Highlights and hot topics in GPCR research from 'Down Under'.

LINKED ARTICLES: This article is part of a themed issue Therapeutic Targeting of G Protein-Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc.

Single nucleotide polymorphisms in the cannabinoid CB2 receptor: Molecular pharmacology and disease associations.

Preclinical evidence implicating cannabinoid receptor 2 (CB2) in various diseases has led researchers to question whether CB2 genetics influence aetiology or progression. Associations between conditions and genetic loci are often studied via single nucleotide polymorphism (SNP) prevalence in case versus control populations. In the CNR2 coding exon, ~36 SNPs have high overall population prevalence (minor allele frequencies [MAF] ~37%), including non-synonymous SNP (ns-SNP) rs2501432 encoding CB2 63Q/R. Interspersed are ~27 lower frequency SNPs, four being ns-SNPs. CNR2 introns also harbour numerous SNPs. This review summarises CB2 ns-SNP molecular pharmacology and evaluates evidence from ~70 studies investigating CB2 genetic variants with proposed linkage to disease. Although CNR2 genetic variation has been associated with a wide variety of conditions, including osteoporosis, immune-related disorders, and mental illnesses, further work is required to robustly validate CNR2 disease links and clarify specific mechanisms linking CNR2 genetic variation to disease pathophysiology and potential drug responses.

Evaluating signaling bias for synthetic cannabinoid receptor agonists at the cannabinoid CB2 receptor.

The rapid structural evolution and emergence of novel synthetic cannabinoid receptor agonists (SCRAs) in the recreational market remains a key public health concern. Despite representing one of the largest classes of new psychoactive substances, pharmacological data on new SCRAs is limited, particularly at the cannabinoid CB2 receptor (CB2 ). Hence, the current study aimed to characterize the molecular pharmacology of a structurally diverse panel of SCRAs at CB2 , including 4-cyano MPP-BUT7AICA, 4F-MDMB-BUTINACA, AMB-FUBINACA, JWH-018, MDMB-4en-PINACA, and XLR-11. The activity of SCRAs was assessed in a battery of in vitro assays in CB2 -expressing HEK 293 cells: G protein activation (Gαi3 and GαoB ), phosphorylation of ERK1/2, and ß-arrestin 1/2 translocation. The activity profiles of the ligands were further evaluated using the operational analysis to identify ligand bias. All SCRAs activated the CB2 signaling pathways in a concentration-dependent manner, although with varying potencies and efficacies. Despite the detection of numerous instances of statistically significant bias, compound activities generally appeared only subtly distinct in comparison with the reference ligand, CP55940. In contrast, the phytocannabinoid THC exhibited an activity profile distinct from the SCRAs; most notably in the translocation of ß-arrestins. These findings demonstrate that CB2 is able to accommodate a structurally diverse array of SCRAs to generate canonical agonist activity. Further research is required to elucidate whether the activation of CB2 contributes to the toxicity of these compounds.

Cannabinoid CB2 receptor orthologues; in vitro function and perspectives for preclinical to clinical translation.

Cannabinoid CB2 receptor agonists are in development as therapeutic agents, including for immune modulation and pain relief. Despite promising results in rodent preclinical studies, efficacy in human clinical trials has been marginal to date. Fundamental differences in ligand engagement and signalling responses between the human CB2 receptor and preclinical model species orthologues may contribute to mismatches in functional outcomes. This is a tangible possibility for the CB2 receptor in that there is a relatively large degree of primary amino acid sequence divergence between human and rodent. Here, we summarise CB2 receptor gene and protein structure, assess comparative molecular pharmacology between CB2 receptor orthologues, and review the current status of preclinical to clinical translation for drugs targeted at the CB2 receptor, focusing on comparisons between human, mouse and rat receptors. We hope that raising wider awareness of, and proposing strategies to address, this additional challenge in drug development will assist in ongoing efforts toward successful therapeutic translation of drugs targeted at the CB2 receptor.

