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Chemically Distinct Ligands Promote Differential CB₁ Cannabinoid Receptor-Gi Protein Interactions

Neuroscience of Drug Abuse Research Program, J. L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham, North Carolina

Received August 4, 2004; accepted March 4, 2005

	Abstract

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To understand how structurally distinct ligands regulate CB₁ receptor interactions with Gi1, Gi2, and Gi3, we quantified the Gi and proteins that coimmunoprecipitate with the CB₁ receptor from a detergent extract of N18TG2 membranes in the presence of ligands. A mixture of A, R, G_GDP (or G_), and ARG_GDP (or ARG_) complexes was observed in the presence of aminoalkylindole (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN 55,212-2) for all three RGi complexes, cannabinoid desacetyllevonantradol for Gi1 and Gi2, and eicosanoid (R)-methanandamide for Gi3. Desacetyllevonantradol maintained RGi3 complexes and (R)-methanandamide maintained RGi1 and RGi2 complexes even in the presence of a nonhydrolyzable GTP analog. The biaryl pyrazole antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR141716) maintained all three RGi complexes. G proteins, and to a certain extent G2, exhibited the same association/dissociation pattern as the G proteins. A GDP analog had no influence on any of these association/dissociation reactions and failed to promote sequestration of G proteins. These results can be explained by invoking the existence of an inverse agonist-supported inactive state in the ternary complex equilibrium model. WIN 55,212-2 behaves as an agonist for all three Gi subtypes; SR141716 behaves as an inverse agonist for all three Gi subtypes; desacetyllevonantradol behaves as an agonist for Gi1 and Gi2, and an inverse agonist at Gi3; and (R)-methanandamide behaves as an inverse agonist at Gi1 and Gi2, and an agonist at Gi3. These ligand-selective G protein responses imply that multiple conformations of the receptor could be evoked by ligands to regulate individual G proteins.

Our studies herein examine the molecular mechanism for the agonist-receptor-G protein selectivity for the CB₁ cannabinoid receptor. The CB₁ receptor is a GPCR found abundantly in brain and neuronal cells and is coupled to the Gi/o family of G proteins to regulate effectors such as adenylyl cyclase and ion channels (Howlett et al., 2002). The CB₁ receptor exhibits properties of agonist-independent receptor-G protein precoupling and constitutive activity in both recombinant (Bouaboula et al., 1997; Pan et al., 1998; Vasquez and Lewis, 1999) and native cell models (Pan et al., 1998; Meschler et al., 2000; Sim-Selley et al., 2001). CB₁ receptor-G complexes readily exist in the absence of exogenously added agonist or inverse agonist ligands (Houston and Howlett, 1993; Howlett et al., 1999; Mukhopadhyay et al., 2000; Mukhopadhyay and Howlett, 2001).

We hypothesized that structurally distinct ligands would exhibit differential ability to regulate CB₁ receptor interactions. To test this hypothesis, we used a well-characterized neuronal model for CB₁ cannabinoid receptor-mediated signal transduction, the N18TG2 neuroblastoma cell, which endogenously expresses CB₁ receptors and all three subtypes of Gi (Mukhopadhyay et al., 2002). We quantified the Gi and proteins that coimmunoprecipitate with the CB₁ receptor from a CHAPS extract of N18TG2 cell membranes. We demonstrate here that the aminoalkylindole WIN 55,212-2, the cannabinoid DALN, and the eicosanoid (R)-methanandamide promote a mixture of receptor-Gi complexes and free receptors differentially depending upon the Gi subtype. SR141716 maintained the receptor in a complex with all three Gi subtypes. These results also provide evidence for the differential behavior of these ligands as agonists or inverse agonists, depending on the Gi subtype. A simplified working model is depicted in Fig. 1 as a basis for developing a platform for understanding the emerging data.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Equilibrium ternary complex model of ligand-receptor-G protein regulated by agonists or inverse agonists. In the ternary complex model of agonist action (Leff et al., 1997), the receptor is denoted as R in the ground state and R* in the state that activates the G protein, and the heterotrimer bearing GDP is denoted as GGDP. An agonist (A) can bind either to R or to RGGDP complexes, creating an equilibrium with the "ternary complex" ARGGDP. Consistent with evidence that CB1 receptors exhibit constitutive activity (Bouaboula et al., 1997), a probability exists that a fraction of the RGGDP complexes become spontaneously activated in the absence of agonist. Isomerization of the RGGDP complex or ARGGDP ternary complex to an active state will lead to dissociation of GDP. In an intact cell in which GTP is abundant, the resulting R*G_ or AR*G_ complexes readily bind GTP, and dissociation of the heterotrimer allows GGTP and G proteins to interact with effectors (Waelbroeck, 1999). In an experimental situation in which GTPS is present in high concentrations, the transiently empty G protein (R*G_ or AR*G_) is rapidly filled by GTPS. Steps that would exclude the complex from re-entering the equilibrium (inside the box) would include 1) GDP dissociation in the absence of added exogenous GDP or a GDP analog, and 2) GTPS/GDP exchange and the subsequent dissociation of GGTPS. Addition of the inverse agonist ligand (I) to precoupled RGGDP induces IRoGGDP, originally proposed by Bouaboula and colleagues to describe a mechanism for the CB1 receptor to "sequester" Gi proteins, thereby explaining their data that basal signal transduction was blocked in the presence of SR141716 (Bouaboula et al., 1997). The working model has depicted this complex as existing outside of the equilibrium box; however, evidence suggests that CB1 agonists can compete (Meschler et al., 2000), demonstrating the reversibility of this step.

