vector is based on pMal-p2 plasmid (New England Biolabs), in which the tac promoter region of the plasmid was replaced with the lac promoter and the double ribosome-binding site of the vector pASK40 (Tucker and Grisshammer 1996). Part of the M13 ori in this plasmid is replaced with the wild-type hok/sok gene cassette to prevent plasmid loss during prolonged fermentation (Tucker and Grisshammer 1996).
Spheroplasts of E. coli and a cytoplasmic membrane fraction were prepared according to the protocols by Weiss (1976) and Tai and Kaplan (1985) with the following modifications: E. coli cells were collected by centrifugation, washed twice with 0.1 M Tris-HCl (pH 8.0) buffer, and resuspended in 0.1 M Tris-HCl (pH 8.0) buffer containing 20% (w/v) sucrose, to adjust an optical density of the cell suspension to 10 at 600 nm. A cocktail of protease inhibitors (Roche) was added at this stage. The temperature was adjusted to 37°C, and a solution of lysozyme (2 mg/mL) was added slowly, under constant stirring, until the final concentration of lysozyme of 0.1 mg/mL was reached. The cell suspension was incubated at 37°C for another 15 min with stirring. A solution of 0.1 M EDTA (pH 7.0) was added slowly under continuous stirring until the final concentration of EDTA of 10 mM was reached. The incubation continued for another 10 min.
Spheroplasts were centrifuged at 12,000g for 20 min. The pellet was collected and washed once with 0.1 M Tris-HCl (pH 8.0) containing 20% sucrose. Spheroplasts were centrifuged again at 12,000g for 20 min. The pellet of spheroplasts was resuspended in ice-cold water, which resulted in osmotic lysis. The remaining intact cells were broken up by mild sonication. Immediately, 1 M Tris buffer (pH 8.0) containing protease inhibitors was added to produce a final Tris concentration of 50 mM. A 1 M solution of MgCl2 was added to achieve a final concentration of 1 mM. A solution of DNAse I was added, the cell-free extract was briefly sonicated, and further incubated on ice for 1 h. The extract was centrifuged at 150,000g for 1 h, and the membrane pellet was washed with Tris buffer and centrifuged again at 150,000g for 1 h. The membrane pellet was resuspended in a small volume of Tris buffer, frozen in liquid nitrogen, and stored at −80°C.
Solubilization of CB2 fusion protein
Solubilization was carried out at 4°C. Frozen cells (80 g) were homogenized in 600 mL of solubilization buffer (50 mM Tris-HCl at pH 7.5, 200 mM NaCl, 0.5% [w/v] CHAPS, 0.1% [w/v] CHS, 1% [w/v] DM, 5 mM MgCl2, and 30% glycerol) supplemented with DNAse I (10 μg/mL) and EDTA-free protease-inhibitor cocktail (Roche). The mixture was sonicated for 15 min (Branson Sonifier 250, 1/2-inch flat tip, output 6, duty cycle 50%). After stirring the extract for another 40 min, the cell debris was removed by centrifugation (Beckman 70Ti rotor, 50,000 rpm, 1 h). The sample was passed through a 0.2-μm filter and purified by immobilized metal affinity chromatography.
Purification of the CB2-107 fusion protein
Purification of the CB2-107 fusion protein was performed with a 20-mL Ni-NTA agarose (QIAGEN) column at a flow rate of 0.5 mL/min in buffer A (50 mM Tris-HCl at pH 7.5, 200 mM NaCl, 30% glycerol, 0.5% CHAPS, 0.1% CHS, 0.1% DM), on an AKTA-Purifier 100 system equipped with a sample valve S1 with air sensor and a fraction collector Frac950. After loading of the protein extract, the column was washed with 10 column volumes of 30 mM imidazole in buffer A and recombinant protein eluted with a linear gradient of imidazole in buffer A. Fractions of 4 mL were collected and analyzed for the presence of CB2 fusion protein by Western blot. Fusion CB2 protein was eluted at imidazole concentrations in the range from 80 to 150 mM.
Fractions containing recombinant CB2 were pooled together and concentrated 10-fold on an Apollo 20-mL centrifugal spin concentrator (hydrophilic membrane, 70 kDa molecular mass cutoff; Orbital Biosciences). Concentrated fractions were diluted 10-fold with buffer B (50 mM Tris-HCl at pH 7.5 containing 30% glycerol, 0.5% CHAPS, 0.1% CHS, and 0.1% DM) and loaded onto a Source 30Q column (7.6-mL volume; Amersham). Recombinant CB2 was eluted with a linear gradient of NaCl in buffer B. Fractions (4 mL) were collected and analyzed for the presence of recombinant CB2 by Western blot. Fractions containing CB2 were pooled together and concentrated using a centrifugation filter device.