Novel Cannabinoid Receptor 2 (CB2) Low Lipophilicity Agonists Produce Distinct cAMP and Arrestin Signalling Kinetics without Bias.

Cannabinoid Receptor 2 (CB2) is a promising target for treating inflammatory diseases. We designed derivatives of 3-carbamoyl-2-pyridone and 1,8-naphthyridin-2(1H)-one-3-carboxamide CB2-selective agonists with reduced lipophilicity. The new compounds were measured for their affinity (radioligand binding) and ability to elicit cyclic adenosine monophosphate (cAMP) signalling and ß-arrestin-2 translocation with temporal resolution (BRET-based biosensors). For the 3-carbamoyl-2-pyridone derivatives, we found that modifying the previously reported compound UOSS77 (also known as S-777469) by appending a PEG2-alcohol via a 3-carbomylcyclohexyl carboxamide (UOSS75) lowered lipophilicity, and preserved binding affinity and signalling profile. The 1,8-naphthyridin-2(1H)-one-3-carboxamide UOMM18, containing a cis configuration at the 3-carboxamide cyclohexyl and with an alcohol on the 4-position of the cyclohexyl, had lower lipophilicity but similar CB2 affinity and biological activity to previously reported compounds of this class. Relative to CP55,940, the new compounds acted as partial agonists and did not exhibit signalling bias. Interestingly, while all compounds shared similar temporal trajectories for maximal efficacy, differing temporal trajectories for potency were observed. Consequently, when applied at sub-maximal concentrations, CP55,940 tended to elicit sustained (cAMP) or increasing (arrestin) responses, whereas responses to the new compounds tended to be transient (cAMP) or sustained (arrestin). In future studies, the compounds characterised here may be useful in elucidating the consequences of differential temporal signalling profiles on CB2-mediated physiological responses.

A unique melanocortin-4-receptor signaling profile for obesity-associated constitutively active variants.

The melanocortin-4 receptor (MC4R) plays a critical role in regulating energy homeostasis. Studies on obesogenic human MC4R (hMC4R) variants have not yet revealed how hMC4R maintains body weight. Here, we identified a signaling profile for obesogenic constitutively active H76R and L250Q hMC4R variants transfected in HEK293 cells that included constitutive activity for adenylyl cyclase (AC), cyclic adenosine monophosphate (cAMP) response element (CRE)-driven transcription, and calcium mobilization but not phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) activity. Importantly, the signaling profile included impaired α-melanocyte-stimulating hormone-induced CRE-driven transcription but not impaired α-melanocyte-stimulating hormone-induced AC, calcium, or pERK1/2. This profile was not observed for transfected H158R, a constitutively active hMC4R variant associated with overweight but not obesity. We concluded that there is potential for α-melanocyte-stimulating hormone-induced CRE-driven transcription in HEK293 cells transfected with obesogenic hMC4R variants to be the key predictive tool for determining whether they exhibit loss of function. Furthermore, in vivo, α-melanocyte-stimulating hormone-induced hMC4R CRE-driven transcription may be key for maintaining body weight.

RAMP and MRAP accessory proteins have selective effects on expression and signalling of the CB1 , CB2 , GPR18 and GPR55 cannabinoid receptors.