	Materials and Methods

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CB₁ Receptor Antibody and Affinity Matrix Preparation. Rabbit polyclonal antibodies were raised against the N-terminal 14 amino acids of the CB₁ receptor as described previously (Howlett et al., 1998; Mukhopadhyay and Howlett, 2001). Anti-CB₁(1–14) was affinity-purified using a peptide comprising the N-terminal 14 amino acid residues of the rat CB₁ receptor as the affinity ligand attached to agarose matrix using the SulfoLink Immobilization procedure (Pierce, Rockford, IL). An affinity resin for the rat CB₁ cannabinoid receptor was prepared by coupling affinity-purified anti-CB₁(1–14) to Affi-Prep-Hz matrix (Bio-Rad) according to the manufacturer's instructions. This method binds periodate-oxidized carbohydrate moieties on the antibody heavy chain to hydrazide-activated methacrylate matrix (O'Shannessy and Hoffman, 1987).

Membrane Preparation, Detergent Solubilization, and Treatments. N18TG2 neuroblastoma cells were grown in Dulbecco's modified Eagle's medium with 10% heat-inactivated calf serum and 1% penicillin-streptomycin to 90% confluence. Cells were then harvested with phosphate-buffered saline/EDTA, sedimented, and the cell pellet was homogenized in a glass homogenizer in ice-cold HME buffer (20 mM sodium-HEPES, pH 8.0, 2 mM MgCl₂, and 1 mM EDTA). After sedimentation at 1000g for 5 min at 4°C to remove unbroken cells and nuclei, the supernatant was collected and sedimented at 17,000g for 20 min at 4°C. The pellet (P2 membrane fraction) was resuspended in HME, and the protein concentration was determined (Bradford, 1976). For solubilization, 5 mg of membrane protein was sedimented at 17,000g, resuspended in 500 µl of solubilization TM buffer (30 mM Tris-Cl, pH 7.4, and 5 mM MgCl₂) containing 4 mg of CHAPS and 20% glycerol according to the method described by Houston and Howlett (1993). CHAPS extracts were treated with the indicated CB₁ receptor ligands at varying concentrations (10 nM to 1 µM) in the presence or absence of 100 µM GTPS, GppNHp, or GDPS in a final volume of 100 µl of TM buffer for 20 min at 30°C. Control samples were treated with the vehicle for the ligands (TM buffer) under identical conditions. The ligands and guanine nucleotides were present throughout the immunoprecipitation procedure.

Immunoprecipitation. After the incubation, the immunoprecipitation of the CB₁ receptor and associated proteins from ligand- or guanine nucleotide-treated CHAPS extracts was performed by following the method used in this laboratory previously (Mukhopadhyay et al., 2000; Mukhopadhyay and Howlett, 2001). A 100-µl aliquot of the ligand- or guanine nucleotide-treated CHAPS extract was incubated under constant rotation with Sepharose bead-coupled anti-CB₁ antibody (20 µl) for 6 h at 4°C. Thus, the addition of antibody-coupled matrix to the solubilized preparation resulted in a 20% dilution of the ligands or guanine nucleotides. The anti-CB₁ affinity matrix was then sedimented at 17,000g for 5 min, and matrix was washed three times with 500 µl of TBST buffer (20 mM Tris-Cl, pH 7.4, 140 mM NaCl, and 0.1% Tween 20). Immunoprecipitated protein was eluted from the matrix with 50 µl of glycinechloride, pH 2.5 (100 mM), and the eluate was immediately neutralized with 450 µl of Tris-Cl, pH 8.0 (1.5 M). The protein from the neutralized eluate was precipitated by the addition of 8 volumes of CHCl₃/CH₃OH/H₂O (1:4:3), dissolved in Laemmli sample buffer containing 5 mM EDTA, and heated at 65°C for 5 min. Samples were subjected to polyacrylamide gel electrophoresis on 10% polyacrylamide/0.1% SDS/6 M urea gels.

