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Journal ArticleDOI

Structure of a cannabinoid receptor and functional expression of the cloned cDNA

09 Aug 1990-Nature (Nature Publishing Group)-Vol. 346, Iss: 6284, pp 561-564

TL;DR: The cloning and expression of a complementary DNA that encodes a G protein-coupled receptor that is involved in cannabinoid-induced CNS effects (including alterations in mood and cognition) experienced by users of marijuana are suggested.
Abstract: Marijuana and many of its constituent cannabinoids influence the central nervous system (CNS) in a complex and dose-dependent manner. Although CNS depression and analgesia are well documented effects of the cannabinoids, the mechanisms responsible for these and other cannabinoid-induced effects are not so far known. The hydrophobic nature of these substances has suggested that cannabinoids resemble anaesthetic agents in their action, that is, they nonspecifically disrupt cellular membranes. Recent evidence, however, has supported a mechanism involving a G protein-coupled receptor found in brain and neural cell lines, and which inhibits adenylate cyclase activity in a dose-dependent, stereoselective and pertussis toxin-sensitive manner. Also, the receptor is more responsive to psychoactive cannabinoids than to non-psychoactive cannabinoids. Here we report the cloning and expression of a complementary DNA that encodes a G protein-coupled receptor with all of these properties. Its messenger RNA is found in cell lines and regions of the brain that have cannabinoid receptors. These findings suggest that this protein is involved in cannabinoid-induced CNS effects (including alterations in mood and cognition) experienced by users of marijuana.
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Journal ArticleDOI
William A. Devane1, Lumir Hanus1, Aviva Breuer1, Roger G. Pertwee2  +6 moreInstitutions (3)
18 Dec 1992-Science
Abstract: Arachidonylethanolamide, an arachidonic acid derivative in porcine brain, was identified in a screen for endogenous ligands for the cannabinoid receptor. The structure of this compound, which has been named "anandamide," was determined by mass spectrometry and nuclear magnetic resonance spectroscopy and was confirmed by synthesis. Anandamide inhibited the specific binding of a radiolabeled cannabinoid probe to synaptosomal membranes in a manner typical of competitive ligands and produced a concentration-dependent inhibition of the electrically evoked twitch response to the mouse vas deferens, a characteristic effect of psychotropic cannabinoids. These properties suggest that anandamide may function as a natural ligand for the cannabinoid receptor.

5,025 citations


Journal ArticleDOI
02 Sep 1993-Nature
TL;DR: The cloning of a receptor for cannabinoids is reported that is not expressed in the brain but rather in macrophages in the marginal zone of spleen, which helps clarify the non-psychoactive effects of cannabinoids.
Abstract: THE major active ingredient of marijuana, Δ9-tetrahydrocannabi-nol (Δ9-THC), has been used as a psychoactive agent for thousands of years. Marijuana, and Δ9-THC, also exert a wide range of other effects including analgesia, anti-inflammation, immunosuppression, anticonvulsion, alleviation of intraocular pressure in glaucoma, and attenuation of vomiting1. The clinical application of cannabinoids has, however, been limited by their psychoactive effects, and this has led to interest in the biochemical bases of their action. Progress stemmed initially from the synthesis of potent derivatives of δ9-THC4,5, and more recently from the cloning of a gene encoding a G-protein-coupled receptor for cannabinoids6. This receptor is expressed in the brain but not in the periphery, except for a low level in testes. It has been proposed that the non-psychoactive effects of cannabinoids are either mediated centrally or through direct interaction with other, non-receptor proteins1,7,8. Here we report the cloning of a receptor for cannabinoids that is not expressed in the brain but rather in macrophages in the marginal zone of spleen.

