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BACKGROUND AND PURPOSE CB1 cannabinoid receptor signalling is canonically mediated through inhibitory Gαi proteins, but signalling through other G proteins occurs under some circumstances; Gαs being the most characterised secondary pathway. Determinants of this signalling switch identified to date include Gαi blockade, CB1/D2 dopamine receptor co-stimulation, CB1 agonist class, and cell background. The aim of this study was to examine the effects of receptor number and ligand dependence on CB1 signalling. EXPERIMENTAL APPROACH CB1 was expressed in HEK cells at different levels, and signalling characterisation was performed for cAMP by real-time BRET biosensor – CAMYEL – and for phospho-ERK by AlphaScreen. Characterisation of a novel irreversible antagonist of CB1, AM6544, was performed by homogenate and whole cell radioligand binding assays. KEY RESULTS In HEK cells expressing high levels of CB1, agonist treatment resulted in cAMP stimulation; a response not previously known to be mediated by receptor number. Δ9-THC and BAY59-3074 increased cAMP only in high-expressing cells that had been pre-treated with pertussis toxin, and agonists demonstrated more diverse signalling profiles in the stimulatory pathway than they elicited in the canonical inhibitory pathway. CB1 pharmacological knockdown and Gαi1 supplementation were sufficient to restore canonical Gαi signalling to high expressing cells. Constitutive signalling in both lower- and higher-expressing cells was Gαi-mediated. CONCLUSION AND IMPLICATIONS CB1 coupling to opposing G proteins is determined by both receptor and G protein number, and this underpins a mechanism for non-canonical signalling in a fashion consistent with Gαs signalling. CB1 is known to mediate opposite consequences in endpoints such as tumour viability receptor number-dependently, and our study may help to explain such effects at the level of G protein coupling.

Background and purpose N-arachidonyl dopamine (NADA) has been identified as a putative endocannabinoid, but there is little information about which signalling pathways it activates. The purpose of this study was to identify the signalling pathways activated by NADA in vitro. Experimental approach Human or rat cannabinoid CB1 receptors were expressed in AtT20, CHO or HEK 293 cells. NADA displacement of radiolabelled cannabinoids, and CB1 receptor mediated activation of K channels or ERK phosphorylation, release of intracellular calcium ([Ca]i ) and modulation of adenylyl cyclase were measured in addition to NADA effects on CB1 receptor trafficking. Key results At concentrations up to 30 μM, NADA failed to activate any signalling pathways via CB1 receptors, with the exception of mobilization of [Ca]i . The elevations of [Ca]i were insensitive to pertussis toxin, and reduced or abolished by blockers of Gq /11 -dependent processes including U73122, thapsigargin and a peptide antagonist of Gq /11 activation. Prolonged NADA incubation produced modest loss of cell surface CB1 receptors. The prototypical cannabinoid agonist CP55940 signalled as expected in all assays. Conclusions and implications NADA is an ineffective agonist at most canonical cannabinoid receptor signalling pathways, but did promote mobilization of [Ca]i via Gq -dependent processes and some CB1 receptor trafficking. This signalling profile is distinct from that of any known cannabinoid, and suggests that NADA may have a unique spectrum of effects in vivo. Our results also indicate that it may be possible to identify highly biased CB1 receptor ligands displaying a subset of the pharmacological or therapeutic effects usually attributed to CB1 ligands.

ORG27569 (1) is an allosteric modulator of CB1. 1 produces a distinct cAMP temporal fingerprint with complex time-dependent modulation of agonist-mediated responses. The aim of this study was to characterize the cAMP signaling response of indole-2-carboxamides structurally correlated to 1 for both CB1 and CB2. We show that at CB1 1, 10, 13, and 18 display a delay in inhibiting CP55,940-mediated cAMP inhibition, whereas compounds 7, 14, 15, 16, 20, and 22 act immediately. To further characterize this, compounds 1, 10, 13, 14, 15, 18, and 20 were tested for their influence on CP55,940-mediated hyperpolarization in AtT20-hCB(1) cells. Intriguingly, all compounds generated a response similar to that of 1, producing no decrease in CB1-mediated peak hyperpolarization at concentrations up to 10 mu M but enhancing the rate at which the channel repolarizes. Additionally, we show that compounds 5, 10, and 20 indole-2-carboxamides modulate cAMP signaling through CB2.