Western Immunoblot Analysis. Electrophoretic transfer of proteins from the gel to polyvinylidene difluoride membranes was carried out in 10 mM CAPS buffer with 0.01% SDS, pH 11, for 16 h (0–4°C) at 20 V using a Bio-Rad Trans-Blot Cell equipped with a cooling coil. Blots were rinsed with TBS buffer and were incubated with blocking buffer (5% nonfat dry milk plus 5% normal goat serum in TBS) at room temperature for 1 h to eliminate nonspecific binding. Blots were then incubated with affinity-purified anti-CB₁(1–14) combined with the indicated antibodies to Gi (1:1000), G (subtypes 1–4), or G2 in blocking buffer for 90 min at room temperature, followed by washing three times with TBS containing 0.1% Tween 20. Control experiments were performed using separate incubations with individual antibodies, and the results were the same as experiments stained with combined antibodies. Blots were incubated with horseradish peroxidase-coupled anti-rabbit and anti-mouse IgG sequentially for 1 h at room temperature, followed by one rinse with TBS, seven rinses with TBST, and four rinses with water. Immunoreactive bands were detected by ECL reaction and exposure of Hyperfilm. Densitometric scanning was analyzed using a modified version (version 1.59) of the Scion Image software (Scion Corporation, Frederick, MD) or using Alpha Innotech software (Alpha Innotech, San Leandro, CA). Data analysis and figures were produced using Prism 3 (GraphPad Software Inc., San Diego, CA).

	Results

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Ligand-Mediated Redistribution of the CB₁ Receptor and Specific Gi Proteins. CB₁ receptors solubilized from the membrane in CHAPS detergent exist in a state that is associated with various subtypes of the Gi protein family (Gi1, Gi2, and Gi3) in the absence of exogenously added agonists (Fig. 2). It is particularly interesting to note that a significant fraction of the Gi proteins present in the CHAPS extract are coimmunoprecipitated with the CB₁ receptors [compare lane 1 (Load) with lane 2 (Immunoprecipitated)]. Only a limited fraction of residual Gi proteins remained in the supernatant fraction (lane 3) or in any of the subsequent washes of the affinity matrix-bound CB₁ receptor-G protein complex. This indicates that the CB₁ receptor preferentially exists as a receptor-G protein complex in detergent solution under these experimental conditions. This association can be disrupted by incubation with pertussis toxin, demonstrating that the receptors and G proteins exist in a dynamic association/dissociation reaction mixture in detergent solution (Howlett et al., 1999; Mukhopadhyay and Howlett, 2001). If these receptor-G protein complexes are functional, then they should be targets for functional interaction with CB₁ receptor ligands. Experimental conditions were chosen in which GTP and GDP are absent so that association/dissociation reactions could proceed in which free agonist, receptor, and G protein could coexist with ternary complexes. In the absence of GTP, the G protein cycle would not be able to continue through GTPase-dependent hydrolysis and reassociation of Gi_GDP with G. The coimmunoprecipitation method can provide a quantitative measure of the ability of ligands to modify the distribution of free versus complexed receptors and G proteins.

View larger version (60K): [in this window] [in a new window]   Fig. 2. Coimmunoprecipitation of CB1 receptor-Gi complexes from CHAPS-solubilized N18TG2 cell membranes. CHAPS extracts were prepared and the immunoprecipitation procedure was followed with care to preserve equivalent volumes at each step. Lanes were as follows: 1, load: CHAPS extract from 50 µg of N18TG2 membrane protein (400 µl) mixed with 100 µl of TM buffer as control; 2, immunoprecipitate: CHAPS extract from N18TG2 membranes (400 µl) mixed with 100 µl of Sepharose-anti-CB1 antibody affinity matrix. Proteins were eluted from the affinity matrix, neutralized, and sedimented as described in the text; immunoprecipitated proteins were dissolved in 500 µl of TBST; 3, supernatant: CHAPS extract remaining after the affinity matrix-bound protein was removed (approximately 475 µl); 4 to 6, washings 1 to 3: supernatants remaining after the affinity matrix-bound protein was washed with 500 µl of TBST as described in the text. For each of these fractions, a 25-µl aliquot was added to 25 µlof2x Laemmli sample buffer containing EDTA, and 35 µl of this mixture was loaded on the lane. Western blot analysis was carried out by costaining with both anti-CB1 receptor antibody and anti-G: Gi1 (top), Gi2 (middle), or Gi3 (bottom). Immunoreactive bands were visualized by ECL as described in the text.

