Introduction

Top Abstract Introduction Methods Results Discussion References

The cannabinoid receptor is the initial site of action for the most widely abused street drug, marijuana. Marijuana has prominent effects on the central nervous system as well as numerous peripheral effects, including immunomodulation. The primary psychoactive constituent in marijuana, and prototypical cannabinoid compound is ⁹-THC. ⁹-THC and structurally related cannabinoids are extremely lipophilic molecules, and for many years it was difficult to prove that the unique profile of pharmacological effects produced by these drugs was receptor-mediated, for instance, by demonstration of high-affinity specific binding with ⁹-THC (reviewed in Martin, 1986). However, studies in neuroblastoma cells had shown an inhibition of adenylyl cyclase activity that was specific for psychoactive cannabinoids, implicating a GPCR-mediated process (Howlett and Fleming, 1984). The development of [³H]CP-55,940, a highly potent synthetic analog of ⁹-THC, allowed the identification of specific cannabinoid binding sites in the brain (Devane et al., 1988; Herkenham et al., 1990). Then, a rat brain cDNA clone isolated by homology to GPCRs was identified as the cannabinoid receptor (CB1) by virtue of its ability to induce cannabinoid-mediated inhibition of cAMP accumulation in transfected cells and the similarities in its expression pattern to that of [³H]CP-55,940 binding sites (Matsuda et al., 1990). Shortly thereafter, the cloning of a human CB1 receptor cDNA was reported (Gerard et al., 1991). This CB1 receptor is one of the most abundantly expressed of the neuronal receptors. A second cannabinoid receptor subtype (CB2) was discovered by a PCR-based strategy designed to isolate GPCRs in differentiated myeloid cells (Munro et al., 1993). The CB2 receptor, which has been found in the spleen and cells of the immune system, has 44% amino acid identity with the brain clones. The affinities for several cannabinoids at the CB2 receptor are distinct from that of the brain receptor (Showalter et al., 1996). The CB2 receptor also mediates inhibition of cAMP accumulation (Felder et al., 1995; Slipetz et al., 1995).

A family of endogenous ligands has been identified for these receptors, of which arachidonic acid ethanolamide (anandamide) was the first (Devane et al., 1992). The isolation of endogenous ligands has provided additional evidence supporting the role of cannabinoid receptors as important neurochemical and immune system modulators. In addition, the recent development of a selective antagonist to the CB1 receptor, SR141716A, provides a tool for determining the receptor-mediated vs. the non-receptor-mediated effects of the cannabinoids (Rinaldi-Carmona et al., 1994).

In vitro mutagenesis of cloned cDNAs provides a means of examining the specific functions of the proteins they encode. Selected mutations can be introduced into regions of the receptor cDNAs believed to be critical to receptor recognition or second messenger function. The aspartic acid residue in the second transmembrane domain is highly conserved among GPCRs. Mutational studies have shown that this residue is important for ligand recognition, cation selectivity and/or coupling to G proteins in various receptors in this family (summarized in Ceresa and Limbird, 1994). To determine the role of this residue in the function of the human cannabinoid receptors (CB1 and CB2), aspartate 163 (CB1) and aspartate 80 (CB2) were replaced with glutamic acid or asparagine. Our data suggest that this aspartate residue is not generally important for ligand recognition in the cannabinoid receptors, but rather is involved in G-protein coupling and, thereby, signal transduction.

	Methods

Top Abstract Introduction Methods Results Discussion References

Materials. [³H]CP-55,940 and [³H]WIN-55,212-2 were purchased from DuPont-NEN (Wilmington, DE). [³H]SR141716A was purchased from Amersham (Arlington Heights, IL). ⁹-THC and anandamide were obtained from the National Institutes on Drug Abuse (Rockville, MD). CP-55,940 initially was provided by Dr. Larry Melvin (Pfizer Inc., Groton, CT). SR141716A was synthesized by Dr. John Lowe (Pfizer Inc., Groton, CT). WIN-55,212-2 originally was provided by Dr. Susan Ward (Sterling-Winthrop Research Institute, Rensselaer, NY). 11-Hydroxy-⁸-THC-dimethylheptyl was provided generously by Dr. Raphael Mechoulam (Hebrew University, Jerusalem, Israel). Human embryonic kidney HEK 293 cells were obtained from American Type Culture Collection. The human CB1 cDNA was provided by Dr. Marc Parmentier (Universite Libre de Bruxelles, Belgium). The human CB2 cDNA was provided by Dr. Sean Munro (MRC, Cambridge, England).

