The cannabinoid receptors are members of the rhodopsin-like family of 7-transmembrane spanning receptors that are believed to bind their agonist ligands within the central core formed by the interaction of the seven transmembrane helices. Cannabinoid receptors are associated with G proteins of the Gi/o family (Gi_1,2 and ₃, and Go₁ and ₂) via the intracellular loops of the protein. Two receptor subtypes have been characterized: CB₁ receptors found predominantly in the nervous system and neuronal cells, and CB₂ receptors found predominantly in immune tissue. An alternative receptor that is stimulated by anandamide has been pharmacologically described, and it is coupled to the Gi family of signal transduction proteins. Both CB₁ and CB₂ cannabinoid receptors inhibit adenylyl cyclase via Gi, and both subtypes stimulate p42/p44 mitogen-activated protein kinase (MAPK) activity. Only the CB₁ receptor has been shown to signal via ion channels. CB₁ and CB₂ receptors influence other signal transduction pathways as well. The molecular mechanism for agonist activation of the cannabinoid receptors involves modification of the conformation of the third and fourth intracellular loops of the receptor such that associated G proteins can be activated.

CB₁ Receptor and Signal Transduction Pathways

CB₂ Receptor and Signal Transduction Pathways

The inhibition of cyclic AMP production has been observed in human lymphocytes, mouse spleen cells expressing CB₂ receptors, and in host cell lines expressing CB₂ receptors.^29,59,60 CB₂ receptor-mediated inhibition of adenylyl cyclase was blocked by pertussis toxin, implicating Gi/o proteins as transducers.²⁹ The inhibition of adenylyl cyclase by CB₂ receptors probably occurs in only those cells that express adenylyl cyclase isoform families 5, 6 and 1, 3, 8.¹⁴ 2-Arachidonoylglycerol was a full agonist, and anandamide and (R)-methanandamide were partial, low-efficacy agonists for the inhibition of forskolin-stimulated cyclic AMP accumulation in CHO cells expressing recombinant CB₂receptors.^29,59–60
MAPK was activated by cannabinoid receptor agonists in cultured human promyelocytic HL60 cells possessing endogenous CB₂ receptors, and in CHO cells expressing recombinant CB₂ receptors.⁶²However, cannabinoid agonists failed to activate PKB/Akt in HL60 cells, suggesting that a PI3K mechanism may not be regulated by CB₂ receptors in this model.⁴⁷ Krox-24 expression was stimulated via CB₂ receptors in HL60 promyelocytes.⁶²
A novel signal transduction pathway regulated by CB₂ receptors appears to be directed by the synthesis of ceramide. Long-term (days) treatment with ceramide, which binds to and activates raf-1, led to the sustained activation of p42/p44 MAPK and the ensuing apoptosis in sensitive strains of rat C6 glioma cells.⁶³ Cannabinoid agonists (Δ⁹-THC, CP55940, HU210, WIN55212-2 and endocannabinoids anandamide and 2-arachidonoylglycerol) could promote the antiproliferative effects in C6 cells by contributions from both CB₁ and CB₂ receptors,^63,64 although in U373MG astrocytoma cells, stimulation of the CB₁ receptor was protective against ceramide-induced apoptosis.⁴⁶ The primary mechanism was related to the continued synthesis of ceramide over a period of days.^63,65 The importance of the CB₂receptors in regulation of serine palmitoyltransferase, the rate-limiting enzyme in ceramide synthesis, was demonstrated using enzyme inhibitors and the selective CB₂ agonists JWH015 and JWH133 and antagonist SR144528.^64,66 These data demonstrate the importance of ceramide as a cellular modulator, and suggest that the duration of the activation of p42/p44 MAPK is critical to the apoptotic response in transformed glioma cells.⁶³
Several signal transduction pathways that have been characterized for the CB₁ receptor have not been identified for the CB₂ receptor. PLC activation in response to anandamide or WIN55212-2 was not able to be detected in CHO cells expressing recombinant CB₂ receptors.^7,28,29 The inhibition of Q-type Ca²⁺currents in AtT-20 cells expressing CB₂ receptors could not be stimulated by WIN55212-2 or anandamide.²⁹