Three structurally different CB₁ receptor agonist classes were tested to determine their effects on CB₁ receptor-Gi (Gi1, Gi2, or Gi3) complexes in CHAPS-solubilized N18TG2 cell membranes. Representative Western immunoblots depicting the effects of ligands and the nonhydrolyzable GTP analog, GTPS, are shown in Fig. 3. The immunoblots depict the CB₁ receptor monomer found in cultured neuronal cells and the G subunits coimmunoprecipitated with the receptor in the same lane (Fig. 3, A–C; each lane 1, top, middle, and bottom). The ratio of the densities of the G protein band compared with the CB₁ receptor band were calculated from multiple experiments, and the means and standard errors from multiple experiments are shown in Figs. 4 and 5. The aminoalkylindole ligand WIN 55,212-2 evoked partial dissociation of all three subtypes of Gi proteins from the receptor, reaching a maximum dissociation of only 50% of the control amount of receptor-Gi complexes (Figs. 3A and 4A). WIN 55,212-2 was relatively more potent in dissociating the receptor-Gi1 complex, achieving a maximal dissociation at 10 nM. In contrast, the dissociation of Gi2 and Gi3 from the receptor occurred between 10 and 100 nM. The cannabinoid ligand DALN (Figs. 3B and 4B) dissociated Gi1 and Gi2 from the CB₁ receptor-Gi complex in a dose-dependent manner. Gi2 was dissociated completely from the receptor at 1 µM DALN (Fig. 4B). CB₁ receptor-Gi1 dissociation reached a maximum of approximately 50% at 100 nM, with no further dissociation with increasing agonist concentrations. DALN had no effect on CB₁ receptor-Gi3 complexes. The eicosanoid (R)-methanandamide evoked dissociation of only CB₁ receptor-Gi3 (Figs. 3C and 4C), and this disruption was nearly complete at 100 nM. Unlike WIN 55,212-2 or DALN, (R)-methanandamide failed to produce any dissociation of CB₁ receptor-Gi1 or Gi2 complexes.

View larger version (34K): [in this window] [in a new window]   Fig. 3. Western blot analysis of the effects of CB1 receptor agonists and GTPS on Gi protein association with the CB1 receptor. CHAPS detergent extracts of N18TG2 cell membranes were incubated for 20 min at 30°C with vehicle (lanes 1 and 5), 10 nM (lanes 2 and 6), 100 nM (lanes 3 and 7), or 1 µM (lanes 4 and 8) concentrations of the aminoalkylindole WIN 55,212-2 (A), cannabinoid DALN (B), or (R)-methanandamide (C) in the absence (lanes 1–4) or presence (lanes 5–8) of 100 µM GTPS. The CB1 receptor and associated proteins were immunoprecipitated with affinity-purified anti-CB1(1–14), and Western blot analysis was performed as described in the text. Each blot was costained for both CB1 receptor and either Gi1 (top), Gi2 (middle), or Gi3 (bottom) proteins. Immunoreactive bands were visualized by ECL. Results are shown from a single representative of at least three experiments. The lower mobility band is the CB1 receptor at 64 kDa, and the higher mobility band is the Gi protein at approximately 40 kDa.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Agonist-evoked dissociation of Gi subtypes from the CB1 receptor by WIN 55,212-2 (A), DALN (B), and (R)-methanandamide (C). Western blots were performed as described in Fig. 3, and the densities of the bands on the film were quantified. For each lane, the ratio of the density of the Gi band compared with the CB1 receptor was calculated to normalize the data and correct for potential sample loading differences, transfer, or staining variability. For each experiment, the ratio of Gi/CB1 band density for the control (vehicle) was defined as 100%, and the ratios of Gi/CB1 band densities for the experimental samples were expressed in relation to the control (% Control). The mean and S.E.M. values from three separate experiments were determined. Where error bars are not showing, the bars were smaller than the symbol. Data points were plotted, and spline or straight lines were drawn to connect the points for ease of visualization.

View larger version (43K): [in this window] [in a new window]   Fig. 5. Interactions of agonist-occupied CB1 receptor and guanine nucleotide-occupied Gi. CHAPS extracts from N18TG2 cell membranes were incubated with 100 µM GTPS (A–C) or GDPS (D–F) in the absence or presence of various concentrations of WIN 55,212-2 (A and D), DALN (B and F), or (R)-methanandamide (C and F) as indicated. Immunoprecipitation, Western blotting, and data analyses were carried out as described in the text and in legends to Figs. 3 and 4. A to C, data are the mean and S.E.M. from n = 3 independent experiments. A two-way ANOVA was used to determine the contribution of variance. A significant difference from GTPS alone is indicated by +, p < 0.05, and by *, p < 0.001. D to F, data are the mean and S.D. from n = 2 (WIN 55,212-2) or n = 3 (DALN or (R)-methanandamide) independent experiments. Data were analyzed by one-way ANOVA followed by Bonferroni's multiple comparison test. A significant difference (p < 0.05) from control is indicated by + and from GDPS is indicated by #. ND, not determined.