Cannabinoid receptors, like other GPCRs, signal via a spectrum of related signaling pathways. Recently, monitoring GPCR-mediated cAMP signaling has become significantly easier with the development of genetically encoded, transfectable cAMP biosensors. Cell lines transfected with these biosensors can be monitored continuously, allowing the analysis of receptor-mediated signaling in unprecedented detail. Here, we describe a protocol for transfectable biosensors which report cellular cAMP concentrations by bioluminescence resonance energy transfer (BRET). This assay system has been utilized to elucidate the temporal nature of agonists and allosteric modulators of the cannabinoid receptor CB1. In particular, the CB1 allosteric modulator ORG27569 has been shown to modify receptor agonism in a time-dependent fashion; a characteristic which would not have been observed via traditional endpoint methods of detecting cAMP signaling. BRET cAMP biosensors are suitable for miniaturization and automation, and as such are valuable and cost-effective tools for moderate- to high-throughput experimental protocols.

Background and Purpose The cannabinoid receptor type 1 (CB1) has an allosteric binding site. The drugs ORG27569 {5-chloro-3-ethyl-N-[2-[4-(1-piperidinyl)phenyl]ethyl]-1H-indole-2-carboxamide} and PSNCBAM-1 {1-(4-chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea} have been extensively characterized with regard to their effects on signalling of the orthosteric ligand CP55,940 {(−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol}, and studies have suggested that these allosteric modulators increase binding affinity but act as non-competitive antagonists in functional assays. To gain a deeper understanding of allosteric modulation of CB1, we examined real-time signalling and trafficking responses of the receptor in the presence of allosteric modulators. Experimental Approach Studies of CB1 signalling were carried out in HEK 293 and AtT20 cells expressing haemagglutinin-tagged human and rat CB1. We measured real-time accumulation of cAMP, activation and desensitization of potassium channel-mediated cellular hyperpolarization and CB1 internalization. Key Results ORG27569 and PSNCBAM-1 produce a complex, concentration and time-dependent modulation of agonist-mediated regulation of cAMP levels, as well as an increased rate of desensitization of CB1-mediated cellular hyperpolarization and a decrease in agonist-induced receptor internalization. Conclusions and Implications Contrary to previous studies characterizing allosteric modulators at CB1, this study suggests that the mechanism of action is not non-competitive antagonism of signalling, but rather that enhanced binding results in an increased rate of receptor desensitization and reduced internalization, which results in time-dependent modulation of cAMP signalling. The observed effect of the allosteric modulators is therefore dependent on the time frame over which the signalling response occurs. This finding may have important consequences for the potential therapeutic application of these compounds.

Understanding the molecular basis of drug action can facilitate development of more potent and selective drugs. Here, we explore the molecular basis for action of a unique small molecule ligand that is a type 1 cholecystokinin (CCK) receptor agonist and type 2 CCK receptor antagonist, GI181771X. We characterize its binding utilizing structurally related radioiodinated ligands selective for CCK receptor subtypes that utilize the same allosteric ligand-binding pocket, using wild-type receptors and chimeric constructs exchanging the distinct residues lining this pocket. Intracellular calcium assays were performed to determine biological activity. Molecular models for docking small molecule agonists to the type 1 CCK receptor were developed using a ligand-guided refinement approach. The optimal model was distinct from the previous antagonist model for the same receptor and was mechanistically consistent with the current mutagenesis data. This study revealed a key role for Leu(7.39) that was predicted to interact with the isopropyl group in the N1 position of the benzodiazepine that acts as a "trigger" for biological activity. The molecular model was predictive of binding of other small molecule agonists, effectively distinguishing these from 1065 approved drug decoys with an area under curve value of 99%. The model also selectively enriched for agonist compounds, with 130 agonists identified by ROC analysis when seeded in 2175 non-agonist ligands of the type 1 CCK receptor (area under curve 78%). Benzodiazepine agonists in this series docked in consistent pose within this pocket, with a key role played by Leu(7.39), whereas the role of this residue was less clear for chemically distinct agonists.