Endothelial Anandamide Receptor: Pharmacology and Signal Transduction Pathways

A distinct non-CB₁/non-CB₂ receptor for anandamide has recently been characterized in endothelial cells.^67–60 Vasorelaxation is the physiological response regulated by this receptor, and this was demonstrated in a rodent mesenteric vascular preparation^67–71 as well as a rabbit aortic ring preparation.⁶⁸ The response required endothelial cell-intact preparations, suggesting an endothelial location for the receptor.^67–68 This notion has subsequently been confirmed by signal transduction studies in endothelial cells.^69–72 The observation that vasorelaxation could be reproduced in preparations from CB₁/CB₂ (-/-) transgenic mice is evidence that these genetically defined receptors are not responsible for this biological response.⁶⁷ The pharmacology of the response is distinct from that of either CB₁ or CB₂ receptors. The agonists that evoked vasodilation include anandamide, (R)-methanandamide and abnormal cannabidiol. It is noteworthy that potent cannabinoid receptor agonists including Δ⁹-THC, CP55940, WIN55212-2 and HU210 failed to elicit this response.^67–69,73 Although the CB₁ antagonist SR141716 could competitively antagonize the vasorelaxation response, higher concentrations were required than would be expected for a CB₁ receptor-mediated response.^67,68Cannabidiol, which exhibits particularly poor efficacy as a CB₁ receptor agonist, antagonized the anandamide-mediated vasorelaxation.^67,74 Subsequently, a novel antagonist for this receptor, O-1918, a congener of cannabidiol, was developed and demonstrated to behave as a competitive antagonist against anandamide and abnormal cannabidiol in the vasorelaxation response.⁶⁹ In intact anesthetized mice, O-1918 blocked the hypotensive action of abnormal cannabidiol but not that of the cannabinoid receptor agonist HU210.⁶⁹ Neither abnormal cannabidiol nor O-1918 were able to compete for the binding of [³H]CP55940 to the CB₁ or CB₂ receptor, confirming that these ligands are not acting via these receptors.
The signal transduction pathway by which this endothelial anandamide receptor mediates its response has been examined. Both (R)-methanandamide-mediated vasorelaxation of aortic rings and abnormal cannabidiol-mediated vasorelaxation of rat mesenteric artery segments were pertussis toxin-sensitive, indicating that the receptor is coupled to the Gi/o family of signal transducing proteins.^68,69 The vasorelaxation in the rabbit aortic ring preparation was blocked by the NOS inhibitor L-NAME, indicating that NO production is an intrinsic part of the pathway.⁶⁸ However, this appeared not to be the case in the rat mesenteric artery preparation⁶⁹ or perfused mesenteric bed preparations.^67,75 The vasorelaxation response to anandamide in coronary or mesenteric artery preparations and to abnormal cannabidiol in rat mesenteric artery segments was blocked by charybdotoxin.^69,75–77 This implicates the involvement of either a delayed rectifier K+ channel or a large conductance calcium-activated K⁺ (BK_Ca) channel. Further analysis of the signal transduction pathways was performed in cultured endothelial cells. In human umbilical vein endothelial cells (HUVEC), abnormal cannabidiol could evoke the phosphorylation of p42/44 MAPK and PKB/Akt. The latter could be blocked by a PI3K inhibitor (LY294002 or wortmannin), indicating the mediation of this response via PI3K.⁶⁹ In cultured rabbit aortic endothelial cells, (R)-methanandamide elicited NO production, which was blocked by pertussis toxin and LY294002, indicating the involvement of Gi/o proteins and the PI3K pathway, respectively.⁷² Thus, the endothelial anandamide receptor presents distinct pharmacological and signal transduction properties compared with the two characterized cannabinoid receptors.

Mechanism of Receptor-Mediated G Protein Coupling

Cannabinoid receptors are 7-transmembrane spanning receptors associated with intracellular G proteins (fig. 1). When the CB₁ receptor-G protein complex is solubilized using the mild nonionic detergent CHAPS, the protein-protein interactions remain stable even as the lipids surrounding the proteins are supplanted by the detergent.^78,79 The CB₁ receptor must remain precoupled with G proteins even in the absence of an exogenously added agonist, inasmuch as it is unlikely that any endogenous agonist can remain bound to the receptor during the solubilization and immunoprecipitation processes.