Effect of Guanine Nucleotides on the CB₁ Receptor-Gi Complex. Incubation of the CHAPS extract of N18TG2 membranes with the nonhydrolyzable GTP analog GTPSat 100 µM resulted in 85 to 100% dissociation of all three CB₁ receptor-Gi complexes (Fig. 3, A–C, lane 5 for each Gi subtype; Fig. 5, A–C). The addition of GppNHp (100 µM) also resulted in complete dissociation of all CB₁ receptor-Gi complexes (data not shown). The observation of complete receptor-Gi dissociation suggests that the GDP-GTPS exchange seems to have gone to completion under the assay conditions used in the present study. In the absence of agonist ligands, this would represent spontaneous dissociation of GDP from the receptor-G_GDP complex, perhaps as a result of the spontaneous isomerization to the activated state, exchange of GDP for GTPS, and dissociation of the heterotrimer to free receptor and Gi_GTPS. This process could have been facilitated by the absence of exogenous Na⁺ in the assay solutions.

The ability of GTPS to promote dissociation of the CB₁ receptor-Gi proteins was influenced differentially depending on the ligand and the Gi subtype. One sees little influence of WIN 55,212-2 on any of the three Gi_GTPS dissociated states, consistent with the relative nonselectivity for any of the Gi subtype (Figs. 3A, lanes 5–8, and 5A). DALN had no influence on the ability of GTPS to promote dissociation of the CB₁ receptor-Gi2 complex and only limited influence on the CB₁ receptor-Gi1 complex (Figs. 3B, lanes 5–8, and 5B). In similar experiments using an alternative GTP analog, GppNHp, dissociation of CB₁ receptor-Gi1 and Gi2 complexes was complete in the presence of DALN (data not shown). (R)-Methanandamide had little influence on the Gi_GTPS-dissociated state for Gi3 (Figs. 3C, lanes 5–8, and 5C). In contrast, the cannabinoid ligand DALN precluded the Gi3_GTPS dissociation and partially attenuated the Gi1_GTPS dissociation (Figs. 3B and 5B). (R)-Methanandamide potently (10 nM) attenuated the Gi1_GTPS dissociation, and concentrations between 100 and 1000 nM attenuated the Gi2_GTPS dissociation (Figs. 3C and 5C).

To assess the possible spontaneous GDP release in the association/dissociation reaction, the CB₁ receptor-Gi complexes were incubated in the presence of a high concentration (100 µM) of the GDP analog GDPS. The addition of GDPS to the detergent extract of N18TG2 membranes neither increased nor decreased the ratio of any of the Gi subtypes to CB₁ receptor in the immunoprecipitate (Fig. 5, D–F, bars 1 versus 2). If there existed any unoccupied Gi_ in the extract, it would have been predicted that GDPS would bind, thereby promoting formation of additional heterotrimer (Gi_GDPS-) that would have been able to associate with the CB₁ receptor. The failure of the GDP analog to promote a greater abundance of CB₁R-Gi complexes than in control extracts suggests that the CB₁ receptor-G protein association was at its maximum as it existed in the CHAPS extract. The addition of GDPS failed to alter the CB₁R-Gi complex when incubated with cannabinoid receptor ligands (Fig. 5, D–F, bars 3 versus 4). This observation would support predictions from the ternary complex model that the GDP analog should not promote dissociation of the agonist-bound CB₁ receptor-G heterotrimer complexes.

Inverse Agonist Influence on CB₁R-Gi Complexes. SR141716 is a CB₁ receptor-selective competitive antagonist that has been shown to exhibit inverse agonist activity in signal transduction assays in recombinant cell models (Bouaboula et al., 1997). It may be predicted that if free Gi proteins exist in solution under control conditions, then a greater population of Gi proteins could be found in a CB₁ receptor-Gi complex in the presence of SR141716. However, as shown in Fig. 6A, SR141716 exhibited little or no effect (<10% decrease in the amount of Gi associated with receptors) on the amount of receptor-Gi complex for any of the Gi subtypes. A similar finding was reported earlier for the CB₁ receptor associated with Go in solubilized preparations from rat brain (Mukhopadhyay et al., 2000). If a significantly greater population of unliganded Gi_ were present in solution, one would predict that in the presence of high concentrations of GDPS, SR141716 would stabilize a greater amount of coimmunoprecipitatable CB₁ receptor-Gi_GDPS complexes. This was not the case for any of the Gi subtypes at any of the concentrations of SR141716 tested (Fig. 6B).

