In this review, we, on behalf of the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology, describe criteria for assessing the evidence for pairing receptors with endogenous/physiological ligands for formal receptor deorphanization. This process is illustrated through consideration of the class A G protein-coupled receptors (GPCRs) not yet formally paired with an endogenous/physiological ligand by the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology. Of the 67 orphan class A GPCRs considered, 25 class A GPCRs have no identified endogenous agonists, although 5 (GPR21, GPR27, GPR52, GPR85, and GPR88) have synthetic ligands that have the potential to be used as tools for uncovering physiological roles and further pharmacological properties of these receptors. Surprisingly, 6 orphan GPCRs (GPR135, GPR152, GPR153, MRGPRF, MRGPRG, and MRGPRX3) have no clear pharmacology or phenotype reported following genetic disruption. Thirty-two orphan GPCRs have been paired with at least 1 endogenous agonist (mainly lipids and their derivatives, peptides, and other metabolites), but further characterization is required from the scientific community to validate these results. We identify 10 orphan class A GPCRs for which there are plausible grounds for considering deorphanization: GPR4 (protons), GPR15 (GPR15L), GPR31 (12S-hydroxyeicosatetraenoic acid), GPR39 (zinc divalent ions, Zn2+), GPR65 (protons), GPR68 (protons), GPR132 (9-hydroxyoctadecadienoic acid), GPR183 (7α,25-dihydroxycholesterol), MRGPRD (β-alanine), and MRGPRX1 (bovine adrenal medulla peptide 8-22). The issue of nomenclature for these 10 GPCRs will be considered by further subcommittees of the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology. We hope this review will prompt further investigations into these members of the currently most widely clinically exploited protein superfamily. SIGNIFICANCE STATEMENT: The use of systematic, rational nomenclature for drug targets provides a framework to ensure consistent identification and rapid recognition. Given that G protein-coupled receptors have fundamental physiological roles and are widespread targets of drugs in current clinical use, we hope the target summary and deorphanization criteria provided here will prompt renewed efforts to investigate these orphan receptors as regulators of physiology and as opportunities for future therapeutic exploitation.

Synthetic cannabinoid receptor agonists (SCRAs) are a major health challenge. Indazole-based SCRAs continue to evolve through modification of tail and linked groups, although they have mostly been tested under different conditions by different laboratories and their pharmacokinetic properties have been little studied. We prepared and pharmacologically characterized a library of 57 indazole SCRAs combining varying aliphatic N-alkyl tail groups along with common linked groups (e.g., naphthyl, cumyl, adamantyl, and amino-acid-derived motifs). Compounds were profiled for CB1/CB2 binding affinity, functional activity in calcium mobilization assays, and β-arrestin-2 recruitment, followed by human and mouse liver microsomal stability. Extending short aliphatic tails to n-butyl/n-pentyl generally increased CB1 affinity and functional potency, while linkedgroups contributed toward subtype selectivity and signaling, yielding multiple high-efficacy agonists at both receptors. Microsomal studies revealed species differences, with substantially faster clearance in mice than humans for many analogs. Selected potent compounds were advanced to mouse pharmacokinetic studies. Many compounds demonstrated rapid systemic exposure and brain penetrance. Together, these results define structure-activity relationships for indazole SCRAs and support prioritization of emerging analogs with high CB1 efficacy and favorable CNS exposure for further characterization in vivo.