Mutagenesis. The Altered Sites (Promega Corp., Madison, WI) in vitro mutagenesis system was used to mutate the CB1 receptor. The human CB1 cDNA was subcloned into the pALTER phagemid, and with the helper phage R408, single-stranded templates were produced. The desired mutation was produced by annealing a complementary mutagenic oligonucleotide as well as an oligonucleotide which confers ampicillin resistance to the single-stranded template followed by elongation with T4 DNA polymerase and ligation. The heteroduplex DNA was used to transform the repair-minus Escherichia coli strain BMH 71-18 mut S and the cells grown in the presence of ampicillin. A second round of transformation in JM109 ensured proper segregation of mutant and wild-type plasmids. The mutations were confirmed by sequencing, and the mutated cDNA subcloned into the mammalian expression vector pcDNA3 (Invitrogen, San Diego, CA) for expression. To mutate D163 to E163 of CB1, the mutagenic oligonucleotide (5' CGG TGG CAG AAC TCC TGG GGA) containing the desired mutation (GAC to GAA) was used. The entire cDNA insert was then sequenced to confirm the absence of additional mutations. To make the D163N mutation, the mutagenic oligonucleotide (5' CGG TGG CAA ACC TCC TGG GGA) containing the desired mutation (GAC to AAC) was used.

Cell culture and transfection. Human embryonic kidney 293 cells were maintained in DMEM with 10% fetal clone II (HyClone, Logan UT) and 5% CO₂ at 37°C in a Forma incubator. Cell lines were created by transfection of wild-type or mutant CB1pcDNA3 or CB2pcDNA3 into 293 cells by the Lipofectamine reagent (Life Technologies, Gaithersburg, MD). Stable transformants were selected in growth medium containing geneticin (1 mg/ml, reagent, Life Technologies, Gaithersburg, MD). Colonies of about 500 cells were picked (about 2 weeks post-transfection) and allowed to expand, then tested for expression of receptor mRNA by Northern blot analysis. Cell lines containing moderate to high levels of receptor mRNA were tested for receptor-binding properties. Transfected cell lines (including CB2-CHO, previously described in Showalter et al., 1996) were maintained in DMEM with 10% fetal clone II plus 0.3 to 0.5 mg/ml geneticin and 5% CO₂ at 37°C in a Forma incubator.

[³H]Cannabinoid binding in cells. The current assay is a modification of Compton et al. (1993). Cells were harvested in phosphate-buffered saline containing 1 mM EDTA and centrifuged at 500 × g. The cell pellet was homogenized in 10 ml of solution A (50 mM Tris-HCl, 320 mM sucrose, 2 mM EDTA, 5 mM MgCl₂, pH 7.4). The homogenate was centrifuged at 1,600 × g (10 min), the supernatant saved and the pellet washed three times in solution A with subsequent centrifugation. The combined supernatants were centrifuged at 100,000 × g (60 min). The (P₂ membrane) pellet was resuspended in 3 ml of buffer B (50 mM Tris-HCl, 1 mM EDTA, 3 mM MgCl₂, pH 7.4) to yield a protein concentration of approximately 1 mg/ml. The tissue preparation was divided into equal aliquots, frozen on dry ice and stored at 70°C. Binding was initiated by the addition of 40 to 50 µg membrane protein to silanized tubes containing [³H]CP-55,940 (102.9 Ci/mmol), [³H]WIN-55,212-2 (45.5 Ci/mmol) or [³H]SR141716A (55 Ci/mmol) and a sufficient volume of buffer C (50 mM Tris-HCl, 1 mM EDTA, 3 mM MgCl₂ and 5 mg/ml fatty acid-free BSA, pH 7.4) to bring the total volume to 0.5 ml. The addition of 1 µM unlabeled CP-55,940 was used to assess nonspecific binding. After incubation (30°C for 1 hr), binding was terminated by the addition of 2 ml of ice-cold buffer D (50 mM Tris-HCl, pH 7.4, plus 1 mg/ml BSA) and rapid vacuum filtration through Whatman GF/C filters [pretreated with polyethyleneimine (0.1%) for at least 2 hr]. Tubes were rinsed with 2 ml of ice-cold buffer D, which was also filtered, and the filters subsequently rinsed twice with 4 ml of ice-cold buffer D. Before radioactivity was quantitated by liquid scintillation spectrometry, filters were shaken for 1 hr in 5 ml of scintillation fluid.

