Activation of Cannabinoid Receptors in Rat Brain by WIN 55212-2 Produces Coupling to Multiple G Protein a-Subunits with Different Potencies

Previous studies had shown that the amplification factors for cannabinoid receptors, defined as the number of total G proteins activated per occupied receptor, differs between several rat brain regions. In this study, we sought to determine which specific Gi/Goa subunits were activated by CB1 receptors in several rat brain regions and if this coupling might explain the regional differences in receptor/G protein amplification factors. Furthermore, we examined whether cannabinoid agonists might activate different subtypes of Ga subunits with varying degrees of efficacy and/or potency. Activation of specific G proteins by cannabinoid receptors was evaluated by the ability of the agonist WIN 55212-2 to stimulate incorporation of [a-P]azidoanilido-GTP into Ga subunits in membranes. Photolabeled G proteins were either directly resolved using urea/ SDS-polyacrylamide gel electrophoresis or first immunoprecipitated with specific antisera for different Ga subunits before electrophoresis. Individual Ga subunits were separated into distinct bands on a single gel and the amount of agonistinduced increase in radioactivity was quantified by densitometry. Stimulation of CB1 receptors by WIN 55212-2 resulted in the activation of a distinct pattern of at least five different Gia/Goa subunits in several brain regions. Furthermore, although the pattern of G proteins activated by WIN 55212-2 appeared to be similar across brain regions, slight differences were observed in both the percentage of increase and the amount of the individual Ga subunits activated. Most importantly, the amount of WIN 55212-2 required to half-maximally activate individual G proteins in the cerebellum varied over a 30-fold range for different Ga subunits. These results suggest that cannabinoid receptors activate multiple G proteins simultaneously in several brain regions and both the efficacy and potency of cannabinoid agonists to activate individual Ga subunits may vary considerably. D-Tetrahydrocannabinol is the principal psychoactive ingredient found in the plant Cannabis sativa (marijuana) and it produces its effects by interacting with CB1 and CB2 cannabinoid receptors (Dewey, 1986; Howlett, 1995). CB1 receptors (CB1 and CB1A) are located primarily in the central nervous system, whereas CB2 receptors are found principally in the periphery (Munro et al., 1993; Shire et al., 1995). All subtypes of cannabinoid receptors belong to the large superfamily of G protein-coupled receptors (GPCRs) that traverse the plasma membrane seven times and activate intracellular G proteins. Heterotrimeric G proteins are composed of three distinct subunits, a (39–50 kDa), b (35–36 kDa), and g (6–10 kDa) and their activation by GPCRs produces an exchange of GTP for GDP on the a-subunits. This results in the dissociation of the G protein from the receptor and the separation of the a-GTP from the bg-subunits. Both the free a-GTP and bg-subunits then proceed to regulate various downstream effectors (Gudermann et al., 1997). Pertussis toxin (PTX)-sensitive G proteins (i.e., Gia and Goa subtypes) appear to mediate the physiological effects of cannabinoids (Howlett, 1995), although recent studies also suggest a possible role for Gsa (Glass and Felder, 1997; Maneuf and Brotchie, 1997; Felder et al., 1998). Regulation of intracellular effectors by cannabinoid receptors includes inhibition of adenylyl cyclase (Howlett, 1984), inhibition of voltage-gated Ca channels (Mackie et al., 1995), activation of inwardly rectifying K channels (Mackie et al., 1995), and activation of mitogen-activated protein kinase (Bouaboula et al., 1995). CB1 receptors are widely distributed throughout the mammalian central nervous system in relatively high density (Herkenham et al., 1990), particularly in areas involved in This study was supported in part by National Institute on Drug Abuse Grants DA10936 (to P.L.P.) and DA06784 and DA06634 (to S.R.C.). ABBREVIATIONS: GCPR, G protein-coupled receptor; PTX, pertussis toxin; GTPgS, guanosine-59-O-(3-thio)triphosphate; AA-GTP, azidoanilidoguanosine-59-O-(3-thio)triphosphate; PAGE, polyacrylamide gel electrophoresis; IP, immunoprecipitation; AR, autoradiography; ECL, enhanced chemiluminescence. 