4,503 citations


Journal ArticleDOI
Raphael Mechoulam1, Shimon Ben-Shabat1, Lumir Hanus1, Moshe Ligumsky1  +11 moreInstitutions (6)
TL;DR: Upon intravenous administration to mice, 2-Ara-Gl caused the typical tetrad of effects produced by THC: antinociception, immobility, reduction of spontaneous activity, and lowering of the rectal temperature.
Abstract: In this study, we report the isolation from canine intestines of 2-arachidonyl glycerol (2-Ara-Gl). Its structure was determined by mass spectrometry and by direct comparison with a synthetic sample. 2-Ara-Gl bound to membranes from cells transiently transfected with expression plasmids carrying DNA of either CB 1 or CB 2 —the two cannabinoid receptors identified thus far—with K i values of 472 ± 55 and 1400 ± 172 nM, respectively. In the presence of forskolin, 2-Ara-Gl inhibited adenylate cyclase in isolated mouse spleen cells, at the potency level of Δ 9 -tetrahydrocannabinol ( Δ 9 -THC). Upon intravenous administration to mice, 2-Ara-Gl caused the typical tetrad of effects produced by THC: antinociception, immobility, reduction of spontaneous activity, and lowering of the rectal temperature. 2-Ara-Gl also shares the ability of Δ 9 -THC to inhibit electrically evoked contractions of mouse isolated vasa deferentia; however, it was less potent than Δ 9 -THC.

2,610 citations


Journal ArticleDOI
TL;DR: It is considered premature to rename cannabinoid receptors after an endogenous agonist as is recommended by the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification, because pharmacological evidence for the existence of additional types of cannabinoid receptor is emerging and other kinds of supporting evidence are still lacking.
Abstract: Two types of cannabinoid receptor have been discovered so far, CB(1) (2.1: CBD:1:CB1:), cloned in 1990, and CB(2) (2.1:CBD:2:CB2:), cloned in 1993. Distinction between these receptors is based on differences in their predicted amino acid sequence, signaling mechanisms, tissue distribution, and sensitivity to certain potent agonists and antagonists that show marked selectivity for one or the other receptor type. Cannabinoid receptors CB(1) and CB(2) exhibit 48% amino acid sequence identity. Both receptor types are coupled through G proteins to adenylyl cyclase and mitogen-activated protein kinase. CB(1) receptors are also coupled through G proteins to several types of calcium and potassium channels. These receptors exist primarily on central and peripheral neurons, one of their functions being to inhibit neurotransmitter release. Indeed, endogenous CB(1) agonists probably serve as retrograde synaptic messengers. CB(2) receptors are present mainly on immune cells. Such cells also express CB(1) receptors, albeit to a lesser extent, with both receptor types exerting a broad spectrum of immune effects that includes modulation of cytokine release. Of several endogenous agonists for cannabinoid receptors identified thus far, the most notable are arachidonoylethanolamide, 2-arachidonoylglycerol, and 2-arachidonylglyceryl ether. It is unclear whether these eicosanoid molecules are the only, or primary, endogenous agonists. Hence, we consider it premature to rename cannabinoid receptors after an endogenous agonist as is recommended by the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. Although pharmacological evidence for the existence of additional types of cannabinoid receptor is emerging, other kinds of supporting evidence are still lacking.

2,453 citations


Cites background from "Structure of a cannabinoid receptor..."

  • ...For 9-THC, careful purification led to a ( )-enantiomer with activity less than 1% of the ( )-enantiomer (Herkenham et al., 1990; Matsuda et al., 1990; Felder et al., 1992; Pertwee, 1997)....

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  • ...8-THC has affinities for CB1 and CB2 receptors that are similar to those of 9-THC (Table 2) and also resembles 9-THC in behaving as a partial agonist at CB1 receptors (Matsuda et al., 1990; Gérard et al., 1991)....

    [...]

  • ...5 to 6 kb, but an alternatively polyadenylated cDNA sequence was reported (Matsuda et al., 1990), which is 2....

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  • ...The finding that cultured cell lines that express recombinant CB1 or CB2 receptors lead to inhibition of cyclic AMP production is supportive evidence that these receptor types can initiate this response (Matsuda et al., 1990; Felder et al., 1992; Vogel et al., 1993; Slipetz et al., 1995)....