View larger version (59K): [in this window] [in a new window]   Fig. 6. Effect of SR141716 and guanine nucleotides on the CB1 receptor-Gi interaction. A, CHAPS-solubilized extracts of N18TG2 cell membranes were incubated in the presence of 1 µM SR141716, 100 µM GTPS, or both as indicated. B, CHAPS-solubilized extracts of N18TG2 cell membranes were incubated in the presence of 100 µM GDPS in the absence or presence of 10 nM, 100 nM, or 1 µM SR141716. Immunoprecipitation and Western blotting for individual Gi proteins and the CB1 receptor were performed as described in the text and in previous figure legends. Band densities were determined and reported as a ratio of Gi density to the CB1 receptor density. Data are mean and S.D. from n = 2 experiments. Data were analyzed by two-way ANOVA and a Bonferroni post hoc test. Significant differences (p < 0.05) from control are indicated by * and from SR141716 alone are indicated by #. Significant differences (p < 0.05) between GTPS and GTPS plus SR141716 are indicated by a connecting bracket.

GTPS

Agonist and Guanine Nucleotide Effects on CB₁ Receptor-G Complexes. The interaction of the CB₁ receptor with the G dimer was examined in Fig. 7. G and G proteins were both detected in the protein complex immunoprecipitated by the CB₁ antibody. Upon incubation with agonist ligands at concentrations that promoted dissociation of those selective Gi proteins, 40 to 70% of the G (isoforms 1–4) was dissociated. G2 did not show a pattern of dissociation from the CB₁ receptor. This may be caused by the profile of G subtypes that are present in the N18TG2 cell membranes and associated with the Gi proteins as a heterotrimer. This antibody does not recognize all G subtypes that may potentially be present and/or associated with the CB₁ receptor. G2 is only one of several G subtypes that would be expected to be present in neuronal cells (Downes and Gautam, 1999).

View larger version (52K): [in this window] [in a new window]   Fig. 7. Effect of ligands and guanine nucleotides on the CB1 receptor interaction with G (A) and G (B). CHAPS-solubilized extracts of N18TG2 membranes were incubated in the absence or presence of 100 nM WIN 55,212-2, 100 nM DALN, 100 nM (R)-methanandamide, 1 µM SR141716, 100 µM GTPS, 100 µM GDPS, or combinations as indicated. Immunoprecipitation and Western blotting was performed with SDS-polyacrylamide gel electrophoresis conditions modified to allow for the detection of low-molecular-weight proteins. G and G proteins and the CB1 receptor band densities were quantified as described in the text and quantified as a ratio compared with the CB1 receptor band density. Data are mean and S.D. of n = 2 experiments. Each group of data were analyzed by one-way ANOVA and a Bonferroni post hoc test. Significant differences in control versus drug for each group are indicated as *, p < 0.05, and **, p < 0.01. ND, not determined.

GTPS

	Discussion

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Our present studies have examined the stability of CB₁ receptor complexes with three subtypes of Gi proteins in detergent solution to gain insight regarding the role that agonists and inverse agonists play in the ternary complex equilibrium and G protein activation cycle models. Stable ternary ARG complexes in detergent solution were promoted by agonists for somatostatin, -opioid, and ₂-adrenergic receptors in the absence of GTP or GTPS (Law and Reisine, 1992, 1997; Brown and Schonbrunn, 1993; Lachance et al., 1999). In the present investigation using CHAPS extracts from cultured N18TG2 neuronal cell membranes, and studies that we reported previously using rat brain membranes (Houston and Howlett, 1993, 1998; Mukhopadhyay et al., 2000), a significant fraction of the total Gi was found to be associated with immunoprecipitatable CB₁ cannabinoid receptor in the absence of exogenous agonists. The fraction of receptors having high affinity for agonists (believed to be the fraction of receptors in RG complexes) was approximately 20% in rat brain membranes and 35% for WIN 55,212-2 and 50% for DALN in CHAPS extracts (Houston and Howlett, 1998). Constitutive activity is readily observed in recombinant cell systems (Bouaboula et al., 1997; Pan et al., 1998; Vasquez and Lewis, 1999) and native cell systems under favorable experimental conditions (Pan et al., 1998; Meschler et al., 2000; Sim-Selley et al., 2001). Thus, a facile RG_GDP association is likely to occur in vivo. The model in Fig. 1 can be used to conceptualize the data regarding alterations in the equilibrium between G proteins bound to immunoprecipitatable receptors (RG_GTP or RG_) and free CB₁ receptors.