Cannabinoid receptors, like other GPCRs, signal via a spectrum of related signaling pathways. Recently, monitoring GPCR-mediated cAMP signaling has become significantly easier with the development of genetically encoded, transfectable cAMP biosensors. Cell lines transfected with these biosensors can be monitored continuously, allowing the analysis of receptor-mediated signaling in unprecedented detail. Here, we describe a protocol for transfectable biosensors which report cellular cAMP concentrations by bioluminescence resonance energy transfer (BRET). This assay system has been utilized to elucidate the temporal nature of agonists and allosteric modulators of the cannabinoid receptor CB1. In particular, the CB1 allosteric modulator ORG27569 has been shown to modify receptor agonism in a time-dependent fashion; a characteristic which would not have been observed via traditional endpoint methods of detecting cAMP signaling. BRET cAMP biosensors are suitable for miniaturization and automation, and as such are valuable and cost-effective tools for moderate- to high-throughput experimental protocols.

Since the first description of allosterism in the mid-1900s a number of G protein-coupled receptor allosteric sites have been described. The first description of an allosteric site for CB1 was in 2005, with a series of compounds from Organon described, followed shortly thereafter by the structurally similar PSNCBAM-1. Since then several other ligands have been suggested to act allosterically at CB1, including lipoxin A4, pregnenolone, and pepcans. This chapter will describe the pharmacology of allosteric modulators of CB1 with a main focus on the binding and efficacy modulation induced by ORG27569 and PSNCBAM-1. Additionally this chapter will describe how heterodimerization is another form of CB1 allosterism and highlight the therapeutic potential of allosteric modulation of CB1.

CBCBIn HEK cells expressing high levels of CBCB

Allosteric binding pockets in peptide-binding G protein-coupled receptors create opportunities for the development of small molecule drugs with substantial benefits over orthosteric ligands. To gain insights into molecular determinants for this pocket within type 1 and 2 cholecystokinin receptors (CCK1R and CCK2R), we prepared a series of receptor constructs in which six distinct residues in TM2, -3, -6, and -7 were reversed. Two novel iodinated CCK1R- and CCK2R-selective 1,4-benzodiazepine antagonists, differing only in stereochemistry at C3, were used. When all six residues within CCK1R were mutated to corresponding CCK2R residues, benzodiazepine selectivity was reversed, yet peptide binding selectivity was unaffected. Detailed analysis, including observations of gain of function, demonstrated that residues 6.51, 6.52, and 7.39 were most important for binding the CCK1R-selective ligand, whereas residues 2.61 and 7.39 were most important for binding CCK2R-selective ligand, although the effect of substitution of residue 2.61 was likely indirect. Ligand-guided homology modeling was applied to wild type receptors and those reversing benzodiazepine binding selectivity. The models had high predictive power in enriching known receptor-selective ligands from related decoys, indicating a high degree of precision in pocket definition. The benzodiazepines docked in similar poses in both receptors, with C3 urea substituents pointing upward, whereas different stereochemistry at C3 directed the C5 phenyl rings and N1 methyl groups into opposite orientations. The geometry of the binding pockets and specific interactions predicted for ligand docking in these models provide a molecular framework for understanding ligand selectivity at these receptor subtypes. Furthermore, the strong predictive power of these models suggests their usefulness in the discovery of lead compounds and in drug development programs.

Receptor ligands, identified as antagonists, based on the absence of stimulation of signaling, can rarely stimulate receptor internalization. d-Tyr-Gly-[(Nle28,31,d-Trp30)CCK-26–32]-2-phenylethyl ester (d-Trp-OPE) is such a ligand that binds to the cholecystokinin (CCK) receptor and stimulates internalization. Here, the molecular basis of this trafficking event is explored, with the assumption that ligand binding initiates conformational change, exposing an epitope to direct endocytosis. Ligand-stimulated internalization was studied morphologically using fluorescent CCK and d-Trp-OPE. d-Trp-OPE occupation of Chinese hamster ovary cell receptors stimulated internalization into the same region as CCK. Arrestin-biased action was ruled out using morphological translocation of fluorescent arrestin 2 and arrestin 3, moving to the membrane in response to CCK, but not d-Trp-OPE. Possible roles of the carboxyl terminus were studied using truncated receptor constructs, eliminating the proline-rich distal tail, the serine/threonine-rich midregion, and the remainder to the vicinal cysteines. None of these constructs disrupted d-Trp-OPE-stimulated internalization. Possible contributions of transmembrane segments were studied using competitive inhibition with peptides that also had no effect. Intracellular regions were studied with a similar strategy using coexpressing cell lines. Peptides corresponding to ends of each loop region were studied, with only the peptide at the carboxyl end of the third loop inhibiting d-Trp-OPE-stimulated internalization but having no effect on CCK-stimulated internalization. The region contributing to this effect was refined to peptide 309–323, located below the recognized G protein-association motif. While a receptor in which this segment was deleted did internalize in response to d-Trp-OPE, it exhibited abnormal ligand binding and did not signal in response to CCK, suggesting an abnormal conformation and possible mechanism of internalization distinct from that being studied. This interpretation was further supported by the inability of peptide 309–323 to inhibit its d-Trp-OPE-stimulated internalization. Thus the 309–323 region of the type 1 CCK receptor affects antagonist-stimulated internalization of this receptor, although its mechanism and interacting partner are not yet clear.