0026-895X/00/051000-11$3.00/0 MOLECULAR PHARMACOLOGY Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics MOL 57:1000–1010, 2000 /12884/821226 1000 at A PE T Jornals on M ay 2, 2017 m oharm .aspeurnals.org D ow nladed from mediating the processes affected by marijuana. These brain regions include the cortex (cognition), hippocampus (memory), hypothalamus (body temperature), and cerebellum and basal ganglia (motor function) (Breivogel et al., 1997; Breivogel and Childers, 1998). Although the correlation between regional cannabinoid receptor number and the observed physiological effects is striking, the signal transduction mechanisms underlying these actions have not been determined. Cannabinoid agonists stimulate the binding of the hydrolysis-resistant GTP analog [S]guanosine-59-O-(3-thio) triphosphate (GTPgS), to G protein a-subunits and this can be used as a measure of receptor activation. We have recently developed a method to measure the number of G proteins activated per occupied receptor (i.e., receptor/transducer amplification factors) by calculating the ratio of the apparent Bmax of net agonist-stimulated [ S]GTPgS binding to the Bmax of receptor binding (Breivogel et al., 1997). Using this technique, we found that the amplification factors for cannabinoid receptors differed between several rat brain regions, ranging from 2.0 in the frontal cortex to 7.5 in the hypothalamus. This suggests that although some brain regions contain lower densities of cannabinoid receptors (i.e., hypothalamus), CB1 stimulation results in the activation of an equivalent or greater number of G proteins relative to regions containing higher densities of receptors (i.e., frontal cortex). Although agonist-induced [S]GTPgS binding provides valuable information concerning activation of total G proteins in a particular brain region or tissue, this technique cannot be used to examine the coupling of GPCRs to individual Ga subunits. One approach to measure the activation of specific G proteins by receptors is to use agonist-stimulated incorporation of [P]azidoanilido-GTP (AA-GTP) into Ga subunits, followed by immunoprecipitation (IP) and separation with urea/SDS-polyacrylamide gel electrophoresis with subsequent autoradiography (AR) (Prather et al., 1994a,b, 1995; Chakrabarti et al., 1995). With this technique, individual Ga subunits can be separated into distinct bands on a single gel and thus receptor coupling to individual G proteins simultaneously can be evaluated. In this study, we sought to determine which specific Ga subunits were activated by CB1 receptors in several rat brain regions and if this coupling might explain the previously observed regional differences in receptor/G protein amplification factors. Furthermore, we examined whether cannabinoid agonists might activate different subtypes of Gia/Goa subunits with varying degrees of efficacy and/or potency. Our results demonstrate that stimulation of CB1 receptors by maximally effective concentrations of WIN 55212-2 results in the activation of a distinct pattern of at least five different Gia/Goa subunits in several brain regions. Furthermore, although the pattern of G proteins activated by WIN 55212-2 appears to be similar across brain regions, slight differences are observed in both the percentage of increase and the amount of the individual Ga subunits activated. Most importantly, the amount of WIN 55212-2 required to half-maximally activate individual G proteins in the cerebellum varies over a 30-fold range for different Ga subunits. Experimental Procedures