BACKGROUND AND PURPOSE: Receptor activity-modifying proteins (RAMPs) and melanocortin receptor accessory proteins (MRAPs) modulate expression and signalling of calcitonin and melanocortin GPCRs. Interactions with other GPCRs have also been reported. The cannabinoid receptors, CB1 and CB2 , and two putative cannabinoid receptors, GPR18 and GPR55, exhibit substantial intracellular expression and there are discrepancies in ligand responsiveness between studies. We investigated whether interactions with RAMPs or MRAPs could explain these phenomena. EXPERIMENTAL APPROACH: Receptors and accessory proteins were co-expressed in HEK-293 cells. Selected receptors were studied at basal expression levels and also with enhanced expression produced by incorporation of a preprolactin signal sequence/peptide (pplss). Cell surface and total expression of receptors and accessory proteins were quantified using immunocytochemistry. Signalling was measured using cAMP (CAMYEL) and G protein dissociation (TRUPATH Gα13 ) biosensors. KEY RESULTS: MRAP2 enhanced surface and total expression of GPR18. Pplss-GPR18 increased detection of cell surface MRAP2. MRAP1α and MRAP2 reduced GPR55 surface and total expression, correlating with reduced constitutive, but not agonist-induced, signalling. GPR55, pplss-CB1 and CB2 reduced detection of MRAP1α at the cell surface. Pplss-CB1 agonist potency was reduced by MRAP2 in Gα13 but not cAMP assays, consistent with MRAP2 reducing pplss-CB1 expression. Some cannabinoid receptors increased RAMP2 or RAMP3 total expression without influencing surface expression. CONCLUSIONS AND IMPLICATIONS: Mutual influences on expression and/or function for specific accessory protein-receptor pairings raises the strong potential for physiological and disease-relevant consequences. Sequestration and/or hetero-oligomerisation of cannabinoid receptors with accessory proteins is a possible novel mechanism for receptor crosstalk.

Utilizing PET and MALDI Imaging for Discovery of a Targeted Probe for Brain Endocannabinoid α/ß-Hydrolase Domain 6 (ABHD6).

Multimodal imaging provides rich biological information, which can be exploited to study drug activity, disease associated phenotypes, and pharmacological responses. Here we show discovery and validation of a new probe targeting the endocannabinoid α/ß-hydrolase domain 6 (ABHD6) enzyme by utilizing positron emission tomography (PET) and matrix-assisted laser desorption/ionization (MALDI) imaging. [18F]JZP-MA-11 as the first PET ligand for in vivo imaging of the ABHD6 is reported and specific uptake in ABHD6-rich peripheral tissues and major brain regions was demonstrated using PET. A proof-of-concept study in nonhuman primate confirmed brain uptake. In vivo pharmacological response upon ABHD6 inhibition was observed by MALDI imaging. These synergistic imaging efforts used to identify biological information cannot be obtained by a single imaging modality and hold promise for improving the understanding of ABHD6-mediated endocannabinoid metabolism in peripheral and central nervous system disorders.

Developing the Cannabinoid Receptor 2 (CB2) pharmacopoeia: past, present, and future.

Cannabinoid Receptor 2 (CB2) is a G protein-coupled receptor (GPCR) with considerable, though as yet unrealised, therapeutic potential. Promising preclinical data supports the applicability of CB2 activation in autoimmune and inflammatory diseases, pain, neurodegeneration, and osteoporosis. A diverse pharmacopoeia of cannabinoid ligands is available, which has led to considerable advancements in the understanding of CB2 function and extensive preclinical evaluation. However, until recently, most CB2 ligands were highly lipophilic and as such not optimal for clinical application due to unfavourable physicochemical properties. A number of strategies have been applied to develop CB2 ligands to achieve closer to 'drug-like' properties and a few such compounds have now undergone clinical trial. We review the current state of CB2 ligand development and progress in optimising physicochemical properties, understanding advanced molecular pharmacology such as functional selectivity, and clinical evaluation of CB2-targeting compounds.

Delineating the interactions between the cannabinoid CB2 receptor and its regulatory effectors; ß-arrestins and GPCR kinases.

