Cannabinoid Receptors and Ligands

Allyn Howlett, Ph.D. and Jeff Johnson, Ph.D.

In the mid 1980’s, the cannabinoid drugs, such as Δ⁹-tetrahydrocannabinol (THC) derived from Cannabis sativa, were thought to exert their central nervous system (CNS) effects by altering membrane fluidity rather than directly interacting with a protein receptor.^1,2 Such membrane perturbations could influence signal transduction of other neuroreceptors, but did not occur with a potency order or concentration range compatible with the known CNS effects of the classical ABC-tricyclic cannabinoid drugs. Many researchers suspected that a unique receptor for THC must exist based upon the existence of a clearly defined structure-activity relationship profile for the many analogs that were tested in in vivo animal models.^3,4 Largely due to the development of a series of non-classical AC-bicyclic and ACD-tricyclic cannabinoid analgesics by Pfizer, Inc.,⁴ a high potency radiolabelled agonist, [³H]-CP-55940, was produced.⁵ Rat cortical membranes were found to possess a single, saturable, high affinity (K_d = 133 pM) stereoselective binding site for (-)CP-55940.5 From this binding site, the CB₁ receptor could be described to account for the signal transduction in neuronal cells and many CNS actions of cannabinoid drugs.^6-8

The cannabinoid receptors, CB₁ and CB₂, were named based upon the classical and non-classical cannabinoid agonists that bound with high affinity and produced signal tranduction responses via the pertussis toxin-sensitive G_i proteins.^6-8 The CB₁ receptor was found in abundance in the brain. A splice variant of the human receptor, CB_1(b), is modified in the N-terminal extracellular domain, but exhibits the same pharmacological properties as the predominant CB₁ receptor. Shortly after the CB₁ receptor sequence was published, a homologous receptor was cloned from human myeloblast HL60 cells. This receptor, abundant in the immune system, is called the CB₂ receptor. To date, no other closely related receptors have been identified by homology cloning techniques.

Shortly after the determination and characterization of the CB₁ and CB₂ receptors, a series of endogenous eicosanoid ligands were discovered to serve as agonists for these receptors.^9,10 These lipid mediators are now known collectively as endocannabinoids. The first to be identified was arachidonoyl ethanolamide (AEA; anandamide).¹¹ Although a family of ethanolamine amides of fatty acids had been known,¹² only AEA, 5,8,11-eicosatrienoyl (mead) ethanolamide,¹³ N-dihomo-γ-linolenoyl ethanolamine, and N-docosatetraenoyl ethanolamine¹⁴ could bind to the cannabinoid receptors and earn the distinction of being endocannabinoid compounds. The ester of arachidonic acid and ethanolamine, named virodhamine, has been reported in brain and other tissues, and appears to behave as a partial CB₁ agonist and full CB₂ agonist.¹⁵ Eicosanoid adducts of glycerol, notably 2-arachidonoyl glycerol^16,17 and 2-arachidonyl glyceryl ether (noladin ether)¹⁸ were identified and joined the ranks of endocannabinoid compounds that bind to cannabinoid receptors and produce a signal transduction response. AEA can be oxidized by cyclooxygenase-2 and lipoxygenases, and some of these products may have biological activity.¹⁹ Arachidonoyl amides of amino acids such as glycine, alanine, and γ-amino-butyrate exist in animal tissues and appear to exhibit analgesic activity without binding to the CB₁ receptor.^19-21 Arachidonoyl glycine may have biological effects on immune cells¹⁹ but a role for the CB₂ receptor is not clear. Recent studies suggest that an arachidonoyl conjugate of dopamine²² is synthesized in vivo and may produce biological responses.²³ The tally suggests that there may be at least a half-dozen compounds that can be classified as endocannabinoid compounds that stimulate the two identified cannabinoid receptors. This is an unusually high ratio of endogenous agonists to receptor, and might suggest either that these receptors are activated by multiple stimuli in a complex fashion, or else some of these endogenous ligands are not physiologically relevant agonists for this class of receptors.

The complexity is not limited to the unusually large number of endogenous ligands for cannabinoid receptors. Additional complexity is introduced by the ability of the most highly characterized endocannabinoid, AEA, to interact with multiple receptors in addition to those classified as cannabinoid receptors. AEA is able to regulate the vanilloid receptor, VR₁,²⁴ a member of the TRPV ion channel family of receptors that responds to the vanilloid capsaicin.²⁵ AEA has also been shown to interact with L-type Ca²⁺ channels, several ligand-gated ion channels, and gap junctions.²⁶

AEA can regulate two other putative receptors that have been described pharmacologically but not yet identified as gene products. The first receptor is responsible for the antinociceptive and hypoactivity effects of anandamide that cannot be blocked by the CB₁ receptor antagonist SR141716A.^27-29 This putative receptor was apparent in CB₁^(-/-) mice as the mediator of the cannabinoid-like tetrad of responses to AEA. In in vitro studies of signal transduction, both AEA and WIN55212-2 (but not THC or other classical or nonclassical cannabinoid agonists) promoted GTPγ[³⁵S] binding to G proteins in brain membranes from CB₁^(-/-) mice, and this response could not be antagonized by SR141716A. It must be questioned whether a receptor that fails to respond to cannabinoid drugs but does respond to endocannabinoid compounds should be referred to as a cannabinoid receptor.

The second putative receptor for AEA is found in endothelial cells in the vasculature.^30-32 Pharmacologically, AEA and methanandamide produce vasorelaxation in CB₁^(-/-) mice, but classical cannabinoid agonists such as THC and the endocannabinoid 2-arachidonoyl glycerol do not. Abnormal cannabidiol^* and O-1918, which fail to bind to the CB₁ receptor at relevant concentrations, stimulate this response, and cannabidiol can serve as an antagonist. We know that this response is localized to endothelial cells, is pertussis toxin sensitive, and occurs via NO,³² suggesting that a G-protein-coupled receptor associated with the Gi family is mediating the response. The only other commonality with CB₁ receptors is that SR141716A can serve as an antagonist; however, high concentrations must be used.

Lastly, evidence exists for a putative receptor that mediates the antinociceptive effects of palmitoyl ethanolamide in the mouse formalin paw test but not the mouse hot plate test.^33,34 Palmitoyl ethanolamide is an endogenous fatty acid amide that fails to bind to either the CB₁ or CB₂ receptor and thus cannot be referred to as an endocannabinoid compound. Because the response to palmitoyl ethanolamide was antagonized by the CB₂ receptor antagonist SR144528, a putative receptor referred to as CB₂-like has been proposed. Based on the information at hand, it may be inappropriate to apply such a label to a receptor that does not respond to cannabinoid or endocannabinoid compounds.

In conclusion, numerous endogenous lipid mediators of cannabinoid receptor responses that occur via the CB₁ or CB₂ receptors have been identified. At least one of these compounds, AEA, appears to serve as an agonist for multiple receptors in addition to the CB₁ and CB₂ receptors. Future research will likely seek to determine whether the other endocannabinoid compounds also mediate their effects via receptors other than CB₁ and CB₂. Should the trend continue, the nomenclature of the endocannabinoids and the receptors to which they bind will undoubtedly require modest revision.

Article References

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* ‘Abnormal cannabidiol’ is an unnatural synthetic regioisomer of cannabidiol.³⁰

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