Materials. Male Sprague-Dawley rats were purchased from Zivic Miller (Zeleinople, PA). [P]GTP (3000 Ci/mmol), [S]GTPgS (1250 Ci/mmol), and antisera (EC2 and GC2) were purchased from NEN (Boston, MA). GDP for membrane [S]GTPgS binding assays and unlabeled GTPgS were purchased from Boehringer Mannheim (New York, NY). Antiserum LEP4 was a generous gift from Dr. Ping-Yee Law (University of Minnesota, Minneapolis, MN). Enhanced chemiluminescence (ECL) reagents and Hyperfilm-ECL were purchased from Amersham (Arlington Heights, IL). WIN 55212-2 and AM 281 were obtained from Tocris Cookson, Inc. (Ballwin, MO). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Membrane Preparations. Brain regions were dissected from fresh rat brains on ice. Tissue samples were pooled and homogenized with a Tissumizer (Tekmar, Cincinnati, OH) in cold assay buffer (50 mM Tris-HCl, pH 7.4; 3 mM MgCl2; 0.2 mM EGTA; and 100 mM NaCl) and centrifuged at 31,000g for 10 min at 4°C. Pellets were resuspended in membrane buffer, then centrifuged at 31,000g for 10 min at 4°C. Pellets were homogenized in membrane buffer, assayed for protein content (Bradford, 1976), and stored in aliquots at 280°C until being assayed. Photoaffinity Labeling of Ga Subunits with [ P]AA-GTP. The method for synthesis and purification of [P]AA-GTP can be found in Prather et al. (1994a). The photoaffinity labeling of Ga subunits with [P]AA-GTP also has been recently reported (Prather et al., 1994a,b, 1995; Chakrabarti et al., 1995). Plasma membranes (25 mg per assay) were incubated in the presence or absence of agonist for 6 min at 30°C in 100 ml of buffer I (50 mM HEPES, pH 7.4; 0.1 mM EDTA; 10 mM MgCl2; 30 mM NaCl; 30 mM GDP; and 0.04 U/ml adenosine deaminase). After agonist incubation, [P]AA-GTP (1 mCi /assay) was added, and samples were incubated for an additional 6 min at 30°C. The reaction was terminated by placing samples on ice. Membranes were then collected by centrifugation at 12,000g for 10 min and resuspended in 100 ml of buffer II (50 mM HEPES, pH 7.4; 0.1 mM EDTA; 10 mM MgCl2; 30 mM NaCl; and 2 mM dithiothreitol). Resuspended pellets (droplets) were then irradiated at 4°C with 240 milliJoules from an ultraviolet lamp (254 nm; 150 W) at a distance of 15 cm. Samples were centrifuged as before, resuspended in electrophoresis sample buffer, and separated by SDS-PAGE (see below). In cases where G proteins were immunoprecipitated after photoaffinity labeling, membrane pellets (100 mg/assay) were solubilized in 80 ml of 4% SDS for 10 min at room temperature. Immediately following, 560 ml of buffer A (1% Nonidet P-40, 1% desoxycholate, 0.5% SDS, 150 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, and 10 mM TrisHCl, pH 7.4) was added. Samples were then centrifuged for 10 min (12,000g) and pellets were discarded. Antiserum (10 ml) was added to supernatants and samples were constantly rotated at 0°C for 2 h. Antisera used were GC2 for Goa (Spiegel, 1990) and LEP4 for Gia1 and Gia2 (Prather et al., 1994). After incubation, 120 ml of a 12.5% suspension of protein A-Sepharose beads was added and samples were constantly rotated overnight at 0°C. The next day, samples were pelleted (12,000g for 10 min) and washed twice with 1 ml of buffer B (600 mM NaCl; 50 mM Tris-HCl, pH 7.4; 0.5% SDS; and 1% Nonidet P-40), followed by a final wash with 1 ml of buffer C (300 mM NaCl; 100 mM Tris-HCl, pH 7.4; and 10 mM EDTA). Samples were then centrifuged as before and protein A-Sepharose beads were resuspended in 100 ml of electrophoresis sample buffer. Samples were heated at 100°C for 10 min and centrifuged (12,000g for 10 min). Finally, supernatants were subjected to SDS-PAGE as described below. After electrophoresis, SDS-PAGE gels were dried and [P]AAGTP-labeled Ga subunits were visualized autoradiographically by a Molecular Dynamics Inc. PhosphorImager 445 SI (Sunnyvale, CA). Autoradiographic bands were quantified by densitometry with the National Institutes of Health