BACKGROUND AND PURPOSE: The cannabinoid CB2 receptor (CB2 ) is a promising therapeutic target for modulating inflammation. However, little is known surrounding the mechanisms underpinning CB2 desensitisation and regulation, particularly the role of GPCR kinases (GRKs). Here, we evaluated the role of six GRK isoforms in ß-arrestin recruitment to CB2 . Mutagenesis of several distal C-terminal aspartic acid residues was also performed in an attempt to delineate additional structural elements involved in the regulation of CB2 . EXPERIMENTAL APPROACH: In CB2 -expressing HEK 293 cells, ß-arrestin translocation was measured using real-time BRET assays. G protein dissociation BRET assays were performed to assess the activation and desensitisation of CB2 in the presence of ß-arrestin 2. KEY RESULTS: Overexpression of GRK isoforms 1-6 failed to considerably improve translocation of either ß-arrestin 1 or ß-arrestin 2 to CB2 . Consistent with this, inhibition of endogenous GRK2/3 did not substantially reduce ß-arrestin 2 translocation. Mutagenesis of C-terminal aspartic acid residues resulted in attenuation of ß-arrestin 2 translocation, which translated to a reduction in desensitisation of G protein activation. CONCLUSION AND IMPLICATIONS: Our findings suggest that CB2 does not adhere to the classical GPCR regulatory paradigm, entailing GRK-mediated and ß-arrestin-mediated desensitisation. Instead, C-terminal aspartic acid residues may act as phospho-mimics to induce ß-arrestin activation. This study provides novel insights into the regulatory mechanisms of CB2 , which may aid in our understanding of drug tolerance and dependence.

Cannabinoid receptor CB1 and CB2 interacting proteins: Techniques, progress and perspectives.

Cannabinoid receptors 1 and 2 (CB1 and CB2) are implicated in a range of physiological processes and have gained attention as promising therapeutic targets for a number of diseases. Protein-protein interactions play an integral role in modulating G protein-coupled receptor (GPCR) expression, subcellular distribution and signaling, and the identification and characterization of these will not only improve our understanding of GPCR function and biology, but may provide a novel avenue for therapeutic intervention. A variety of techniques are currently being used to investigate GPCR protein-protein interactions, including Förster/fluorescence and bioluminescence resonance energy transfer (FRET and BRET), proximity ligation assay (PLA), and bimolecular fluorescence complementation (BiFC). However, the reliable application of these methodologies is dependent on the use of appropriate controls and the consideration of the physiological context. Though not as extensively characterized as some other GPCRs, the investigation of CB1 and CB2 interacting proteins is a growing area of interest, and a range of interacting partners have been identified to date. This review summarizes the current state of the literature regarding the cannabinoid receptor interactome, provides commentary on the methodologies and techniques utilized, and discusses future perspectives.

Blood-spinal cord barrier leakage is independent of motor neuron pathology in ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving progressive degeneration of upper and lower motor neurons. The pattern of lower motor neuron loss along the spinal cord follows the pattern of deposition of phosphorylated TDP-43 aggregates. The blood-spinal cord barrier (BSCB) restricts entry into the spinal cord parenchyma of blood components that can promote motor neuron degeneration, but in ALS there is evidence for barrier breakdown. Here we sought to quantify BSCB breakdown along the spinal cord axis, to determine whether BSCB breakdown displays the same patterning as motor neuron loss and TDP-43 proteinopathy. Cerebrospinal fluid hemoglobin was measured in living ALS patients (n = 87 control, n = 236 ALS) as a potential biomarker of BSCB and blood-brain barrier leakage. Cervical, thoracic, and lumbar post-mortem spinal cord tissue (n = 5 control, n = 13 ALS) were then immunolabelled and semi-automated imaging and analysis performed to quantify hemoglobin leakage, lower motor neuron loss, and phosphorylated TDP-43 inclusion load. Hemoglobin leakage was observed along the whole ALS spinal cord axis and was most severe in the dorsal gray and white matter in the thoracic spinal cord. In contrast, motor neuron loss and TDP-43 proteinopathy were seen at all three levels of the ALS spinal cord, with most abundant TDP-43 deposition in the anterior gray matter of the cervical and lumbar cord. Our data show that leakage of the BSCB occurs during life, but at end-stage disease the regions with most severe BSCB damage are not those where TDP-43 accumulation is most abundant. This suggests BSCB leakage and TDP-43 pathology are independent pathologies in ALS.