    [...]

  • ...The CB1 mRNA is typically 5.5 to 6 kb, but an alternatively polyadenylated cDNA sequence was reported (Matsuda et al., 1990), which is 2.6-kb shorter in the rat....

    [...]


Journal ArticleDOI
Miles Herkenham1, A B Lynn1, M R Johnson1, L S Melvin1  +2 moreInstitutions (1)
TL;DR: The results suggest that the presently characterized cannabinoid receptor mediates physiological and behavioral effects of natural and synthetic cannabinoids, because it is strongly coupled to guanine nucleotide regulatory proteins and is discretely localized to cortical, basal ganglia, and cerebellar structures involved with cognition and movement.
Abstract: A potent, synthetic cannabinoid was radiolabeled and used to characterize and precisely localize cannabinoid receptors in slide-mounted sections of rat brain and pituitary. Assay conditions for 3H-CP55,940 binding in Tris-HCl buffer with 5% BSA were optimized, association and dissociation rate constants determined, and the equilibrium dissociation constant (Kd) calculated (21 nM by liquid scintillation counting, 5.2 nM by quantitative autoradiography). The results of competition studies, using several synthetic cannabinoids, add to prior data showing enantioselectivity of binding and correlation of in vitro potencies with potencies in biological assays of cannabinoid actions. Inhibition of binding by guanine nucleotides was selective and profound: Nonhydrolyzable analogs of GTP and GDP inhibited binding by greater than 90%, and GMP and the nonhydrolyzable ATP analog showed no inhibition. Autoradiography showed great heterogeneity of binding in patterns of labeling that closely conform to cytoarchitectural and functional domains. Very dense 3H-CP55,940 binding is localized to the basal ganglia (lateral caudate-putamen, globus pallidus, entopeduncular nucleus, substantia nigra pars reticulata), cerebellar molecular layer, innermost layers of the olfactory bulb, and portions of the hippocampal formation (CA3 and dentate gyrus molecular layer). Moderately dense binding is found throughout the remaining forebrain. Sparse binding characterizes the brain stem and spinal cord. Densitometry confirmed the quantitative heterogeneity of cannabinoid receptors (10 nM 3H-CP55,940 binding ranged in density from 6.3 pmol/mg protein in the substantia nigra pars reticulata to 0.15 pmol/mg protein in the anterior lobe of the pituitary). The results suggest that the presently characterized cannabinoid receptor mediates physiological and behavioral effects of natural and synthetic cannabinoids, because it is strongly coupled to guanine nucleotide regulatory proteins and is discretely localized to cortical, basal ganglia, and cerebellar structures involved with cognition and movement.

1,967 citations


Cites background or methods from "Structure of a cannabinoid receptor..."

  • ...Recently, Matsuda et al. (1990) have used the adenylate cyclase assay and structure-activity relationships to show that a cloned G-protein-coupled receptor is a cannabinoid receptor in transfected cells....

    [...]

  • ...The cellular elements that contain the receptors are not known at present, though subpopulations of dentate granule cells have been shown to express cannabinoid receptor mRNA (Matsuda et al., 1990)....

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References
More filters

Journal ArticleDOI
Piotr Chomczynski1, Nicoletta Sacchi1Institutions (1)
TL;DR: A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described, providing a pure preparation of undegraded RNA in high yield and can be completed within 4 h.
Abstract: A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.