Image software program (version 1.56). To determine the relative amount of radioactivity incorporated by individual G proteins, the area of each band was traced and multiplied by its mean optical density. Cannabinoid Receptor Coupling to G Proteins 1001 at A PE T Jornals on M ay 2, 2017 m oharm .aspeurnals.org D ow nladed from SDS-PAGE and Immunoblotting. To identify Ga subunits, membranes were separated on 20-cm separating gels containing 10% acrylamide and 6 M urea (Prather et al., 1994a,b, 1995; Chakrabarti et al., 1995). Before separation, samples were resuspended in 80 ml of electrophoresis loading buffer (65 mM Tris HCl, pH 6.8; 2% SDS; 10% glycerol; and 5% 2-mercaptoethanol), and heated at 90°C for 2 min. The ECL method of immunoblotting was used (Amersham). Gels were transferred to Hybond-ECL nitrocellulose membranes and incubated overnight at 4°C with 10% milk in blotting buffer (TBS0.1%; 25 mM Tris HCl, pH 7.6; 154 mM NaCl; and 0.1% Tween 20). Blots were then washed three times (5 min each) with TBS-0.1% and incubated with primary antibodies (1:1000) for 1 h at room temperature while shaking. The primary antibodies were then removed and blots were washed as described previously. Secondary antibody (donkey anti-rabbit immunoglobin horseradish peroxidase, 1:5000) was then added and incubated for 30 min, with shaking. The secondary antibody was removed and blots were washed 33 5-min with TBS0.3%, followed by 33 5-min with TBS-0.1%. Blots were then incubated for 1 min with equal volumes of ECL detection reagents 1 and 2, wrapped in plastic wrap, and exposed to Hybond-ECL X-ray film for periods varying between 30 s and 10 min. The Ga antisera used were EC2 selective for Gia3/Goa (Simonds et al., 1989), GC2 for Goa (Spiegel, 1990), and LEP4 for Gia1/Gia2 (Prather et al., 1994b). LEP4 was developed in the laboratory of Dr. Ping-Yee Law (University of Minnesota) by immunizing rabbits with a Gia1/Gia2 C-terminal peptide. Agonist-Stimulated [S]GTPgS Binding Assays. Frozen membranes were thawed and then assayed for protein (Bradford, 1976). All assays included 10 to 20 mg of membrane protein and were conducted at 30°C for 2 h with 0.1% BSA (w/v), 30 mM GDP, and 0.05 nM [S]GTPgS in a final volume of 1 ml. Nonspecific binding was determined with 30 mM unlabeled GTPgS. WIN 55212-2 concentration-effect curves were determined by incubating membranes with various concentrations of WIN 55212-2 (0.03–30,000 nM). Reactions were terminated in all tubes simultaneously by rapid filtration under vacuum through Whatman GF/B glass fiber filters, followed by three washes with cold Tris buffer, pH 7.4. Bound radioactivity was determined by liquid scintillation spectrophotometry at 95% efficiency for S after overnight extraction of the filters in 4 ml of Scintisate Econo 1 scintillation fluid. Data Analysis. Unless otherwise stated, data represent the mean 6 S.E. from at least three separate experiments that were each performed in triplicate. Data obtained from full concentration-effect curves using WIN 55212-2 were subjected to sigmoidal curve fitting with the Sigmaplot computer program. The minimum and maximum plateau values for the amount of Ga subunits activated (expressed in mean optical density units) and the amount of agonist required to produce 50% of maximal activation (ED50) were determined from the best-fit curves. The maximum amount of Ga subunits activated was defined as the difference between the minimum and maximum plateau values. Percentage of increase in G protein activation was defined as the amount of [P]AA-GTP incorporated in the presence of agonist, divided by basal incorporation, times 100%. Net agoniststimulated [S]GTPgS binding values were calculated by subtracting basal binding values (absence of agonist) from agonist-stimulated values. Statistical significance of the data was determined by ANOVA followed by comparison with either the nonpaired two-tailed Student’s t test or Tukey’s method.