The pulmonary administration of cannabidiol (CBD) in dry powder form provides an effective strategy to bypass first-pass metabolism and achieve therapeutic concentrations in both the lungs and the systemic circulation. Formulating a stable CBD dry powder remains challenging and requires appropriate excipients to achieve the desired physicochemical and performance characteristics. Although excipients can improve aerosolization performance, their excessive use may compromise drug loading and introduce potential adverse effects. An alternative strategy involves assessing co-active compounds that could offer additional pharmacological benefits and potentially support formulation properties. In this study, kaempferol (KAM), a non-toxic flavonoid with antioxidant and anti-inflammatory properties, was co-formulated with CBD into an inhalable dry powder via spray drying. A 32-factorial design was employed to evaluate the effects of feed concentration and L-leucine (LEC) concentration on aerosolization performance of the CBD-KAM combination dry powder. The formulation with 0.8% (w/v) feed concentration and 10% (w/w) LEC showed the highest fine particle fraction (FPF), attaining 60.3 ± 1.0% for CBD and 66.3 ± 0.4% for KAM. The cytotoxicity studies on A549 cells confirmed the non-toxic nature of KAM. The CBD-KAM combination exhibited reduced cytotoxicity compared with CBD alone. The anti-inflammatory activity of the CBD-KAM combination was comparable to raw CBD. These findings suggest that the CBD-KAM combination powder formulations exhibit improved aerosolization, reduced cytotoxicity, and maintained efficacy, representing a promising approach for inhaled combination therapy in pulmonary diseases.

ABSTRACT Synthetic cannabinoids remain one of the most prominent classes of New Psychoactive Substances (NPS). The introduction of “blanket bans” has only invigorated the development and emergence of novel compounds. Recently, a putative synthetic cannabinoid, AB‐MDMSBA, was identified in Australia and New Zealand, with little to no pharmacological data on this compound. This current study, therefore, aimed to determine the potential activity of AB‐MDMSBA at the human cannabinoid CB1 receptor. Results revealed AB‐MDMSBA failed to activate cAMP and β‐arrestin 2 pathways, or to induce a conformational change in a CB1 biosensor, unlike the known synthetic agonist AMB‐FUBINACA, suggesting the compound is not a synthetic cannabinoid. These findings may aid in drug classification and the identification of structurally similar compounds.

This chapter will review the basic pharmacology of the canonical cannabinoid receptors. The endocannabinoid system is a complex signalling network involved in a wide range of physiological processes, including pain modulation, appetite regulation, and synaptic plasticity. The canonical cannabinoid receptors, CB1 and CB2, are central in orchestrating this system. CB1 is highly enriched in the central nervous system (CNS), where it plays a crucial role in modulating neurotransmitter release and synaptic plasticity. In contrast, CB2 is predominantly expressed in peripheral tissues and immune cells, participating in anti-inflammatory processes. Here, we focus on cannabinoid receptor distribution, intracellular signalling, and receptor regulation. We describe the intracellular signalling pathways activated by CB1, including the modulation of ion channels, second messengers, and protein kinases. Overall, this chapter provides an overview of the canonical cannabinoid receptors and their role in the regulation of neuronal signalling and plasticity, highlighting the molecular and cellular mechanisms underlying their effects in the CNS.

Cannabinoid Receptor 2 (CB2) is a promising therapeutic target for modulating inflammation. Canonical signalling responses to receptor ligands are critically dependent on cell surface receptor expression. However, it is also now appreciated that intracellular G protein-coupled receptors can contribute to signalling responses and influence functional outcomes. Therefore, understanding how the subcellular distribution of receptors is controlled is also highly pertinent. CB2 is observed to be expressed at the cell surface as well as having a considerable proportion expressed intracellularly. Despite this distribution being well established, little is known about the regulation of CB2 anterograde trafficking and subcellular distribution. We report that sustained treatment with a range of CB2 agonists and inverse agonists stimulates a distinct population of CB2 to be delivered to the cell surface, at various expression levels and despite agonists concurrently internalising cell surface CB2. We present evidence that this ligand-stimulated anterograde trafficking is a result of CB2 agonists, as well as inverse agonists, acting as pharmacological chaperones. We also report that a di-lysine (KK) motif in the CB2 C-terminal tail is required for basal delivery to the cell surface. Corroborating the hypothesis that CB2 ligands can act as pharmacological chaperones, sustained CB2 ligand stimulation induces cell surface expression of the mutated receptor and alters maturation states as measured by western blotting. Our finding that prolonged exposure to CB2 ligands can induce CB2 cell surface delivery via pharmacological chaperoning may well have important implications for optimal design of CB2-targeted therapeutics.