65,324 citations


Journal ArticleDOI
TL;DR: A simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation of mammalian cells are described and linear DNA is almost inactive in mammalian cells.
Abstract: We describe a simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation of mammalian cells. In this protocol, the calcium phosphate-DNA complex is formed gradually in the medium during incubation with cells and precipitates on the cells. The crucial factors for obtaining efficient transformation are the pH (6.95) of the buffer used for the calcium phosphate precipitation, the CO2 level (3%) during the incubation of the DNA with the cells, and the amount (20 to 30 micrograms) and the form (circular) of DNA. In sharp contrast to the results with circular DNA, linear DNA is almost inactive. Under these conditions, 50% of mouse L(A9) cells can be stably transformed with pcDneo, a simian virus 40-based neo (neomycin resistance) marker vector. The NIH3T3, C127, CV1, BHK, CHO, and HeLa cell lines were transformed at efficiencies of 10 to 50% with this vector and the neo marker-incorporated pcD vectors that were used for the construction and transduction of cDNA expression libraries as well as for the expression of cloned cDNA in mammalian cells.

5,445 citations


Journal ArticleDOI
TL;DR: The potencies of a series of natural and synthetic cannabinoids as competitors of [3H]CP 55,940 binding correlated closely with their relative potencies in several biological assays, suggesting that the receptor characterized in the in vitro assay is the same receptor that mediates behavioral and pharmacological effects of cannabinoids, including human subjective experience.
Abstract: [3H]CP 55,940, a radiolabeled synthetic cannabinoid, which is 10-100 times more potent in vivo than delta 9-tetrahydrocannabinol, was used to characterize and localize a specific cannabinoid receptor in brain sections. The potencies of a series of natural and synthetic cannabinoids as competitors of [3H]CP 55,940 binding correlated closely with their relative potencies in several biological assays, suggesting that the receptor characterized in our in vitro assay is the same receptor that mediates behavioral and pharmacological effects of cannabinoids, including human subjective experience. Autoradiography of cannabinoid receptors in brain sections from several mammalian species, including human, reveals a unique and conserved distribution; binding is most dense in outflow nuclei of the basal ganglia--the substantia nigra pars reticulata and globus pallidus--and in the hippocampus and cerebellum. Generally high densities in forebrain and cerebellum implicate roles for cannabinoids in cognition and movement. Sparse densities in lower brainstem areas controlling cardiovascular and respiratory functions may explain why high doses of delta 9-tetrahydrocannabinol are not lethal.