As depicted in the model, the demonstration that GTPS alone promoted dissociation of the G proteins from the CB₁ receptor indicates that the RG_GDP complexes can become spontaneously activated in the absence of agonist, permitting GDP release and a transiently empty R*G_ state. Once GTPS binds, the G_GTPS dissociates and can no longer participate in the association/dissociation reaction (Fig. 1). The model depicts the ability of agonists to facilitate this association/dissociation reaction, leading to mixtures in the absence of GTP or GTPS comprising equal amounts of the receptor in an ARG_GDP complex and in the dissociated state as AR plus G_GDP. WIN 55,212-2 promoted development of this mixture for all three Gi subtypes and promoted complete dissociation of the three RGi complexes in the presence of GTPS. This same behavior appeared in the presence of DALN for Gi1 and Gi2 and in the presence of the (R)-methanandamide for Gi3. The complete dissociation of G proteins from the CB₁ receptor evoked by DALN for Gi2 and by (R)-methanandamide for Gi3 suggests that an isomerization to AR*G may have been induced. AR*G_ would exist as a very transient complex in intact cells that possess an abundance of GTP to fill the guanine nucleotide binding site. Under the present experimental conditions, with no GTP present to promote G_GTP dissociation, the AR*G complex may be susceptible to protein denaturation, as has been observed for conformationally relaxed constitutively active mutants of GPCRs (Gether et al., 1997). In our experimental model, a denatured receptor that is unable to bind G would not be discernible from a functionally dissociated receptor.

Inverse agonist SR141716 maintained all three RGi complexes in the absence of GTP analogs and exerted a very small effect on the GTPS-promoted dissociation of G proteins from receptors. These results can be explained by invoking the existence of an inverse agonist-supported inactive state (IR^oG_GDP) in the ternary complex equilibrium model (Fig. 1). This state was originally proposed by Bouaboula and colleagues (1997) to describe a mechanism for the CB₁ receptor to "sequester" Gi proteins, thereby explaining their data that basal signal transduction through the mitogen-activated protein kinase or adenylyl cyclase pathways was blocked in the presence of SR141716. We propose that inverse agonist sequestration of G proteins with CB₁ receptors in an IR^oG_GDP complex would reduce the fraction of RG_GDP complex that could spontaneously convert to R*G_ or become available to interact with agonists to induce the AR*G_ complex.

The conversion of the RG_GDP complex to a sustainable IR^oG_GDP complex by inverse agonist SR141716 was mimicked by DALN for Gi3 and by (R)-methanandamide for Gi1 and Gi2. The property of these ligands to behave as inverse agonists for these G protein subtypes was manifest as the failure of these RG_GDP complexes to participate in the reversible dissociation to R + G_GDP. This would explain the ability of DALN or (R)-methanandamide to preclude the ability of GTPS to drive forward the dissociation of Gi3, or Gi1 and Gi2, respectively. In previous studies (Houston and Howlett, 1998), GTPS converted the majority of the high-affinity WIN 55,212-2 binding sites (ARG_GDP or AR*G_) to the low-affinity state (AR). In contrast, the fraction of receptors remaining in the high-affinity state for DALN was never reduced less than 25% even in the presence of GTPS and Na⁺ (Houston and Howlett, 1998). These findings are consistent with our current observation that in the presence of WIN 55,212-2, GTPS was able to promote dissociation all three Gi subtypes from the CB₁ receptor, but that in the presence of DALN, GTPS failed to dissociate Gi3.

An alternative mechanism might be that the inverse agonist-occupied receptors serve as guanine nucleotide-exchange factors that act on Gi_GTPS to exchange GDP for GTPS. This mechanism is not likely, because our studies indicated that G was dissociated from the CB₁ receptor, and there is a smaller probability that Gi_GTPS would be able to interact with the receptor in the absence of G (Clark et al., 2001). Furthermore, the studies with GDPS failed to support the notion that SR141716 could increase the population of receptor-G protein complexes by filling the guanine nucleotide-binding site of unoccupied G proteins in the presence of an excess of the GDP analog. It is interesting that the effects of SR141716 on all three Gi subtypes, and DALN on Gi1, were only partially disruptive of the GTPS-driven dissociation of Gi_GTPS, suggesting that these ligands do not possess as great an inverse agonist efficacy to promote the isomerization to IR^oG_GDP as does DALN for Gi3 or (R)-methanandamide for Gi1 and Gi2.

Under the present assay conditions, G was dissociated from the CB₁ receptor in parallel with Gi, supporting the notion that the heterotrimer dissociation allows the release of both components of the heterotrimer from the receptor. Agonists, but not SR141716, could facilitate dissociation of a fraction of the population of G (multiple isoforms) from the CB₁ receptors. In the presence of GTPS, agonists promoted the dissociation of a fraction of the G isoforms consistent with the AR*G_ AR + G + Gi_GTPS forward reaction. Protein-interaction studies by others have demonstrated that G can interact with both R and AR in the absence of G in detergent solution and reconstituted lipid vesicles (Heithier et al., 1992). In surface-plasmon resonance studies of immobilized rhodopsin, G binding was transient but was required to facilitate binding of G (Clark et al., 2001).

