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The cannabinoid receptors CB1 and CB2 mediate a variety of physiological processes and continue to be explored as desirable drug targets. Both receptors are activated by the endogenous endocannabinoids and the psychoactive components of marijuana. Over the years, many efforts have been made to make selective ligands; however, the high degree of homology between cannabinoid receptor subtypes introduces challenges in studying either receptor in isolation. Recent advancements in structure biology have resulted in a surge of high-resolution structures, enriching our knowledge and understanding of receptor structure and function. In this review, of recent cannabinoid receptor structures, key features of the inactive and active state CB1 and CB2 are presented. These structures will provide additional insight into the modulation and signaling mechanism of cannabinoid receptors CB1 and CB2 and aid in the development of future therapeutics., (© 2023 The Author(s).)

GPCRs modulate a plethora of physiological processes and mediate the effects of one-third of FDA-approved drugs. Depending on which ligand activates a receptor, it can engage different intracellular transducers. This 'biased signalling' paradigm requires that we now characterize physiological signalling not just by receptors but by ligand-receptor pairs. Ligands eliciting biased signalling may constitute better drugs with higher efficacy and fewer adverse effects. However, ligand bias is very complex, making reproducibility and description challenging. Here, we provide guidelines and terminology for any scientists to design and report ligand bias experiments. The guidelines will aid consistency and clarity, as the basic receptor research and drug discovery communities continue to advance our understanding and exploitation of ligand bias. Scientific insight, biosensors, and analytical methods are still evolving and should benefit from and contribute to the implementation of the guidelines, together improving translation from in vitro to disease-relevant in vivo models., (© 2022 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

The ability of a ligand to preferentially promote engagement of one signaling pathway over another downstream of GPCR activation has been referred to as signaling bias, functional selectivity, and biased agonism. The presentation of ligand bias reflects selectivity between active states of the receptor, which may result in the display of preferential engagement with one signaling pathway over another. In this study, we provide evidence that the G protein-biased mu opioid receptor (MOR) agonists SR-17018 and SR-14968 stabilize the MOR in a wash-resistant yet antagonist-reversible G protein-signaling state. Furthermore, we demonstrate that these structurally related biased agonists are noncompetitive for radiolabeled MOR antagonist binding, and while they stimulate G protein signaling in mouse brains, partial agonists of this class do not compete with full agonist activation. Importantly, opioid antagonists can readily reverse their effects in vivo. Given that chronic treatment with SR-17018 does not lead to tolerance in several mouse pain models, this feature may be desirable for the development of long-lasting opioid analgesics that remain sensitive to antagonist reversal of respiratory suppression., Competing Interests: Competing interest statement: The Scripps Research Institute has filed for a patent on the SR compounds used in this study.

Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with G i , as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Biased agonists of G protein-coupled receptors may present a means to refine receptor signaling in a way that separates side effects from therapeutic properties. Several studies have shown that agonists that activate the κ-opioid receptor (KOR) in a manner that favors G protein coupling over β-arrestin2 recruitment in cell culture may represent a means to treat pain and itch while avoiding sedation and dysphoria. Although it is attractive to speculate that the bias between G protein signaling and β-arrestin2 recruitment is the reason for these divergent behaviors, little evidence has emerged to show that these signaling pathways diverge in the neuronal environment. We further explored the influence of cellular context on biased agonism at KOR ligand-directed signaling toward G protein pathways over β-arrestin-dependent pathways and found that this bias persists in striatal neurons. These findings advance our understanding of how a G protein-biased agonist signal differs between cell lines and primary neurons, demonstrate that measuring [ 35 S]GTPγS binding and the regulation of adenylyl cyclase activity are not necessarily orthogonal assays in cell lines, and emphasize the contributions of the environment to assessing biased agonism., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

G protein-coupled receptors, such as the cannabinoid type 1 receptor (CB1R), have been shown to interact with multiple binding partners to transmit signals. In both transfected cell systems and in endogenously expressing cell lines, CB1R signaling has been described as multifaceted. The question remains as to how this highly widely expressed receptor signals in a given cell at a given time in vivo. The concept of functional selectivity, or biased agonism, describes the ability of an agonist to engage the receptor in a manner that preferentially engages certain signaling interactions (e.g., G proteins) over others (e.g., β-arrestins), presumably by stabilizing certain receptor conformations. There is growing interest in using such properties of ligands to direct signaling downstream of CB1R toward desirable therapeutic outcomes and to avoid adverse side effects. While it is not currently clear what pathways should be engaged and which should be avoided, the development of biased agonist tool compounds will aid in answering these questions. In this chapter, we discuss the approaches and caveats to assessing biased agonism at the CB1R., (© 2017 Elsevier Inc. All rights reserved.)