The success in screening for drug candidates is highly dependent on the power of the strategy implemented. In this work, we report and characterize a novel fluorescent benzodiazepine antagonist of the type 1 cholecystokinin receptor (3-(3-(7-fluoro-1-(2-isopropyl(4-methoxyphenyl)amino)-2-oxoethyl)-2,4-dioxo-5-phenyl-2,3,4,5-tetrahydro-1H-benzo[b][1,4]-diazepin-3-yl)ureido)benzoic acid) that can be used as a receptor ligand in a fluorescence polarization assay, which is ideally suited for the identification of small molecule allosteric modulators of this physiologically important receptor. By binding directly to the small molecule-docking region within the helical bundle of this receptor, this indicator can be displaced by many small molecule candidate drugs, even those that might not affect the binding of an orthosteric cholecystokinin-like peptide ligand. The biological, pharmacological, and fluorescence properties of this reagent are described, and proof-of-concept is provided in a fluorescence polarization assay utilizing this fluorescent benzodiazepine ligand.

Cholecystokinin (CCK) is a physiologically important gastrointestinal and neuronal peptide hormone, with roles in stimulating gallbladder contraction, pancreatic secretion, gastrointestinal motility and satiety. CCK exerts its effects via interactions with two structurally related class I guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs), the CCK1 receptor and the CCK2 receptor. Here, we focus on the CCK1 receptor, with particular relevance to the broad spectrum of signalling initiated by activation with the natural full agonist peptide ligand, CCK. Distinct ligand-binding pockets have been defined for the natural peptide ligand and for some non-peptidyl small molecule ligands. While many CCK1 receptor ligands have been developed and have had their pharmacology well described, their clinical potential has not yet been fully explored. The case is built for the potential importance of developing more selective partial agonists and allosteric modulators of this receptor that could have important roles in the treatment of common clinical syndromes. This article is part of a themed section on Molecular Pharmacology of GPCR. To view the editorial for this themed section visit http://dx.doi.org/10.1111/j.1476-5381.2010.00695.x

N-arachidonyl dopamine (NADA) has been identified as a putative endocannabinoid, but there is little information about which signalling pathways it activates. The purpose of this study was to identify the signalling pathways activated by NADA in vitro.Human or rat cannabinoid CB1 receptors were expressed in AtT20, CHO or HEK 293 cells. NADA displacement of radiolabelled cannabinoids, and CB1 receptor mediated activation of K channels or ERK phosphorylation, release of intracellular calcium ([Ca]i ) and modulation of adenylyl cyclase were measured in addition to NADA effects on CB1 receptor trafficking.At concentrations up to 30 μM, NADA failed to activate any signalling pathways via CB1 receptors, with the exception of mobilization of [Ca]i . The elevations of [Ca]i were insensitive to pertussis toxin, and reduced or abolished by blockers of Gq /11 -dependent processes including U73122, thapsigargin and a peptide antagonist of Gq /11 activation. Prolonged NADA incubation produced modest loss of cell surface CB1 receptors. The prototypical cannabinoid agonist CP55940 signalled as expected in all assays.NADA is an ineffective agonist at most canonical cannabinoid receptor signalling pathways, but did promote mobilization of [Ca]i via Gq -dependent processes and some CB1 receptor trafficking. This signalling profile is distinct from that of any known cannabinoid, and suggests that NADA may have a unique spectrum of effects in vivo. Our results also indicate that it may be possible to identify highly biased CB1 receptor ligands displaying a subset of the pharmacological or therapeutic effects usually attributed to CB1 ligands.