Preparation of CB2 labeled with fluorescent dye Alexa Fluor 532
Recombinant CB2 protein (1 mg) in buffer A, purified by chromatography on Ni-NTA- and Source 30Q columns as described above, was incubated with 2 mL of a Ni-NTA agarose slurry (QIAGEN) overnight on a shaker. Resin retaining immobilized protein was packed into a 10-mL EconoPack column, and the buffer was exchanged by passing 30 column volumes of 50 mM sodium phosphate (pH 7.5) containing 30% glycerol, 0.5% CHAPS, and 0.1% DM. The resin was resuspended in 2 mL of sodium phosphate buffer with detergents, and 100 μL of Alexa Fluor 532 reactive dye (succinimidyl ester, Molecular Probes) in 0.1 M Na-bicarbonate buffer was added. After incubation for 2 h on a shaker, the resin was transferred into a small EconoPack column and washed with 50 column volumes of buffer A to remove the nonreacted dye. Labeled protein was eluted with 200 mM imidazole in buffer A and concentrated in a centrifugal spin concentrator (Orbital Bioscience). All steps were performed at 4°C. Efficiency of labeling was determined by measuring absorbance of the conjugate solution at 280 nm and 530 nm, according to the protocol recommended by the manufacturer (Molecular Probes).
Recombinant CB2 fusion protein synthesized in E. coli was detected by Western blot with anti-MBP sera (1:2000; New England Biolabs), anti-CB2 polyclonal antibody (1:2000; Cayman Chemical), or with anti-His INDIA-HRP reagent (Pierce). The Western blot was developed with anti-rabbit IgG conjugated with fluorescein (Amersham) and scanned on a Typhoon 8600 fluorescent scanner (Amersham).
Protein concentration in the presence of high detergent concentrations was determined using a Dc protein assay reagent (BioRad).
CB2 binding assays
The ligand-binding analysis with [3H] CP-55,940 (Perkin-Elmer) on E. coli membranes was performed essentially as described in Felder et al. (1992) using a Brandel cell-harvesting device. Briefly, E. coli membranes were incubated with 0.5 nM [3H] CP-55,940 and different concentrations of unlabeled ligands for 60 min at 30°C. The incubation (binding) buffer consisted of 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 3 mM MgCl2, and 0.14 % (w/v) bovine serum albumin. Nonspecific binding was determined in the presence of 1 μM unlabeled CP-55,940. Free and bound radioligands were separated by rapid filtration through pre-soaked Whatman GF/B filters. The filters were washed three times with 4 mL of cold binding buffer, and the amount of radioisotope retained on the filter was determined by liquid scintillation counting. Data from ligand binding were analyzed using GraphPad Prizm Software. IC50 and Ki values were determined through nonlinear regression analysis. Kd and Bmax values were determined from saturation binding experiments.
Ligand-binding assay on Anopore filters
Anopore filters (Whatman) were used as an alternative method to perform the ligand-binding assay on the E. coli membranes expressing recombinant cannabinoid receptor. Briefly, the procedure used to perform the binding assay was as follows. Anopore filters (25 mm diameter, 0.2 μm pore size) were pre-loaded with the E. coli BL21-107 membranes expressing CB2 by slowly passing a membrane suspension in 50 mM Tris-HCl (pH 7.5) buffer through the filter on a Millipore vacuum-filtration manifold. Filters were washed by passing 10 mL of binding buffer containing 50 mM Tris (pH 7.5) buffer, 3 mM MgCl2, and 0.1% bovine serum albumin. Then 1 mL of the solution of radiolabeled ligand and variable concentrations (from 1 × 10−11 M to 1 × 10−5 M) of unlabeled ligand (CP-55,940) in binding buffer was applied to the Anopore filter and slowly passed through the filter after vacuum was applied. Filters were briefly washed three times with 2 mL of binding buffer and immersed in the scintillation liquid, and activity was counted.