Do gabapentin or pregabalin directly modulate the µ receptor?

BACKGROUND: Pregabalin and gabapentin improve neuropathic pain symptoms but there are emerging concerns regarding their misuse. This is more pronounced among patients with substance use disorder, particularly involving opioids. Co-ingestion of gabapentinoids with opioids is increasingly identified in opioid related deaths, however, the molecular mechanism behind this is still unclear. We have sought to determine whether pregabalin or gabapentin directly modulates acute µ receptor signaling, or µ receptor activation by morphine. METHODS: The effects of pregabalin and gabapentin were assessed in HEK 293 cells stably transfected with the human µ receptor. Their effect on morphine induced hyperpolarization, cAMP production and ERK phosphorylation were studied using fluorescent-based membrane potential assay, bioluminescence based CAMYEL assay and ELISA assay, respectively. Pregabalin/gabapentin effects on morphine-induced hyperpolarization were also investigated in AtT20 cells. RESULTS: Pregabalin or gabapentin (1 µM, 100 µM each) did not activate the µ receptor or affect K channel activation or ERK phosphorylation produced by morphine. Neither drug affected the desensitization of K channel activation produced by prolonged (30 min) application of morphine. Gabapentin (1 µM, 100 µM) and pregabalin (1 µM) did not affect inhibition of forskolin-stimulated cAMP production by morphine. However, pregabalin (100 µM) potentiated forskolin mediated cAMP production, although morphine still inhibited cAMP levels with a similar potency to control. DISCUSSION: Pregabalin or gabapentin did not activate or modulate µ receptor signaling in three different assays. Our data do not support the hypothesis that gabapentin or pregabalin augment opioid effects through direct or allosteric modulation of the µ receptor. Pregabalin at a high concentration increases cAMP production independent of morphine. The mechanism of enhanced opioid-related harms from co-ingestion of pregabalin or gabapentin with opioids needs further investigation.

Development of High-Specificity Fluorescent Probes to Enable Cannabinoid Type 2 Receptor Studies in Living Cells.

Pharmacological modulation of cannabinoid type 2 receptor (CB2R) holds promise for the treatment of numerous conditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this receptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CB2R signaling, especially in cell-type and tissue-dependent contexts. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CB2R fluorescent probes, used successfully across applications, species, and cell types. These include flow cytometry of endogenously expressing cells, real-time confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based kinetic and equilibrium binding assays. High CB2R specificity was demonstrated by competition experiments in living cells expressing CB2R at native levels. The probes were effectively applied to FACS analysis of microglial cells derived from a mouse model relevant to Alzheimer's disease.

Low intrinsic efficacy for G protein activation can explain the improved side effect profiles of new opioid agonists.

Biased agonism at G protein-coupled receptors describes the phenomenon whereby some drugs can activate some downstream signaling activities to the relative exclusion of others. Descriptions of biased agonism focusing on the differential engagement of G proteins versus ß-arrestins are commonly limited by the small response windows obtained in pathways that are not amplified or are less effectively coupled to receptor engagement, such as ß-arrestin recruitment. At the µ-opioid receptor (MOR), G protein-biased ligands have been proposed to induce less constipation and respiratory depressant side effects than opioids commonly used to treat pain. However, it is unclear whether these improved safety profiles are due to a reduction in ß-arrestin-mediated signaling or, alternatively, to their low intrinsic efficacy in all signaling pathways. Here, we systematically evaluated the most recent and promising MOR-biased ligands and assessed their pharmacological profile against existing opioid analgesics in assays not confounded by limited signal windows. We found that oliceridine, PZM21, and SR-17018 had low intrinsic efficacy. We also demonstrated a strong correlation between measures of efficacy for receptor activation, G protein coupling, and ß-arrestin recruitment for all tested ligands. By measuring the antinociceptive and respiratory depressant effects of these ligands, we showed that the low intrinsic efficacy of opioid ligands can explain an improved side effect profile. Our results suggest a possible alternative mechanism underlying the improved therapeutic windows described for new opioid ligands, which should be taken into account for future descriptions of ligand action at this important therapeutic target.