cAMP accumulation assay. Intracellular cAMP levels were measured with a competitive protein binding assay (Diagnostic Products, Inc., Los Angeles, CA) (Abood and Tao, 1995). Cells were harvested at 70 to 90% confluence by mechanical disruption in phosphate-buffered saline containing 1 mM EDTA and counted with a hemacytometer. After centrifugation at 500 × g, the cell pellet was resuspended at a concentration of 1 × 10⁶ cells/ml in DMEM containing 20 mM HEPES, pH 7.3, 0.1 mM RO-20-1724 and 1 mM isobutylmethylxanthine and incubated for 30 min at 37°C. Aliquots of cells (90 µl) were added to polypropylene microfuge tubes containing 0.5 µM forskolin ± cannabinoids + 5 mg/ml fatty acid-free BSA, in a final volume of 100 µl and incubated for 5 min at 37°C. Because the cannabinoids were dissolved in ethanol, all tubes contained an equivalent amount of ethanol (0.5%). The reactions were terminated by boiling for 4 min, followed by centrifugation and removal of 50 µl of the supernatant which was assayed for cAMP levels. The results are expressed as percent inhibition of forskolin-stimulated cAMP accumulation. The levels of forskolin-stimulated cAMP accumulation (expressed in pmol/10⁶ cells/min) for the cell lines tested were: 7.22 ± 1.66 (CB1); 9.39 ± 1.44 (D163E); 5.50 ± 1.05 (D163N); 8.55 ± 2.78 (CB2); 6.38 ± 0.83 (D80E); 9.72 ± 2.50 (D80N). EC₅₀ curves were generated with the use of the GraphPad Prism program (GraphPad, San Diego, CA).

Statistical analysis. Statistical analysis of binding and EC₅₀ data were compared by ANOVA or the student's t test, where suitable. Bonferroni post hoc analyses were conducted when appropriate. Statistical significance was defined as P < .05.

	Results

Top Abstract Introduction Methods Results Discussion References

Expression of the wild-type human CB1 and CB2 receptors. Stable transformants of 293 cells were established which expressed the human CB1 or CB2 receptors. No specific [³H]CP 55,940 or [³H]SR141716A cannabinoid binding to 293 cells was found before transfection (data not shown). With [³H]CP 55,940 as a radioligand in the cell line expressing wild-type CB1, K_d and B_max values of 1.21 ± 0.27 nM and 0.95 ± 0.16 pmol/mg protein, respectively, were obtained (fig. 1, table 1). These values are similar to a CB1-expressing CHO cell line described previously (K_d = 0.65 ± 0.09 nM; B_max = 0.83 ± 0.16 pmol/mg protein, table 1) (Showalter et al., 1996). In the 293 cell line stably expressing the CB2 receptor, K_d and B_max values of 0.88 ± 0.09 nM and 1.55 ± 0.39 pmol/mg protein, respectively, were obtained (table 1). These values are similar to a CB2-expressing CHO cell line described previously (K_d = 0.61 ± 0.14 nM; B_max = 3.1 ± 0.9 pmol/mg protein, table 1) (Showalter et al., 1996). The binding characteristics of the cannabinoid receptors are the same whether they are expressed in 293 cells or in CHO cells (tables 1-3 and Showalter et al., 1996).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Representative Scatchard and saturation (inset) analysis of [3H]CP-55,940 binding to membranes prepared from transfected 293 cells expressing human CB1. In this experiment, Kd and Bmax values were 1.02 nM and 898 fmol/mg protein for CB1. These data are representative of three experiments performed in triplicate.

View larger version (9K): [in this window] [in a new window]   Fig. 2.   Agonist-induced inhibition of cAMP accumulation in wild-type CB1 (A) and CB2 (B) receptor-expressing cell lines. EC50 values were 1.64 ± 1.5 nM for WIN-55,212-2 and 4.17 ± 3.1 nM for CP-55,940 for CB1-293 cells (A). EC50 values were 12.3 ± 3.3 nM for WIN-55,212-2 and 52.1 ± 6.2 nM for CP-55,940 for the CB2 cell line (B). Data are expressed as percent forskolin stimulation versus log molar drug concentration. Points, mean ± S.E. of three or more independent experiments performed in triplicate. Curves were generated as described under "Methods."