Figure 1

The rat CB₁ receptor in two-dimensional representation. Three extracellular (E1, E2, and E3) and intracellular (C1, C2, and C3) regions are represented. The most conserved residues within each helix is indicated by an arrow (see ref. ). The palmitoylation(more…)

When the CB₁ receptor was immunoprecipitated from CHAPS-solubilized rat brain or N18TG2 neuroblastoma cell membranes, specific Gα proteins associated with the CB₁ receptor were found to be in the same proportions as those found in particular brain regions or cell types.^79,80 For example, Gαo was extremely abundant in rat brain membranes, but barely detectable in N18TG2 membranes, and thus CB₁ receptors from rat brain coimmunoprecipitated with an abundance of Gαo. In contrast, Gαi1, Gαi2 and Gαi3 were much more abundant in N18TG2 membranes than in rat brain. These CB₁ receptor-Gα complexes represent a functional association in dynamic equilibrium both in CHAPS as well as in intact membranes, as demonstrated by the finding that pertussis toxin treatment disrupted the receptor-Gα association.^79,81 The observation that many different signal transduction pathways are possible suggests that the cell-type specific G proteins and the stoichiometry of these various Gα and Gβγ dimers with the receptor are factors in determining which signal transduction pathways are operative.
The signal transduction response is inititiated by the binding of agonist ligands to the receptor as it exists in a receptor-G protein complex. Moieties on classical and nonclassical cannabinoid ligands that are important for binding to the CB₁ cannabinoid receptor have been identified by structure-activity relationship (SAR) studies (see reviews refs. 2,4,82,83 for original references). It was proposed that lys192 in TM3 might be a key residue for binding because this residue in the cannabinoid receptor is homologous to key amino acids important for agonist binding to rhodopsin and the aminergic neurotransmitter receptors.^84,85 When tested by site-directed mutagenesis studies, lys₁₉₂ was critical for binding of [³H]CP55940 but not [³H]WIN55212-2, and the inhibition of cyclic AMP production in response to CP55940, HU210 and anandamide was attenuated.^84,85 The similarly charged arg could substitute, but not gln or glt,⁸⁵ lending credence to the hypothesis that lys₁₉₂ may serve as a hydrogen bond donor to the oxygen of the phenolic hydroxyl known to be critical from classical and nonclassical cannabinoid SAR studies.^86,87
SAR studies for CB₂ agonists have not been as extensive as for CB₁ receptor ligands (see ref. 2 for review). Ligands having notable selectivity for the CB₂ receptor include AAI derivatives and cannabinoid structures that lack the phenolic hydroxyl or possess hydroxymethyl groups at that position.^29,88–92Studies of CB₁/2(TM3) mutant receptors have suggested that the TM3 ser in CB₂ receptors may contribute to greater affinity for AAI ligands than the gly in the comparable position in CB₁ receptors.⁹³
The domains of the CB₁ receptor that interact with G proteins are on the cytoplasmic surface. Peptides representing defined regions of the C3 loop and the juxtamembrane C-terminal region were used to mimic the intracellular domain(s) of the CB₁ receptor.⁹⁴ The juxtamembrane C-terminal peptide promoted G protein activation in rat brain membranes and the inhibition of hormone- or forskolin-stimulated adenylyl cyclase in N18TG2 membranes, so it is believed that this peptide mimics the receptor domain that recognizes and activates G proteins.⁹⁵ The requirement for G proteins in the inhibition of adenylyl cyclase by this peptide mimic was demonstrated by its sensitivity to pertussis toxin.^81–95 The juxtamembrane C-terminal peptide competed for the interaction between the CB₁receptor with Gαo or Gαi3⁷⁹ in solubilized brain preparations, and the CB₁ receptor with Gαi3 in solubilized N18TG2 membrane extracts.⁸⁰ However, this peptide failed to disrupt the CB₁ receptor interaction with Gαi1 or Gαi2 in rat brain or N18TG2 cell membranes. These data strongly suggest that the juxtamembrane C-terminal domain interacts with Gαo or Gαi3 proteins, and therefore would direct signal transduction pathways mediated by these G protein subtypes. The C-terminal juxtamembrane peptide from the CB₂ receptor failed to compete for Gαo or Gαi,^79,80 suggesting that this domain does not perform the same function in the CB₂ receptor as it does in the CB₁ receptor.
Under environmental conditions that would mimic the anionic phospholipid headgroups of the lipid bilayer or a negative charge-patch on the Gα surface, the C-terminal juxtamembrane peptide is able to form a helical structure.⁹⁵ It can be hypothesized that changes in TM7 that might alter the positions of the cationic residues in the C-terminal domain may promote activation of these G proteins. There is no evidence to suggest that the C-terminal distal to this region is required for Go-mediated responses, because truncation beyond this domain fails to alter the coupling of the CB₁ receptor to inward rectifier K⁺ currents.⁹⁶
Palmitoylation of a cys residue anchors the C-terminal domain to the plasma membrane distal to the putative helical intracellular domain.97 Mutants of the receptor that are truncated two residues but not 23 residues beyond this cys, resulted in a loss of desensitization, suggesting that critical sites of phosphorylation facilitated by G protein receptor kinase-3 and subsequent association with β-arrestin2 occur within this region.96 Studies of site-mutations of ser₄₂₆ and ser₄₃₀ indicated that these residues are required for desensitization, but not internalization.⁹⁶ Internalization of the CB₁ receptor involved the distal region 14 residues from the C-terminal (val₄₆₀-thr-met-ser₄₆₃), and apparently did not require coupling with functional Gi proteins.⁹⁸