Model simplification is a process to simplify large-scale mathematical models to enable easy applications such as simulation and parameter estimation. A novel heuristic machine analogy method of model simplification was developed and applied to a motivating example of a model for cAMP signaling switch induced by Gi/Gs pathway competition for the CB1 receptor (consisting of 31 species and 76 parameters) to enable its use in estimation. The method first acquired an understanding of the mechanism by full model simulation, and then the mechanism was abstracted to a machine analogy. The machine analogy included signal start, signal mode selector, signal size regulator, and final effector, representing functions of different parts of the full model. The simplified minimal model (consisting of 11 species and 13 estimated parameters) was used for parameter estimation for Gi/Gs signaling of six CB1 agonists. The results of the minimal model suggested that six CB1 agonists have similar ratios of Gi/Gs activation, indicating Gi/Gs preference was more of a system effect rather than a ligand-specific effect. In conclusion, the novel machine analogy method can be used to heuristically simplify a larger-scale model while maintaining the important mechanisms. In the example here, the full Gi/Gs model of CB1 was successfully simplified, and the results indicated Gi/Gs preference is a system-dependent effect.

This study aimed to develop a chemical structure-independent, sensitive screening platform for detecting novel synthetic cannabinoids using isolated cell membranes. Resonance energy transfer techniques were employed to detect conformational changes in the CB1 receptor upon agonist binding. Eight cannabinoid CB1 conformational biosensors were generated and characterized featuring different tag compositions and placements. The biosensors were initially tested in live cell assays, before conditions were optimized for activity in isolated cell membranes. The optimal sensor developed could successfully distinguish between different agonist efficacy and potency profiles. This biosensor allows for the detection of synthetic cannabinoids without relying on their chemical structure, potentially providing a valuable tool for compliance agencies to rapidly respond to the proliferation of new psychoactive substances. The ability to differentiate efficacy and potency profiles may also contribute to harm reduction strategies upon detection of these compounds.

Background: Inhaled delivery of cannabidiol (CBD) through dry powder inhalers is a promising approach for achieving optimal drug concentrations in the lungs. Spray drying is a commonly employed technique to prepare inhalable powders with particle sizes ideally ranging from 1 to 5 µm, for deep lung deposition. However, formulating aerosolizable CBD dry powders remains challenging due to the thermolabile nature of CBD and the cohesive behaviour of micron-sized particles, which affects powder dispersibility, reduces de-agglomeration during inhalation, and causes inefficient lung deposition. These challenges can be overcome by the inclusion of excipients that can stabilize CBD during processing and enhance the dispersion and aerosolization of the powder. Objectives and methods: This study investigates the role of different amino acids (lysine, cysteine, arginine, and phenylalanine) in combination with inulin, a sugar-based excipient, on the in vitro aerosolization performance, stability, and cytotoxicity of inhalable CBD dry powders. Results and conclusion: The prepared CBD dry powders exhibited a size range of 1–5 µm. Amino-acid-free CBD powder showed an irregular and flaky morphology, while in association with amino acids, CBD dry powder showed spherical morphology with a dimpled surface. The ATR-FTIR spectra confirmed no interactions between CBD and amino acids in the dry powder formulations. CBD dry powder formulations containing amino acids demonstrated a better aerosolization profile compared to amino-acid-free CBD powder, with the lysine-containing formulation achieving the highest fine particle fraction (FPF) of 56.6%. Additionally, all the formulations were stable under low and high humidity (<15% RH and 53% RH) conditions for 28 days. Cytotoxicity studies on A549 alveolar basal epithelial cells showed that the amino acids were non-toxic, while the CBD formulations with/without amino acids showed comparable levels of cytotoxicity.

