Active compounds found in plants of the Cannabis genus as well as endogenous analogs of these substances, and a myriad of synthetic analogs can influence a wide range of biological processes by engaging G protein-coupled receptors (GPCRs) of the endocannabinoid system (Figure 1). This system also includes the enzymes involved in endocannabinoid synthesis, transporters that enable cellular uptake/release, and enzymes that initiate endocannabinoid degradation. Many additional receptors including GPR18, GPR55, and GPR119, peroxisome proliferator-activated receptors (PPARs), and several members of the transient receptor potential (TRP) ion channel superfamily are also closely associated. Parts of this system are present in virtually every cell type of the human body, situating them to influence varied physiological processes.

Figure 1. Cannabinoid system overview.

Cannabinoid Receptor Distribution and Mechanism of Action

The mechanism of actions of cannabinoids are becoming more realized with the understanding of the expression and distribution of the cannabinoid receptors CB₁ and CB₂ (Figure 2). CB₁ receptors, though predominant in the brain, are also expressed in the spleen, lung, thymus, heart, and vasculature. In the CNS, they are most highly expressed by the axons and presynaptic termini of neurons in the amygdala, hippocampus, cortex, basal ganglia, and cerebellum. They are strongly associated with GABAergic (inhibitory) and glutamergic (excitatory) neurons, where they mediate inhibition of ongoing release of inhibitory and excitatory neurotransmitters.

Figure 2. Distribution of CB₁ and CB₂ and their associated functions.

CB₁ receptors are coupled to G_i/G_o proteins in a manner that leads to the inhibition of adenylate cyclase activity, activation of the β-arrestin pathway, regulation of L-, N- and P- or Q-type Ca²⁺ channels and G protein-regulated A-type and inwardly rectifying K⁺ channels, initiation of intracellular Ca²⁺ transients, stimulation of MAPK, and/or induction of immediate early gene expression (Figure 3). These receptors can also signal through G_s proteins, working in opposition to stimulate adenylate cyclase activity, causing a rise in intracellular cAMP levels. Under certain circumstances, CB₁ can also couple to G_q. In a form of biased agonism, the actual ligand determines the signaling preference among these possible second messenger pathways.

Figure 3. G protein coupling of cannabinoid receptors.

Ligand binding to the receptor occurs through lateral insertion via the lipid bilayer rather than directly from solution. CB₁ receptors also possess one or more allosteric sites wherein some ligands interact to enhance or inhibit CB₁ receptor activation by direct agonists. Some CB₁ receptors form heteromers with dopamine D₂ receptors, μ-opioid receptors, and orexin-1 receptors, which can affect CB₁ receptor activation by agonists adding to the complexity of cannabinoid signaling.

CB₂ receptors are most abundant in tissues with immune function both outside of the brain, such as spleen, tonsils, bone marrow, pancreas, peripheral blood leukocytes, and inside of the brain in astrocytes and microglia (Figure 2). They function to modulate immune cell migration and cytokine release, but also have roles in cardiovascular and respiratory systems, bone, the gastrointestinal tract, liver, and the reproductive system.

CB₂ receptors couple to G_i/o proteins to signal a response through inhibition of cAMP production, MAPK activation, and immediate early gene expression (Figure 3). No modulation of ion channels or alterations of intracellular Ca²⁺ has been observed in host cells expressing CB₂ receptors.

Cannabinoid Receptor Structure

Much of the understanding of how ligands physically activate and inactivate CB receptors comes from structural observation of binding with agonists and antagonists wherein the C-terminal intracellular helix adopts an active or inactive conformation. The intracellular loop 2, particularly residue L222, of both CB₁ and CB₂ receptors plays a critical role in G_i and G_s protein coupling and specificity, making the inhibition versus activation of adenylate cyclase ligand specific. The first crystal structure of the human CB₁ receptor in complex with a tight binding antagonist was successfully resolved in 2016. Since then, three more crystal structures and two cryo-electron microscopic (cryo-EM) structures of CB₁ have been obtained and reported (Figure 4). Comparisons between the agonist- and antagonist-bound CB₁ complexes show notable structural rearrangements around an extended twin molecular toggle switch (F200 and W356), whose activation leads to G protein binding and plasticity in the orthosteric binding pocket that can accommodate a variety of ligands with different shapes and sizes. F200 functions as a latch to restrict the movement of W356 and locks CB₁ in the inactive state unable to bind G proteins.

Figure 4. Cryo-EM structure of CB₁-G_i coupling bound with AM841 agonist [PDB ID(6KPG)] as determined by Hua, T., et al. and amino acid map of the receptor. Magenta, CB₁; green, agonist; red, Gα_i; yellow, Gβ; grey, single-chain variable fragment scFv16; cyan, Gγ.