Signalling profiles of a structurally diverse panel of synthetic cannabinoid receptor agonists.

Synthetic cannabinoid receptor agonists (SCRAs) represent the most rapidly proliferating class of "designer drugs" or "new psychoactive substances". SCRAs offer unregulated alternatives to cannabis that evade routine drug tests, but their use is increasingly associated with severe toxicity and death worldwide. Little is currently known about SCRA molecular pharmacology, or the mechanisms underpinning their toxicity, although the effects are believed to be primarily mediated by the type 1 cannabinoid receptor (CB1). In this study, we aimed to characterise the signalling profiles of a structurally diverse panel of novel SCRAs at CB1. We compare SCRAs to traditional reference cannabinoids CP55,940, WIN55,212-2, and THC. The activity of the SCRAs was assessed in key receptor signalling and regulatory pathways, including cAMP production, translocation of ß-arrestin 1 and 2, and receptor internalisation. The activity profiles of the ligands were also evaluated using operational analysis to identify ligand bias. Results revealed that SCRAs activities were relatively balanced in the pathways evaluated (compared to WIN55,212-2), although 5F-CUMYL-P7AICA and XLR-11 possessed partial efficacy in cAMP stimulation and ß-arrestin translocation. Notably, the SCRAs showed distinct potency and efficacy profiles compared to THC. In particular, while the majority of SCRAs demonstrated robust ß-arrestin translocation, cAMP stimulation, and internalisation, THC failed to elicit high efficacy responses in any of these assays. Further study is required to delineate if these pathways could contribute to SCRA toxicity in humans.

Regulating membrane lipid levels at the synapse by small-molecule inhibitors of monoacylglycerol lipase: new developments in therapeutic and PET imaging applications.

Monoacylglycerol lipase (MAGL) is a major endocannabinoid hydrolyzing enzyme and can be regulated to control endogenous lipid levels in the brain. This review highlights the pharmacological roles and in vivo PET imaging of MAGL in brain.

Brodifacoum does not modulate human cannabinoid receptor-mediated hyperpolarization of AtT20 cells or inhibition of adenylyl cyclase in HEK 293 cells.

BACKGROUND: Synthetic cannabinoids are a commonly used class of recreational drugs that can have significant adverse effects. There have been sporadic reports of co-consumption of illicit drugs with rodenticides such as warfarin and brodifacoum (BFC) over the past 20 years but recently, hundreds of people have been reported to have been poisoned with a mixture of synthetic cannabinoids and BFC. We have sought to establish whether BFC directly affects cannabinoid receptors, or their activation by the synthetic cannabinoid CP55940 or the phytocannabinoid Δ9-tetrahydrocannabinol (Δ9-THC). METHODS: The effects of BFC on the hyperpolarization of wild type AtT20 cells, or AtT20 cells stably expressing human CB1- or CB2- receptors, were studied using a fluorescent assay of membrane potential. The effect of BFC on CB1- and CB2-mediated inhibition of forskolin-stimulated adenylyl cyclase (AC) activation was measured using a BRET assay of cAMP levels in HEK 293 cells stably expressing human CB1 or CB2. RESULTS: BFC did not activate CB1 or CB2 receptors, or affect the hyperpolarization of wild type AtT20 cells produced by somatostatin. BFC (1 µM) did not affect the hyperpolarization of AtT20-CB1 or AtT20-CB2 cells produced by CP55940 or Δ9-THC. BFC (1 µM) did not affect the inhibition of forskolin-stimulated AC activity by CP55940 in HEK 293 cells expressing CB1 or CB2. BFC (1 µM) also failed to affect the desensitization of CB1 and CB2 signaling produced by prolonged (30 min) application of CP55940 or Δ9-THC to AtT20 cells. DISCUSSION: BFC is not a cannabinoid receptor agonist, and appeared not to affect cannabinoid receptor activation. Our data suggests there is no pharmacodynamic rationale for mixing BFC with synthetic cannabinoids; however, it does not speak to whether BFC may affect synthetic cannabinoid metabolism or biodistribution. The reasons underlying the mixing of BFC with synthetic cannabinoids are unknown, and it remains to be established whether the "contamination" was deliberate or accidental. However, the consequences for people who ingested the mixture were often serious, and sometimes fatal, but this seems unlikely to be due to BFC action at cannabinoid receptors.