The third intracellular loop (C3) is also important for directing signal transduction through G proteins. The combination of three peptides (N-terminal side, middle or C-terminal side) comprising the C3 of the receptor was able to disrupt the CB₁ receptor association with Gαi1 or Gαi2 in both rat brain and N18TG2 membrane extracts,^79,80 whereas the C3 peptides were unable to compete with Gαo in rat brain extracts or Gαi3 in N18TG2 membrane extracts. None of the C3 peptides alone were able to disrupt the CB₁ receptor-Gα interaction. However, the combination of middle plus C-terminal side was capable of dissociating the CB₁ receptor association with Gαi1 or Gαi2, whereas the combination of N-terminal side plus middle failed to do so. Thus, the C3 (C-terminal side) domain interacts with Gαi1 or Gαi2, but not Gαi3 or Gαo. The C3 peptides activated G proteins in rat brain membranes but to a much lesser extent than did the juxtamembrane C-terminal peptide.⁹⁴ One explanation is that the G protein that is highly predominant in brain is Go. A 9- amino acid C-terminal side peptide, predicted by homology with rhodopsin to be embedded within the membrane-cytosol interface, was able to sustain the GTPase activity of a pure preparation of Gαi1.⁹⁹
In an SDS micelle environment, thought to mimic the lipid bilayer and phospholipid headgroup region, the structure of a peptide encompassing the C3 loop was shown by NMR analysis to be helical for the N-terminal third of the intracellular domain extending beyond TM5.¹⁰⁰ Ulfers and colleagues¹⁰⁰speculate that a highly hydrophobic sequence of three ile residues at the terminal end of this helical domain may anchor this region to the membrane. At the juncture between the helical region and the triple-ile hydrophobic domain resides a ser₃₁₇, which has the potential to be phosphorylated by PKC.¹⁰¹Although phosphorylation of ser₃₁₇ prevented the cannabinoid agonist-induced regulation of ion channels, mutation of ser₃₁₇ to ala did not.¹⁰¹ Thus, negative charge in this region must alter the structural properties. The middle third of C3 has limited structure according to the NMR analyses; however, the NMR suggests that a turn may occur at an intracellular gly residue.¹⁰¹
The C3 was shown to be helical beginning within the C-terminal third approximately two turns proximal to the position that is predicted (by homology with rhodopsin¹⁰²) to enter the lipid bilayer as TM6.¹⁰⁰ NMR analysis of a peptide representing this C-terminal region indicated that this peptide is also helical in the presence of Gαi1.⁹⁹ Within this domain, approximately two helical coils embedded in the membrane, exists a leu-ala-lys-thr sequence that is noteworthy because the leu-ala residues are reversed in the Gs-coupled β-adrenergic receptor. ¹⁰³ Reversal of this leu-ala sequence in the CB₁ receptor resulted in a loss of coupling to Gi thereby attenuating inhibition of cyclic AMP production.¹⁰³ Ulfers and colleagues¹⁰⁰ speculated that this region may be oriented along the plasma membrane surface as the result of TM6 being bent at an intracellular pro residue. Alternatively, this part of the coil may be exposed upon activation of the receptor. The ala-leu sequence may not form the appropriate helical structure.⁹⁹ A stimulation of cyclic AMP production was observed when cells bearing the mutant CB₁receptor ala-leu sequence were treated with pertussis toxin to block interaction of Gi proteins with the receptor. This suggests that in the absence of functional Gi proteins, the mutant CB₁ receptor was able to couple to Gs as might be predicted from the sequence homology with the Gs-coupling receptor.¹⁰³
It is possible to speculate on how binding of the CB₁ receptor agonists within the receptor binding pocket might activate G proteins, based upon analogy with what has been reported for other G protein coupled receptors. One mechanisms has been proposed by which the TM2 and TM7 helices might interact via a hydrogen bonding association between asp₁₆₃ in TM2 and asn₃₉₄ in TM7.^104,105 In the proposed activation scheme, ligand binding would disrupt this association and thereby promote movement in TM7 and the juxtamembrane C-terminal domain. This TM2 asp₁₆₃ is the putative site of the regulation of receptor-G protein coupling by Na⁺ ions and has been implicated in the modulation of signal transduction (see ref. 78 for data and original literature). When expressed in AtT20 host cells, CB₁ receptors having a mutation in TM2 asp₁₆₃ exhibited a disruption of signal transduction through the inwardly rectifying K⁺ channel, but other signal transduction pathways (cyclic AMP inhibition, MAPK activation, Ca²⁺ currents) remained unaffected.¹⁰⁵ Similarly, expression of CB₁ receptors having a mutation of TM2 asp₁₆₃ exhibited an attenuation of cannabinoid-stimulated inwardly rectifying K⁺currents due to GIRK1/4 channel proteins that were coexpressed in Xenopus oocytes.³² In contrast to findings in the AtT20 expression system, CB₁ receptors having this TM2 asp₁₆₃ mutation that were expressed in HEK293 cells were able to attenuate the inhibition of cyclic AMP production,¹⁰⁶suggesting that cell-type differences may be responsible for the discrepancy. The original hypothesis that a TM2 to TM7 interaction my be guiding signal transduction is not well supported by experimental evidence because a reciprocal mutation (TM2 asp₁₆₄asn plus TM7 asn₃₉₄asp) failed to “rescue” the asp₁₆₃ mutation as would have been predicted if these two amino acids were associated by hydrogen bonding.¹⁰⁵ Thus, it is known that the TM2 asp₁₆₃ is important for conferring a signal to G proteins, but there is insufficient evidence to propose a mechanism.
Another proposed mechanism for conferring a signal to G proteins is via a TM3 to TM6 interaction that could modulate protein conformation in the IC3 domain. Barnett-Norris and colleagues¹⁰⁷ have proposed an interaction between TM3 arg₂₁₄ in the highly conserved asp-arg-tyr-x-x-ile sequence of rhodopsin-like G protein-coupled receptors and the TM6 asp338 at the membrane interface. The TM6 possesses a pro kink, allowing for the possibility that movement in that helix could be facilitated by a ligand binding interaction. Based upon helical chain conformation models, Barnett-Norris and colleagues¹⁰⁷ have proposed that a TM6 hydrophobic interaction with the alkyl side chain of the cannabinoid agonists acts as the trigger to move TM6. The CB₁ homology model of Shim and colleagues,¹⁰² based upon the structure of rhodopsin, also depicts hydrophobic interactions with cannabinoid agonists at the TM6; however, different amino acid residues were involved. The motion of the TM6 could alter the conformation of the C-terminal side of IC3 to promote G protein activation.