Reconstitution of CB2 receptor with G-protein subunits
The receptor-catalyzed GDP-[35S]GTPγS exchange was measured by incubation of variable amounts of urea-treated membrane preparations of CB2-107 with the Gαi subunit in the presence of a saturating concentration (1 μM) of Gβγ subunits, essentially as described in Glass and Northup (1999). Urea-treated membranes were prepared as follows. The BL21-CB2 membrane preparation (3 mL of a 20 mg/mL protein concentration) was diluted with 11 mL of 10 mM MOPS (pH 7.5) buffer on ice. After centrifugation at 35,000 rpm for 20 min in a 45 Ti rotor, the supernatant was discarded and the pellet was resuspended in 8 mL of 7 M urea in 10 mM MOPS (pH 7.5) buffer. After incubation on ice for 30 min, the suspension was diluted with 6 mL of 10 mM MOPS buffer, and the membranes were precipitated by centrifugation at 50,000 rpm for 30 min. The pellet was resuspended in 10 mM MOPS buffer and centrifuged at 35,000 rpm for 20 min. The resulting pellet was resuspended on ice in 2 mL of 10 mM MOPS buffer containing 12% sucrose, and stored at −80°C.
For the G-protein-coupled assay, E. coli CB2-107 membrane preparations were resuspended in the reaction buffer (50 mM MOPS buffer at pH 7.5 containing 1 mM EDTA, 1 mM MgSO4, 1 mM DTT, 1 mg/mL bovine serum albumin), and G-protein-binding activity was measured upon activation of recombinant receptor with agonists CP-55,940 or WIN 55,212-2 mesylate (concentration 1 μM). A 30-min incubation was started by supplementing the reaction mixture with 200 nM Gαi and saturating concentrations (1 μM) of Gβγ and terminated by rapid addition of 2 mL of cold wash buffer (20 mM Tris-HCl at pH 8.0, 100 mM NaCl). The reaction mixture was filtered over nitrocellulose membrane on a Millipore vacuum manifold. Experiments were performed in siliconized test tubes to decrease nonspecific binding of the receptor to the tube surface.
CB2-107 ligand-binding studies in the micellar state by NMR
Proton high-resolution NMR experiments were conducted at a resonance frequency of 500.13 MHz on a DMX500 NMR spectrometer (Bruker Biospin) equipped with an inverse triple resonance probe head with actively shielded x,y,z gradient coils. The signal of protonated water was suppressed by a Double Pulsed Field Gradient Spin Echo as described in Hwang and Shaka (1995). Depending on ligand concentration, from 500 to 10,000 scans with a delay time of 6 sec were acquired. Sample temperature was controlled to 4.0 ± 0.1°C using a Bruker BVT2000 variable temperature accessory. Data were analyzed using XWINNMR 3.1 software (Bruker, Biospin).
Experiments were conducted on 600 μL of a CB2 solution in DPC micelles. The ligands CP-55,940 and anandamide were added as stock solution in DPC. The ligand stock solutions contained equimolar amounts of DMSO to provide a standard for calibration of ligand resonance intensity. The ligands were added directly into the NMR tube. Samples were homogenized after every addition of ligand by inverting the sealed tubes repeatedly with a motorized device for 15 min at ambient temperature.
Reconstitution of CB2-107 into lipid bilayers and depositioning into Anopore membranes
The solution of CB2 in 50 mM Tris-HCl buffer containing a mixture of the triple detergents (0.1%DM/0.1% CHAPS/0.1% CHS), 100 mM NaCl, and 30% glycerol was mixed with an appropriate amount of a 20 mg/mL solution of SOPC in 3% OG to achieve a ratio of protein to lipid of 1:1 (w/w). For fluorescence studies, 1 μg of fluorescently labeled lipid (Dil C18, 1 mg/mL solution in methanol) was added per 1 mg of SOPC as well as 1 μg of CB2 labeled with fluorescent dye Alexa Fluor 532 (efficiency of labeling: 3.6 molecules of dye per molecule of CB2) added per 1 mg of unlabeled purified CB2. The CB2 and SOPC mixture was rapidly diluted 20-fold into 50 mM Tris-HCl buffer, incubated at room temperature for 10 min, and passed (twice) through a wet Anopore filter (0.2 μm pore size, 25 mm diameter). Filters were washed with Tris-HCl buffer and scanned on a Typhoon 8600 fluorescence scanner (Amersham Biosciences). DilC18 was excited at 633 nm, and emission was detected at 670 nm (photomultiplier setting 500, normal sensitivity). Alexa Fluor 532 was excited at 532 nm and emission was registered at 610 nm (PMT setting 500, normal sensitivity). Quantitation of the amounts of lipid and protein deposited into the Anopore filter was performed using the computer program ImageQuant.