Our studies can be compared with other investigations of CB₁ receptor activation of G proteins that have detected differences in agonist efficacy to produce a response. Glass and Northup (1999) examined differential agonist activation of G proteins by measuring the ability of recombinant CB₁ receptors in Sf9 cell membranes to activate guanosine 5'-O-(3-[³⁵S]thio)triphosphate binding to purified Gi (all subtypes) and Go proteins. Both Gi and Go proteins were activated to the maximum extent by HU-210 and minimally by ⁹-tetrahydrocannabinol. WIN 55,212-2 and anandamide exhibited maximal or near-maximal activity for Gi but only approximately 70% maximal activity for Go. An inhibition of guanosine 5'-O-(3-[³⁵S]thio)triphosphate binding by SR141716 was observed for both Gi and Go. Prather and colleagues (2000) demonstrated differences in the ED₅₀ value for G protein activation by WIN 55,212-2 using [³²P]azidoanilido-GTP binding as the determinant of G protein activation. The ED₅₀ value for WIN 55,212-2 to activate various G protein subtypes in rat cerebellum membranes ranged from 100 nM for Gi1 and Go3 to 3.7 µM for Go2. It is not easy to compare their specific findings with ours because undifferentiated N18TG2 cells do not express an appreciable amount of Go, and those studies did not quantify [³²P]azidoanilido-GTP incorporation into Gi3.

The studies of Glass and Northup (1999) and Prather and colleagues (2000) both determined the exchange of a GTP analog for GDP on the G subunit under conditions that restrict reversal of the reaction. The present investigation determined receptor-G interaction, with the dissociation of the ternary complex as the measure of G protein activation. It has been proposed that the stability of the ternary complex can be determined by the dissociation rate of the interacting G proteins (Waelbroeck, 1999). It is likely that the agonist-receptor-G protein complex requires a sequence of transitions that must overcome a series of energy barriers to achieve release of G proteins from the receptor and GDP-GTP exchange. Shim and Howlett (2004) have proposed a theoretical model whereby nonclassic cannabinoid compounds such as CP55940 can convert to low-energy states within the binding pocket, providing a "steric trigger" for microconformational changes within the binding domain. Chemically distinct ligands may allow this transition to progress by multiple pathways because of their differential ability to provide the activation energy for microisomerization to unique conformations that can direct the activation of selected G protein subtypes (Kenakin and Onaran, 2002). We determined previously that the CB₁ receptor juxtamembrane C-terminal fourth loop domain was responsible for coupling to Go and Gi3 but not to Gi1 or Gi2 (Mukhopadhyay et al., 2000; Mukhopadhyay and Howlett, 2001). In contrast, the third intracellular loop was important for interaction with Gi1 and Gi2 (Mukhopadhyay and Howlett, 2001). This implies that certain agonists could induce a conformational change that is limited to the third intracellular loop, whereas others could induce alterations predominantly in the juxtamembrane C-terminal fourth loop. Clear clinical implications can be made from these studies in the demonstration that pharmacological selectivity can be determined regarding ligand-directed responses depending on the type of G isoform expressed within cells and the relative abundance of G proteins in the environment coupled to receptors.

	Footnotes

 
This work was supported by National Institute on Drug Abuse grants R01-DA03690, R01-DA06312, K05-DA00182, and U24-DA12385.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.104.003558.

ABBREVIATIONS: GPCR, G protein-coupled receptor; CAPS, 3-[cyclohexylamino]-1-propanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate; DALN, desacetyllevonantradol; ECL, enhanced chemiluminescence; GDPS, guanosine 5'-O-(3-thio)-diphosphate; GppNHp, guanylyl-imidodiphosphate; TBS, Tris-buffered saline; TBST, Tris-buffered saline/Tween 20; GTPS, guanosine 5'-O-(3-thio)-triphosphate; WIN 55,212-2, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone; SR141716, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride; HU-210, (–)-7-OH--6-tetrahydrocannabinol-dimethylheptyl; CP55940, 5-(1,1-dimethylheptyl)-2-(5-hydroxy-2-(3-hydroxypropyl)cyclohexyl)phenol; HME, sodium-HEPES/MgCl₂/EDTA; TM, Tris-Cl/MgCl₂; ANOVA, analysis of variance; IR^oG_GDP, inverse agonist-supported inactive state.

Address correspondence to: Dr. Somnath Mukhopadhyay, Neuroscience of Drug Abuse Research Program, J. L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, 700 George Street, Durham, NC 27707. E-mail: smukhopadhyay{at}nccu.edu

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