Cannabinoid CB1 and CB2 Receptor-Mediated Arrestin Translocation: Species, Subtype, and Agonist-Dependence.

Arrestin translocation and signaling have come to the fore of the G protein-coupled receptor molecular pharmacology field. Some receptor-arrestin interactions are relatively well understood and considered responsible for specific therapeutic or adverse outcomes. Coupling of arrestins with cannabinoid receptors 1 (CB1) and 2 (CB2) has been reported, though the majority of studies have not systematically characterized the differential ligand dependence of this activity. In addition, many prior studies have utilized bovine (rather than human) arrestins, and the most widely applied assays require reporter-tagged receptors, which prevent meaningful comparison between receptor types. We have employed a bioluminescence resonance energy transfer (BRET) method that does not require the use of tagged receptors and thereby allows comparisons of arrestin translocation between receptor types, as well as with cells lacking the receptor of interest - an important control. The ability of a selection of CB1 and CB2 agonists to stimulate cell surface translocation of human and bovine ß-arrestin-1 and -2 was assessed. We find that some CB1 ligands induce moderate ß-arrestin-2 translocation in comparison with vasopressin V2 receptor (a robust arrestin recruiter); however, CB1 coupling with ß-arrestin-1 and CB2 with either arrestin elicited low relative efficacies. A range of efficacies between ligands was evident for both receptors and arrestins. Endocannabinoid 2-arachidonoylglycerol stood out as a high efficacy ligand for translocation of ß-arrestin-2 via CB1. Δ9-tetrahydrocannabinol was generally unable to elicit translocation of either arrestin subtype via CB1 or CB2; however, control experiments revealed translocation in cells not expressing CB1/CB2, which may assist in explaining some discrepancy with the literature. Overexpression of GRK2 had modest influence on CB1/CB2-induced arrestin translocation. Results with bovine and human arrestins were largely analogous, but a few instances of inconsistent rank order potencies/efficacies between bovine and human arrestins raise the possibility that subtle differences in receptor conformation stabilized by these ligands manifest in disparate affinities for the two arrestin species, with important potential consequences for interpretation in ligand bias studies. As well as contributing important information regarding CB1/CB2 ligand-dependent arrestin coupling, our study raises a number of points for consideration in the design and interpretation of arrestin recruitment assays.

Chromenopyrazole-based High Affinity, Selective Fluorescent Ligands for Cannabinoid Type 2 Receptor.

Cannabinoid type 2 receptor (CB2R) is an attractive target for the treatment of pain and inflammatory disorders. Availability of a selective CB2R fluorescent ligand to study CB2R expression and localization in healthy and disease conditions would greatly contribute to improving our understanding of this receptor. Herein, we report a series of chromenopyrazole-based CB2R fluorescent ligands. The highest affinity fluorescent ligand was Cy5-containing 24 (hCB2R pK i = 7.38 ± 0.05), which had 131-fold selectivity over CB1R. In a cAMP BRET assay, 24 behaved as a potent CB2R inverse agonist. Widefield imaging experiments showed that 24 binds to CB2R in live cells with good selectivity and low levels of nonspecific fluorescence. The high affinity, selectivity, and suitable imaging properties of fluorescent ligand 24 make it a valuable tool for studying CB2R.