The first crystal structure of CB₂ bound to high-affinity synthetic antagonist was reported in 2019, and both the crystal structure and cryo-EM structure of agonist-bound CB₂ was revealed in 2020 (Figure 5). The N-terminal sequence has different behaviors between the two receptors. Compared to CB₁, CB₂ only experiences minor conformational changes upon agonist binding. With antagonist bound, CB₁ conforms to a V-shaped loop that inserts into the cannabinoid binding pocket. However, this portion of CB₂ remains extended and does not interact with the pocket, instead hovering over it with no direct contact during antagonist binding. Also, there is only a single toggle switch residue (W258) for CB₂. Interestingly, the antagonist-binding motif in CB₂ looks similar to the agonist-binding motif in CB₁, providing a structural basis for the fact that CB₂ antagonists are often CB₁ agonists. These structural differences also define ligand preference in that CB₁ requires the polycyclic core of a potential ligand to have an alkyl chain of five or more carbons, while CB₂ recognizes much smaller compounds.

Figure 5. Cryo-EM structure of CB₂-G_i coupling bound with (+)-WIN 55,212-2 [PDB ID (6PTO)] and amino acid map of the receptor. Grey, CB₂; green, WIN55,212-2; red, Gα_i; yellow, Gβ; cyan, Gγ.

Receptor Pharmacology

Cannabinoid receptor ligands can be classified into four main chemical groups: classical, nonclassical, aminoalkylindole, and eicosanoid (Figure 6). The classical group contains dibenzopyran derivatives, the most well-known of which are Δ⁹-THC and HU-210, a synthetic analog of Δ⁸-THC. In 2019, the novel phytocannbinoid Δ⁹-THCP was isolated from Cannabis and shown to bind to both CB₁ and CB₂. Its affinity for CB₁ as well as cannabimimetic activity attributed to its elongated alkyl side chain is significantly higher than that of Δ⁹-THC. The nonclassical group consists of bicyclic and tricyclic analogs of Δ⁹-THC that lack a pyran ring, such as (–)-CP 55,940. Aminoalkylindoles (represented by (+)-WIN 55,212-2) and eicosanoids (comprised of endocannabinoids) have structures that are distinctly different from classical and nonclassical cannabinoid receptor agonists and also from each other. A fifth chemical classification has also emerged: peptide-like compounds. The endogenous peptide hemopressin, its derivatives RVD-Hpα and VD-Hpα, and the novel Pep19 have all been shown to function as ligands of cannabinoid receptors.

Figure 6. Types of cannabinoid ligands.

Competitive antagonists that display significant CB₁-selectivity include rimonabant, AM251, AM281, LY320135, and taranabant. These compounds all work as inverse agonists, eliciting responses in some CB₁ receptor-containing tissues that are contradictory from those induced by a CB₁ receptor agonist. NESS 0327 and AM4113, which are structural analogs of rimonabant, behave as neutral antagonists in that they lack any detectable ability to induce signs of inverse agonism at the CB₁ receptor. The CB₂-selective inverse agonists most often used as experimental tools include AM630 and SR 144528. A neutral antagonist that selectively targets the CB₂ receptor has yet to be developed. 

There is interest not only in directly acting cannabinoid receptor agonists and antagonists, but also in compounds that can affect the activity of the endocannabinoid system indirectly by allosterically modulating endocannabinoid-induced activation of cannabinoid receptors or by altering the concentration of endocannabinoids at their receptors through effects on endocannabinoid production. Some cannabinoid receptor agonists and antagonists can also activate or block non-CB1/CB2 GPCRs, ligand- and voltage-gated ion channels, and/or nuclear receptors (PPAR receptors). They do, however, target these channels or receptors at different potencies than that which can activate or block CB₁ and/or CB₂ receptors. For example, arachidonoyl ethanolamide (anandamide) can activate T-type voltage-gated calcium channels, voltage-gated K_V3.1 and K_V4.3 potassium channels, calcium-activated (BK) potassium channels, NMDA receptors, TRPV receptors, glycine receptors, and allosteric sites on serotonin 5-HT₃, and nicotinic acetylcholine receptors. Cannabidiol (CBD), which has weak CB₁ and CB₂ antagonistic effects and behaves as a negative allosteric modulator of CB₁, can act as a FAAH inhibitor, TRPV1 agonist, adenosine A_2A agonist, PPARγ activator, 5-HT_1a agonist, and GPR55 antagonist. The combinatorial actions of these compounds on various receptors and channels likely endow their ultimate functions.