The Concise Guide to Pharmacology 2025/26 marks the seventh edition in this series of biennial publications in the British Journal of Pharmacology. Presented in landscape format, the guide provides a comparative overview of the pharmacology of drug target families. The concise nature of the Concise Guide refers to the style of presentation, being clear, accessible, and well-structured, rather than the scope of the content, which spans approximately 500 pages. The Concise Guide summarises the key pharmacological properties of around 1900 human drug targets, and nearly 7000 interactions, involving around 4400 ligands. While the content is a substantially condensed version of the more detailed information and links available at the www.guidetopharmacology.org website, the printed guide serves as a permanent, citable, point-in-time record, that remains stable despite ongoing updates to the online database. The full contents of this publication can be found at https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.70230. The Concise Guides provide expert-curated recommendations of 'Gold Standard' selective pharmacological tools, available either commercially or as donations, which enable the identification of individual drug targets or families of drug targets. While the Concise Guide offers a more streamlined overview, more comprehensive information, including detailed pharmacological profiles and links to multiple online databases, is available through the Guide to Pharmacology website. The 2025/26 edition of the Concise Guide is based on material current as of mid-2025, and supersedes all previous editions, including the 2023/24 Guide, and earlier Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), and as such provides official IUPHAR classification and nomenclature for human drug targets, where applicable. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. Each section includes nomenclature guidance, concise summaries, information of the best available pharmacological tools, key references, and suggestions for further reading.

Cannabidiol (CBD), a non-psychoactive cannabinoid, has shown therapeutic potential for treating inflammatory respiratory diseases such as chronic obstructive pulmonary disease and asthma. However, the therapeutic efficacy of CBD is limited by extensive hepatic metabolism and low oral bioavailability (approximately 20 %). These problems can be overcome by choosing an appropriate targeted drug delivery system. Delivering CBD to the lungs via a dry powder formulation could be an effective method to achieve adequate concentration and therapeutic efficacy. This study aims to develop a dry powder formulation of CBD with Inulin (INU) and L-leucine (LEC) using spray drying and to characterize its physicochemical and aerodynamic properties. A design of experiments (DOE) approach was used to optimize the formulation by varying feed concentration (0.2 % w/v to 0.8 % w/v), LEC concentration (5 % w/w to 20 % w/w), and CBD concentration (5 % w/w to 20 % w/w). The resulting CBD dry powder formulations exhibited a wrinkled morphology with particle sizes ranging from 1 to 5 µm and displayed a crystalline structure, as determined by powder X-ray diffraction. The response surface method (RSM) showed that increasing the feed concentration correlated with higher yields of the CBD formulations. Specifically, the formulation with a feed concentration of 0.8 % w/v achieved a yield of 61 %. The aerosolization data demonstrated a direct relationship between the Fine Particle Fraction (FPF) and LEC concentration, indicating that FPF increases as the LEC concentration increases. The highest FPF of 62 % was achieved with a 20 % w/w LEC concentration and a feed concentration of 0.2 % w/v. Based on this, LEC plays a crucial role in enhancing aerosolization efficiency. While feed concentration negatively affects FPF, lower feed concentrations lead to an increase in FPF. The Fine Particle Dose (FPD) varied with the concentration of CBD, with higher concentrations resulting in a higher FPD. A 28 days stability study under different humidity conditions (<15 % and 53 %) confirmed the stability of the CBD formulations. INU and LEC exhibited minimal cytotoxicity on A549 cells, while the raw CBD and CBD formulations showed comparable levels of cytotoxicity, pIC50 4.5 ± 0.3 and 4.2 ± 0.2. Interestingly, the CBD dry powder formulations significantly reduced inflammation (pEC50 = 4.9) induced by lipopolysaccharide (LPS). These findings suggest that an inhalable formulation of CBD, incorporating LEC and INU, has been successfully developed. The formulations demonstrated improved aerosolization properties, stability, and promising anti-inflammatory effects, potentially making them a viable therapeutic option for inflammatory lung diseases.