Summary and Future Directions

Cannabinoid receptors are G protein coupled receptors, of which, two receptor types have been characterized: CB₁ receptors found predominantly in the brain and neuronal cells, and CB₂ receptors found predominantly in immune tissue. An alternative receptor that is stimulated by anandamide has been pharmacologically described, and it can be speculated that additional receptors may be described and cloned in the future. It is possible that receptors that respond to endocannabinoid ligands but not classical cannabinoid or aminoalkylindole agonists may have to be named to describe more accurately the endogenous compounds that stimulate those receptors in physiological situations.
Cannabinoid receptors are coupled to the Gi family of signal transduction proteins. Both CB₁ and CB₂receptor types inhibit adenylyl cyclase via Gi, and both types stimulate MAPK activity. Only the CB₁receptor has been shown to signal via ion channels. These signal transduction events can undergo complex regulation between pathways, as evidenced by the PKA phosphorylation of ion channels and MAPK pathway proteins. Ultimately, these pathways regulate important cellular functions necessary for synaptic plasticity and long-term changes in neuronal function. Thus, endocannabinoid agonists may be critical in the formation and maintenance of interneuronal communication.
The molecular mechanism for agonist activation of the cannabinoid receptors involves binding of agonist ligands within the central core formed by the interaction of the seven transmembrane helices and subsequent modification of the conformation of the third and fourth intracellular loops of the receptor such that associated G proteins can be activated. Mutation, peptide and modeling studies should provide greater insight into the nature of these mechanisms, so that novel drugs can be designed that confer greater efficacy to selected G proteins. This should promote greater selectivity for signal transduction pathways that are intrinsic to the cellular mechanisms important for therapeutic benefits.

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