Pharmacotherapeutic Implications

Cannabinoid receptor modulation is involved in cognition, memory, anxiety, control of appetite, emesis, motor behavior, sensory, autonomic and neuroendocrine responses, immune responses, and inflammatory effects as well as liver injury and hepatocellular carcinoma. The pharmacologic regulation of CB₁ has been proposed as a promising therapeutic strategy for pain, inflammation, obesity, neurodegenerative disorders, and cancer. Meanwhile, CB₂ has been explored as a therapeutic target for immune modulators, pain management, osteoporosis, and the treatment of liver diseases. Indeed, the endocannabinoid system functions as a gatekeeper of the immune system, regulating cytokine release and immune cell migration, which has implications in preventing the onset of pathological immune responses through the regulation of inflammation, autoimmunity, antitumor, and antipathogen immune responses.

Many in vitro and in vivo studies aimed at exploiting the putative therapeutic potential of cannabinoids have largely focused on isolating Δ⁹-THC and CBD and the need to develop appropriate formulations to separate any psychoactive effects from therapeutic benefits. This has led to the establishment of the first Δ⁹-THC-containing and CBD-containing FDA-approved therapeutics. Dronabinol (Marinol^®, Syndros^®) is a Δ⁹-THC formulation used as an appetite stimulant and anti-emetic that the FDA has only approved for use in HIV/AIDS-induced anorexia and chemotherapy-induced nausea and vomiting. Several countries have approved the use of nabiximols (Sativex^®), a 1:1 combination of Δ⁹-THC and CBD, for the treatment of pain and/or spasticity in multiple sclerosis. The synthetic THC mimic nabilone (Cesamet^®), which has therapeutic use as an antiemetic and as an adjunct analgesic for neuropathic pain, is in a phase III clinical trial for treating non-motor symptoms in Parkinson’s disease. Epidiolex^®, a prescription oral solution of CBD, is being used to treat seizures associated with certain forms of epilepsy and tuberous sclerosis complex. However, the Cannabis plant contains more than 100 different cannabinoids, including numerous secondary cannabinoid metabolites as well as terpenes and flavonoids that likely contribute synergistically to its therapeutic benefits. Due to this entourage effect, focus will need to shift to these other Cannabis components in order to fully understand which components have pharmacotherapeutic potential. Attention has also focused on anandamide analogs including arachidonoyl 2’-chloroethylamide and arachidonoyl cyclopropylamide, CB₁-selective agonists that have been shown to have antidepressant and antinociceptive effects as well as the ability to potentiate anticonvulsant drugs in animal models of epilepsy. CB₂-selective agonists are a keen focus in the field of therapeutic uses since specific modulation of CB₂ may avoid the CNS-related side effects known to occur with CB₁-selective agonists. With recent revelation of the ligand-bound, cryo-EM structure of the active CB₂ receptor in complex with Gi-protein, new ligands rationally designed to interact with CB₂ as selective agonists will be on the horizon.

Request a Cannabinoid Receptor Wall Poster for a guide to what is currently known about cannabinoid receptor signaling.

Cayman offers a broad range of pharmacological tools to help growers profile the cannabinoid species in their products and to help researchers delineate cannabinoid receptor-mediated activity and will continue to produce novel receptor agonists and antagonists to support a new and better understanding of the various mechanisms of action.

Suggested Reading

An, D., Peigneur, S., Hendrickx, L.A., et al. Targeting cannabinoid receptors: Current status and prospects of natural products. Int. J. Mol. Sci.21(14), 5064 (2020).

Hua, T., Li, X., Wu, L., et al. Activation and signaling mechanism revealed by cannabinoid receptor-G_i complex structures. Cell180(4), 655-665 (2020).

Mallipeddi, S., Janero, D.R., Zvonok, N., et al. Functional selectivity at G-protein coupled receptors: Advancing cannabinoid receptors as drug targets. Biochem. Pharmacol. 128, 1-11 (2017).

Oláh, A, Szekanecz, Z., and Bíró, T. Targeting cannabinoid signaling in the immune system: "High"-ly exciting questions, possibilities, and challenges. Front. Immunol. 8, 1487 (2017).

Shahbazi, F., Grandi, V., Banerjee, A., et al. Cannabinoids and cannabinoid receptors: The story so far. iScience23(7), 101301 (2020).

Xing, C., Zhuang, Y., Xu, T.-H., et al. Cryo-EM structure of the human cannabinoid receptor CB₂-G_i signaling complex. Cell180(4), 645-654 (2020).

Ye, L., Cao, Z., Wang, W., et al. New insights in cannabinoid receptor structure and signaling. Curr. Mol. Pharmacol.12(3), 239-248 (2019).

Zou, S. and Kumar, U. Cannabinoid receptors and the endocannabinoid system: Signaling and function in the central nervous system. Int. J. Mol. Sci.19(3), 833 (2018).