The cannabinoid CB1 receptor (CB1) is a G protein-coupled receptor (GPCR) with widespread expression in the central nervous system. This canonically G⍺i/o-coupled receptor mediates the effects of Δ9-tetrahydrocannabinol (THC) and synthetic cannabinoid receptor agonists (SCRAs). Recreational use of SCRAs is associated with serious adverse health effects, making pharmacological research into these compounds a priority. Several studies have hypothesised that signalling bias may explain the different toxicological profiles between SCRAs and THC. Previous studies have focused on bias between G protein activation measured by cyclic adenosine monophosphate (cAMP) inhibition and β-arrestin translocation. In contrast, the current study characterises bias between G⍺ subtypes of the G⍺i/o family and β-arrestins; this method facilitates a more accurate assessment of ligand bias by assessing signals that have not undergone major amplification. We have characterised G protein dissociation and translocation of β-arrestin 1 and 2 using real-time BRET reporters. The responses produced by each SCRA across the G protein subtypes tested were consistent with the responses produced by the reference ligand AMB-FUBINACA. Ligand bias was probed by applying the operational analysis to determine biases within the G⍺i/o family, and between G protein subtypes and β-arrestins. Overall, these results confirm SCRAs to be balanced, high-efficacy ligands compared to the low efficacy ligand THC, with only one SCRA, 4CN-MPP-BUT7IACA, demonstrating statistically significant bias in one pathway comparison (towards β-arrestin 1 when compared with G⍺oA/oB). This suggests that the adverse effects caused by SCRAs are due to high potency and efficacy at CB1, rather than biased agonism.

Background and purpose: Activation of CB1 by exogenous agonists causes adverse effects in vivo. Positive allosteric modulation may offer improved therapeutic potential and a reduced on-target adverse effect profile compared with orthosteric agonists, due to reduced desensitisation/tolerance, but this has not been directly tested. This study investigated the ability of PAMs/ago-PAMs to induce receptor regulation pathways, including desensitisation and receptor internalisation. Experimental approach: Bioluminescence resonance energy transfer (BRET) assays in HEK293 cells were performed to investigate G protein dissociation, ERK1/2 phosphorylation and beta-arrestin 2 translocation, while immunocytochemistry was performed to measure internalisation of CB1 in response to the PAMs ZCZ011, GAT229 and ABD1236 alone and in combination with the orthosteric agonists AEA, 2-AG, and AMB-FUBINACA. Key results: ZCZ011, GAT229 and ABD1236 were allosteric agonists in all pathways tested. The ago-PAM ZCZ011 induced a biphasic ERK1/2 phosphorylation time course compared to transient activation by orthosteric agonists. In combination with 2-AG but not AEA or AMB-FUBINACA, ZCZ011 and ABD1236 caused the transient peak of ERK1/2 phosphorylation to become sustained. All PAMs increased the potency and efficacy of AEA-induced signalling in all pathways tested; however, no notable potentiation of 2-AG or AMB-FUBINACA was observed. Conclusion and implications: Ago-PAMs can potentiate endocannabinoid CB1 agonism by AEA to a larger extent compared with 2-AG. However, all compounds were found to be allosteric agonists and induce activation of CB1 in the absence of endocannabinoid, including beta-arrestin 2 recruitment and internalisation. Thus, the spatiotemporal signalling of endogenous cannabinoids will not be retained in vivo.

Allosteric modulation of CB1 is therapeutically advantageous compared to orthosteric activation as it potentially offers reduced on-target adverse effects. ORG27569 is an allosteric modulator that increases orthosteric agonist binding to CB1 but decreases functional signalling. ORG27569 is characterised by a delay in disinhibition of agonist-induced cAMP inhibition (lag); however, the mechanism behind this kinetic lag is yet to be identified. We aimed to utilise a mathematical model to predict data and design in vitro experiments to elucidate mechanisms behind the unique signalling profile of ORG27569. The established kinetic ternary complex model includes the existence of a transitional state of CB1 bound to ORG27569 and CP55940 and was used to simulate kinetic cAMP data using NONMEM 7.4 and Matlab R2020b. These data were compared with empirical cAMP BRET data in HEK293 cells stably expressing hCB1. The pharmacometric model suggested that the kinetic lag in cAMP disinhibition by ORG27569 is caused by signal amplification in the cAMP assay and can be reduced by decreasing receptor number. This was confirmed experimentally, as reducing receptor number through agonist-induced internalisation resulted in a decreased kinetic lag by ORG27569. ORG27569 was found to have a similar interaction with CP55940 and the high efficacy agonist WIN55,212-2, and was suggested to have lower affinity for CB1 bound by the partial agonist THC compared to CP55940. Allosteric modulators have unique signalling profiles that are often difficult to interrogate exclusively in vitro. We have used a combined mathematical and in vitro approach to prove that ORG27569 causes a delay in disinhibition of agonist-induced cAMP inhibition due to large receptor reserve in this pathway. We also used the pharmacometric model to investigate the common phenomenon of probe dependence, to propose that ORG27569 binds with higher affinity to CB1 bound by high efficacy orthosteric agonists.