Comparison of the mutant and wild-type receptors. Radioligand binding and cAMP accumulation experiments were performed with transfected cells expressing mutated CB1 or CB2 receptors. The mutation was a single base change that altered an aspartate residue in the second transmembrane domain of CB1 (D163) or CB2 (D80) to asparagine (N) or glutamate (E). The D to N mutation removes a positive charge and has been shown in several GPCRs to be important for receptor G-protein signaling. The D to E mutant retains the charge but introduces an additional methyl group that may result in conformational changes.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Comparison between the wild-type and the mutant cannabinoid receptors for agonist-induced inhibition of cAMP accumulation. (A) Effect of WIN 55,212-2 on wild-type CB1 () and mutant D163E () and D163N () receptor-expressing cell lines. (B) Effect of CP-55,940 on wild-type CB1 () and mutant D163E () and D163N () receptor-expressing cell lines. (C) Effect of WIN 55,212-2 on wild-type CB2 () and mutant D80E () and D80N () receptor-expressing cell lines. (D) Effect of CP-55,940 on wild-type CB2 () and mutant D80E () and D80N () receptor-expressing cell lines. Data are expressed as percent forskolin stimulation versus log molar drug concentration. Points, mean ± S.E. of three or more independent experiments performed in triplicate. Curves were generated as described under "Methods."

Allosteric regulation studies. The functional uncoupling of the mutant receptors also was seen as a loss of the ability of Gpp(NH)p to reduce agonist binding (table 4). In the wild-type CB1- and CB2-receptor expressing cell lines, inhibition of specific binding was observed in the presence of Gpp(NH)p. Addition of 1 mM Gpp(NH)p produced greater than a 38% decrease in percent specific binding of 1 nM [³H]CP 55,940 (P < .05). Mutation of the D to N resulted in disruption of the regulation by Gpp(NH)p (table 4). In the D to E mutants of the CB1 and CB2 receptors, a reduced inhibition by Gpp(NH)p also was observed (table 4).

	Discussion

Top Abstract Introduction Methods Results Discussion References

The present studies demonstrate that the conserved aspartate residue in the second transmembrane domain of the cannabinoid receptors, CB1 and CB2, is required for efficient signal transduction, but generally does not disrupt high-affinity agonist binding. These findings distinguish the cannabinoid receptors from other GPCRs, especially those in which inhibition of adenylyl cyclase is a predominant second messenger response. In several other GPCRs, the aspartate to asparagine mutation resulted in a loss of high-affinity agonist binding (e.g., Ceresa and Limbird, 1994; Kong et al., 1993A). We found that the binding of several cannabinoid ligands was not affected in either the CB1 or CB2 receptor mutations, with the exception of WIN-55,212-2 in the CB1 D163E and D163N mutants.

For several other GPCRs, the aspartate to asparagine mutation also was associated with a loss of cation-sensitivity for agonist (e.g., Ceresa and Limbird, 1994; Kong et al., 1993a). Previous reports have indicated that sodium does not substantially reduce high-affinity agonist binding in the cannabinoid receptors (Herkenham et al., 1991; Showalter et al., 1996). In the present report, the addition of NaCl to the binding buffer resulted in a slight, but significant, reduction of agonist binding in all cell lines except the CB1 D163N mutant. [³H]WIN-55,212-2 binding was not altered significantly by the addition of sodium. Sodium ions have been shown to stabilize "empty", uncoupled, receptors (Costa et al., 1989), which may explain why the effect of sodium was retained in some of the mutant cannabinoid receptors. This was a preliminary attempt to address the regulation by sodium in the mutant receptors and was conducted simply by addition of 150 mM NaCl to the binding buffer. Further studies (e.g., controlling for ionic strength, competition curves assessing presence of multiple affinity states) are warranted to determine the role of sodium in binding to the D163N mutant and to determine the sensitivity of WIN-55,212-2 to sodium.

The cannabinoid receptors showed greatly reduced G-protein coupling, both as measured by inhibition of adenylyl cyclase activity, as well as in the ability of guanine nucleotides to reduce agonist binding. In the alpha-2a adrenergic, SSTR2 somatostatin and delta opioid receptors, the D to N mutant retained its ability to inhibit forskolin-stimulated cAMP accumulation (Ceresa and Limbird, 1994; Kong et al., 1993a, b). On the other hand, mutation of the conserved aspartate in the dopamine D2 receptor to alanine or glutamate resulted in a loss of inhibition of adenylyl cyclase activity (Neve et al., 1991). Also, the ability of Gpp(NH)p to inhibit agonist binding was abolished in the alpha-2a adrenergic receptor D79N mutation as was functional coupling as assessed by loss of receptor-activated potassium currents (Ceresa and Limbird, 1994; Surprenant et al., 1992). Furthermore, when the alpha-2a adrenergic receptor D79N mutation was introduced into the genome of mice, alpha-2 adrenergic agonist mediated hypotension was abolished, which indicates that receptor-activated processes were absent in the gene-targeted mice (MacMillan et al., 1996).