2,050 citations


Journal ArticleDOI
Alfred G. Gilman1Institutions (1)
01 Mar 1984-Cell
TL;DR: It is clear that detailed understanding of the mechanism of regulation of CAMP synthesis will soon be achieved from study of the interactions of purified components that have been reconstituted in lipid bilayers of defined composition.
Abstract: Recent studies have revealed that the hormone-sensitive adenylate cyclase system is far more complicated than originally suspected-consisting of at least three types of proteins embedded in the lipids of the plasma membrane. This complex of proteins has the ability to receive information from several sources and generate a single, integrated response. The decision-making process begins when receptors for a large number of hormones, neurotransmitters, and other regulatory molecules interact with appropriate endogenous ligands and with drugs at the cell surface. These interactions ultimately result in stimulation or inhibition of adenylate cyclase activity; there are subsequent alterations of intracellular phosphorylation as a consequence of the actions of CAMP-dependent protein krnases and counter-regulatory phosphoprotein phosphatases. Stimulatory receptors include those for P-adrenergic agonists, ACTH, gonadotropins, and many others, while Inhibitory control is exerted by such agents as a*-adrenergic and muscarinic agonists and opioids. Receptors communicate with a pair of homologous guanine-nucleotide-brnding regulatory proteins-one (G,) mediates stimulation of adenylate cyclase activity, while the other (G,) is responsible for inhibition. The G proteins control the activity of the actual catalyst of the enzyme system (C) in a complex series of reactions discussed below. Notable in this scheme is the fact that the two G proteins share a common subunit, and the action of this subunit appears to be crucial for the integrative capacity of the system. The G proteins that regulate adenylate cyclase activity are members of a larger family of homologous guanine-nucleotide-binding proteins that includes transducin, a regulatory protein of the outer segment of the retinal rod. The products of the ras genes may also be relatrves. Progress in dissection of the hormone-sensitive adenylate cyclase system was slow through the 1960s and much of the 1970s for a number of valid, and for some time unsuspected, reasons. These include the hydrophobic nature of the components: their multiplicity, lability, and extremely low concentration (1 part in 1 O5 of cell protein is typrcal); and the need for proper orientation of the components in an appropriate membrane for hormonal regulation of enzymatic activity. However, a number of advances have now led to a successful path of experimentation. It is clear that detailed understanding of the mechanism of regulation of CAMP synthesis will soon be achieved from study of the interactions of purified components that have been reconstituted in lipid bilayers of defined composition. G. and G, The two guanine-nucleotide-binding regulatory components of the adenylate cyclase system have been purified and studied in some detail-particularly in detergent-containing solutions (Sternweis et al., JBC 256, 11517-l 1526, 1981; Bokoch et al., JBC 258, 2072-2075, 1983). The table and two figures are included to facilitate the following discussron. The catalyst of adenylate cyclase is essentially inactive with its physiological substrate, MgATP, in the absence of G,. G, is thus most conveniently assayed by its ability to stimulate adenylate cyclase activity; the best source of C for such an assay is obviously one that is free of G,. Such resolved preparations of C can be made biochemically. Alternatively, the membrane of the cycS49 lymphoma cell mutant IS a superb assay vehicle. This mutant is deficient in G, activity, but retains C, and was particularly useful for elucidating the importance and activity of G,. It was originally assumed that cycwas devoid of adenylate cyclase, since it had essentially undetectable levels of this enzymatic activity. However, addition of G, to cycmembranes fully restores adenylate cyclase activity stimulated by hormone, guanine nucleotide, and fluoride. G, has an oligomeric structure with 45,000 and 35,000 dalton subunits, while G, has a similar structure with 41,000 and 35,000 dalton subunits. A third subunit, M, 10,000, also appears to be present in both proteins, although this has not been proved rigorously. The larger (a) subunit of each protein contains a site for NAD-dependent ADPribosylation catalyzed by a bacterial toxin. Cholera toxin ADP-ribosylates G,,, in the presence of a membrane-bound protein cofactor (ARF), while islet-activating protein (IAP; one of the toxins of Bordetella pertussis) ADP-ribosylates G,.,. Such ADP-ribosylation results in characteristic modificatlons of the function of each regulatory protein. The (Y subunits of each G protein also contain a site with highaffinity for guanine-nucleotide binding. The 35,000 dalton (@) subunits of G, and G, are indistinguishable from each other functionally (see below) and by analysis of amino acid composition and maps of proteolytic peptides. Incubation of either G, or G, with nonhydrolyzable guanine nucleotides (e.g. GTPyS) or with fluoride (in the presence of Mg2+ and A$+; see Sternweis and Gilman, PNAS 79, 4888-4891, 1982) results in “activation” of the protein. By activation I mean a state in which the protein is capable of either stimulating or inhibiting the activity of C. In fact, activation of either G, or G, by GTPyS is essentially irreversible (in the presence of Mg’+); free GTPrS can be removed, and the activated state is stable. Activation of either G, or G, has been shown to be the result of or associated with ligand-promoted dissociation of the protein’s subunits, as follows (Northup et al., JBC 258, 11369-I 1376, 1983; Katada et al., JBC, in press):

1,641 citations


Journal ArticleDOI
31 Jul 1987-Science
TL;DR: Analysis of human and rat genomic clones indicates that there are at least four functional muscarinic receptor genes and that these genes lack introns in the coding sequence.
Abstract: Complementary DNAs for three different muscarinic acetylcholine receptors were isolated from a rat cerebral cortex library, and the cloned receptors were expressed in mammalian cells. Analysis of human and rat genomic clones indicates that there are at least four functional muscarinic receptor genes and that these genes lack introns in the coding sequence. This gene family provides a new basis for evaluating the diversity of muscarinic mechanisms in the nervous system.

1,419 citations


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