Cannabinoids are effective analgesics but induce adverse cannabimimetic effects and the development of tolerance. Allosteric ligands of the cannabinoid CB1 receptor (CB1) may harness the pain-relieving effects of cannabinoids with reduced adverse effects. CB1 allosteric ligands bind at a site topographically distinct from the orthosteric binding site. CB1 allosteric ligands have been shown to be effective pain-relieving drugs that do not appear to result in the production of adverse effects or the development of tolerance. While this therapeutic profile indicates that CB1 allosteric ligands could be an effective treatment for chronic pain, their molecular mechanism of action remains unclear.

Background & Purpose The constant emergence and broad toxicological effects of synthetic cannabinoids create a discernible public health threat. The synthetic cannabinoid AMB-FUBINACA (AMB-FUB) is a potent agonist at the CB1 receptor and has been associated with numerous fatalities. Synthetic cannabinoids are commonly abused alongside other drugs and medications, including a "party pill" drug, para-fluorophenylpiperazine (pFPP), and the antipsychotic risperidone. This research aimed to investigate the mechanisms underpinning AMB-FUB toxicity and the impact of clinically relevant co-exposures in vivo. Experimental Approach Male and female C57Bl/6 mice received a single dose of AMB-FUB (3 or 6 mg kg-1), pFPP (10 or 20 mg kg-1) or vehicle intraperitoneally. Mice were co-exposed to AMB-FUB (3 mg kg-1) and pFPP (10 mg kg-1) or risperidone (0.5 mg kg-1) to investigate these drug combinations. To study receptor-dependency and potential rescue of AMB-FUB toxicity, rimonabant (3 mg kg-1) was administered both pre- and post-AMB-FUB. Adverse effects caused by drug administration, including hypothermia and convulsions, were recorded. Key Results AMB-FUB induced CB1-dependent hypothermia and convulsions in mice. The combination of AMB-FUB and pFPP significantly potentiated hypothermia, as did risperidone pre-treatment. Interestingly, risperidone provided significant protection from AMB-FUB-induced convulsions in female mice. Pre- and post-treatment with rimonabant was able to significantly attenuate both hypothermia and convulsions in mice administered AMB-FUB. Conclusion & Implications Factors such as dose, CB1 signalling, and substance co-exposure significantly contribute to the toxicity of AMB-FUBINACA. Mechanistic understanding of synthetic cannabinoid toxicity and fatality can help inform overdose treatment strategies and identify vulnerable populations of synthetic cannabinoid users.

Decreasing responsiveness to repeated visual stimuli (i.e., the inability to sustain attention) in jumping spiders (Salticidae) parallels that found in humans. In humans, drugs affect vigilance, and previous work on salticids has shown that the "vigilance decrement" is unlikely to be sensory habituation and that caffeine ameliorates reductions in attention. We exposed Trite planiceps to delta-9 tetrahydrocannabinol (THC) and methamphetamine before presenting them with a repeated visual stimulus. In the THC experiment, spiders were given a THC solution, water, or a vehicle solution, using a within-subjects design. The orienting response (i.e., "interest") of salticids on a track ball to face a fly stimulus presented peripherally on a monitor was scored, as well as "general movement" (e.g., walking, as a control for physical fatigue) and "no movement." The methamphetamine experiment was identical except that salticids were given methamphetamine solution or water. In both the THC and methamphetamine treatments, general movement dropped over time, while in control treatments, this was constant. Additionally, due to an initial stimulating effect of methamphetamine on interest, the response decrement was significantly steeper when spiders were administered methamphetamine compared with water. Our results suggest that the modulation of sustained attention, and possibly motivation, is likely in salticids. basic local alignment search tool genome queries on a closely related species and pharmacological radioligand experiments suggested that salticids do not possess cannabinoid receptors, but the presence of transient receptor potential proteins may help explain the small behavioral changes observed with THC. In contrast, how methamphetamine affects salticids remains unknown. (PsycInfo Database Record (c) 2025 APA, all rights reserved).