The highly conserved aspartate residue in the second transmembrane domain of many GPCR apparently is associated closely with an asparagine residue in the seventh transmembrane domain. Mutations of one of these residues often disrupt functional coupling, whereas reciprocal mutations restore function (Sealfon et al., 1995; Suryanarayana et al., 1992; Zhou et al., 1994). The cannabinoid receptors also contain an asparagine residue in the seventh transmembrane region. Molecular modeling studies have implicated a hydrogen bonding network which could be involved in receptor activation by agonists (Sealfon et al., 1995; Zhou et al., 1994). Furthermore, when the analogous aspartate residue in the serotonin 5-HT_2A receptor was mutated to asparagine, this resulted in a loss of G-protein coupling, but had no effect on binding of a wide range of ligands (Sealfon et al., 1995; Zhou et al., 1994). Modeling results presented by Sealfon et al. (1995) demonstrated that the mutant receptor undergoes a conformational change (in helixes 5 and 6) upon agonist binding, but in the opposite direction seen with the wild-type receptor. They suggest that this conformational change still may produce a high-affinity state for ligand binding (and thus result in receptor affinities essentially unchanged from those of the wild-type receptor), but that the resulting helix arrangements may not support coupling to the appropriate G protein. A similar mechanism may arise in the cannabinoid receptors in response to agonists such as CP-55,940.

However, a different situation must arise in the CB1 receptors on binding WIN 55,212-2. Although the mutations did not eliminate high-affinity binding for most ligands tested, they did exert differential effects on WIN-55,212-2 binding. The affinity of WIN 55,212-2 but not CP-55,940 was reduced in the CB1 receptor mutants, whereas neither was reduced in the CB2 receptor mutants. WIN 55,212-2 also discriminated between receptor expression in the cell lines and the native tissues. A previous mutation study on the CB1 receptor revealed that the binding site for WIN 55,212-2 was distinct from that of other cannabinoid ligands (Song and Bonner, 1996). Mutation of a lysine residue in the third transmembrane domain of the CB1 receptor resulted in a loss of binding for ⁹-THC, CP-55,940 and anandamide, but not WIN 55,212-2 (Song and Bonner, 1996). Conversely, WIN 55,212-2 may bind in a less energetically favorable site in the D163N and D163E mutant cell lines. It is also possible that WIN 55,212-2 still may bind in the same site in the CB1 mutants as in the wild-type receptor, but it cannot produce the conformational change which results in the high-affinity state.

The mutated receptors were analyzed by creating stably transfected cell lines. Our previous experience in radioligand binding with the cannabinoid receptor has demonstrated the need to express the receptor in excess of 0.5 pmol/mg protein to obtain reasonable specific binding (Abood et al., 1997). One concern may be that overexpression in different cell lines may lead to altered ligand affinities. However, the data showing that similar K_d values were obtained between 293 cell lines expressing CB1 or CB2 receptors as compared with CB1- or CB2-CHO cell lines demonstrate that this is not a concern. In addition, the similar affinities found when comparing the stably transfected cell lines with native tissues strengthens the use of transfected cells as model systems. Another concern with transfected cell lines is that receptor coupling with G proteins may be altered because of overexpression of receptors in excess of G proteins. Our data demonstrating the ability of Gpp(NH)p to reduce agonist binding in the wild-type receptors indicate that the cannabinoid receptors expressed in 293 cells are regulated by G proteins. Furthermore, the results of the cAMP accumulation studies in the wild-type receptor cell lines indicating efficient adenylyl cyclase inhibition both in terms of EC₅₀ values and E_max demonstrate appropriate signal transduction.

The overall aim of this mutagenesis research is to elucidate important molecular components of the cannabinoid pharmacophore. This knowledge may lead to the design of more specific cannabinoid ligands, which could offer increased therapeutic activity and decreased side effects. Additionally, as knowledge emerges regarding the role of the cannabinoid receptor in normal physiological function, identification of aberrations in receptor-effector coupling may be critical in treating conditions arising from disorders of the cannabinoid system. Mutation of the highly conserved aspartate residue in the second transmembrane domain of the CB1 and CB2 receptors provided a separation of ligand binding from signal transduction in both subtypes. Other amino acids presumably are involved in discrimination of ligands between the receptor subtypes and are the targets for future research.