Arrestins are key negative regulators of G Protein-Coupled Receptors (GPCRs) through mediation of G protein desensitisation and receptor internalisation. Arrestins can also contribute to signal transduction by scaffolding downstream signalling effectors for activation. GPCR kinase (GRK) enzymes phosphorylate the intracellular C-terminal domain, or intracellular loop regions of GPCRs to promote arrestin interaction. There are seven different GRK subtypes, which may uniquely phosphorylate the C-terminal tail in a type of 'phosphorylation barcode,' potentially differentially contributing to arrestin translocation and arrestin-dependent signalling. Such contributions may be exploited to develop arrestin-biased ligands. Here, we examine the effect of different GRK subtypes on the ability to promote translocation of arrestin-2 and arrestin-3 to the cannabinoid CB1 receptor (CB1) with a range of ligands. We find that most GRK subtypes (including visual GRK1) can enhance arrestin-2 and -3 translocation to CB1, and that GRK-dependent changes in arrestin-2 and arrestin-3 translocation were broadly shared for most agonists tested. GRK2/3 generally enhanced arrestin translocation more than the other GRK subtypes, with some small differences between ligands. We also explore the interplay between G protein activity and GRK2/3-dependent arrestin translocation, highlighting that high-efficacy G protein agonists will cause GRK2/3 dependent arrestin translocation. This study supports the hypothesis that arrestin-biased ligands for CB1 must engage GRK5/6 rather than GRK2/3, and G protein-biased ligands must have inherently low efficacy.

Introduction: The endocannabinoid system (ECS) is a widespread neurotransmitter system. A key characteristic of the ECS is that there are multiple endogenous ligands (endocannabinoids). Of these, the most extensively studied are arachidonoyl ethanolamide (AEA) and 2-arachidonoyl-glycerol (2-AG), both act as agonists at the cannabinoid CB1 receptor. In humans, three CB1 variants have been identified: hCB1, considered the most abundant G protein-coupled receptor in the brain, alongside the less abundant and studied variants, hCB1a and hCB1b. CB1 exhibits a preference for coupling with inhibitory Gi/o proteins, although its interactions with specific members of the Gi/o family remain poorly characterized. This study aimed to compare the AEA and 2-AG-induced activation of various G protein subtypes at CB1. Furthermore, we compared the response of human CB1 (hCB1, hCB1a, hCB1b) and explored species differences by examining rodent receptors (mCB1, rCB1). Materials and Methods: Activation of individual G protein subtypes in HEK293 cells transiently expressing CB1 was measured with G protein dissociation assay utilizing TRUPATH biosensors. The performance of the TRUPATH biosensors was evaluated using Z-factor analysis. Pathway potencies and efficacies were analyzed using the operational analysis of bias to determine G protein subtype selectivity for AEA and 2-AG. Results: Initial screening of TRUPATH biosensors performance revealed variable sensitivities within our system. Based on the biosensor performance, the G protein subtypes pursued for further characterization were Gi1, Gi3, GoA, GoB, GZ, G12, and G13. Across all pathways, AEA demonstrated partial agonism, whereas 2-AG exhibited full or high-efficacy agonism. Notably, we provide direct evidence that the hCB1 receptor couples to G12 and G13 proteins. Our findings do not indicate any evidence of G protein subtype selectivity. Similar observations were made across the human receptor variants (hCB1, hCB1a, hCB1b), as well as at mCB1 and rCB1. Discussion: There was no evidence suggesting G protein subtype selectivity for AEA and 2-AG at CB1, and this finding remained consistent across human receptor variants and different species.