The striatum of the brain coordinates motor function. Dopamine-related drugs may be therapeutic to patients with striatal neurodegeneration, such as Huntington's disease (HD) and Parkinson's disease (PD), but these drugs have unwanted side effects. In addition to stimulating the release of norepinephrine, amphetamines, which are used for narcolepsy and attention-deficit/hyperactivity disorder (ADHD), trigger dopamine release in the striatum. The guanosine triphosphatase Ras homolog enriched in the striatum (Rhes) inhibits dopaminergic signaling in the striatum, is implicated in HD and L-dopa-induced dyskinesia, and has a role in striatal motor control. We found that the guanine nucleotide exchange factor RasGRP1 inhibited Rhes-mediated control of striatal motor activity in mice. RasGRP1 stabilized Rhes, increasing its synaptic accumulation in the striatum. Whereas partially Rhes-deficient (Rhes +/- ) mice had an enhanced locomotor response to amphetamine, this phenotype was attenuated by coincident depletion of RasGRP1. By proteomic analysis of striatal lysates from Rhes-heterozygous mice with wild-type or partial or complete knockout of Rasgrp1, we identified a diverse set of Rhes-interacting proteins, the "Rhesactome," and determined that RasGRP1 affected the composition of the amphetamine-induced Rhesactome, which included PDE2A (phosphodiesterase 2A; a protein associated with major depressive disorder), LRRC7 (leucine-rich repeat-containing 7; a protein associated with bipolar disorder and ADHD), and DLG2 (discs large homolog 2; a protein associated with chronic pain). Thus, this Rhes network provides insight into striatal effects of amphetamine and may aid the development of strategies to treat various neurological and psychological disorders associated with the striatal dysfunction., (Copyright © 2016, American Association for the Advancement of Science.)

The fact that over 30% of current pharmaceuticals target heptahelical G protein-coupled receptors (GPCRs) attests to their tractability as drug targets. Although GPCR drug development has traditionally focused on conventional agonists and antagonists, the growing appreciation that GPCRs mediate physiologically relevant effects via both G protein and non-G protein effectors has prompted the search for ligands that can "bias" downstream signaling in favor of one or the other process. Biased ligands are novel entities with distinct signaling profiles dictated by ligand structure, and the potential prospect of biased ligands as better drugs has been pleonastically proclaimed. Indeed, preclinical proof-of-concept studies have demonstrated that both G protein and arrestin pathway-selective ligands can promote beneficial effects in vivo while simultaneously antagonizing deleterious ones. But along with opportunity comes added complexity and new challenges for drug discovery. If ligands can be biased, then ligand classification becomes assay dependent, and more nuanced screening approaches are needed to capture ligand efficacy across several dimensions of signaling. Moreover, because the signaling repertoire of biased ligands differs from that of the native agonist, unpredicted responses may arise in vivo as these unbalanced signals propagate. For any given GPCR target, establishing a framework relating in vitro efficacy to in vivo biologic response is crucial to biased drug discovery. This review discusses approaches to describing ligand efficacy in vitro, translating ligand bias into biologic response, and developing a systems-level understanding of biased agonism in vivo, with the overall goal of overcoming current barriers to developing biased GPCR therapeutics., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Ligand-directed signaling, biased agonism, and functional selectivity are terms that describe the propensity of a ligand to drive signaling toward one GPCR pathway over another. Most of the early examples demonstrated to date examine the divergence between GPCR signaling to G protein coupling and βarrestin2 recruitment. As biased agonists begin to become available based on cell-based screening criteria, a need arises to determine if G protein signaling biases will be maintained in the endogenous setting, wherein receptors are functioning to control relevant biological responses. This report presents our method and offers tips for evaluating G protein signaling in endogenous tissues. Predominately, brain tissues are discussed here; optimization points that can be applied to any tissues are highlighted.

At one time, G protein-coupled receptors were envisioned to simply relay either inhibitory or stimulatory binary signals through engaging particular G proteins. These receptors are now viewed as complex, multidimensional triggers of a variety of potential signaling cascades. This review will showcase current attempts to elucidate biased signaling and functional selectivity in tissues and organs as well as in the whole animal. In addition, it will emphasize the challenges that are inherent in attributing bias in a living system as well as offer opinions as to the manner in which these problems may be approached., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The kappa opioid receptor (KOPR) has been identified as a potential drug target to prevent or alter the course of mood, anxiety and addictive disorders or reduce response to stress. In a search for highly potent and selective KOPR partial agonists as pharmacological tools, we have modified 12-epi-salvinorin A, a compound which we have previously observed to be a KOPR partial agonist. Five analogues of 12-epi-salvinorin A were synthesized and their effects on G protein activation as well as β-arrestin2 recruitment were evaluated. Only 12-epi-salvinorin A (1) partially activated signaling through G proteins, yet acted as a full agonist in the β-arrestin 2 DiscoveRx assay. Other salvinorin analogues tested in these functional assays were full agonists in both assays of KOPR activation. By comparison, the non-selective opioid ligand nalbuphine, known to be a partial agonist for G-protein activation, was also a partial agonist for the β-arrestin mediated signaling pathway activated through KOPR., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Serotonin receptors are the product of 15 distinct genes, 14 of which are G protein-coupled receptors. These receptors are expressed in a wide range of cell types, including distinct neuronal populations, and promote diverse functional responses in multiple organ systems. These receptors are important for mediating the in vivo effects of their cognate neurotransmitter, serotonin, as well as the endogenous tryptamines. In addition, the actions of many drugs are mediated, either directly or indirectly, through serotonin receptors, including antidepressants, antipsychotics, anxiolytics, sleep aids, migraine therapies, gastrointestinal therapeutics and hallucinogenic drugs. It is becoming increasingly evident that serotonin receptors can engage in differential signaling that is determined by the chemical nature of the ligand and that ligands that demonstrate a predilection for inducing a particular signaling cascade are considered to have "functional selectivity". The elucidation of the cellular signaling pathways that mediate the physiological responses to serotonin and other agonists is an active area of investigation and will be an onward-looking focal point for determining how to effectively and selectively promote beneficial serotonergic mimicry while avoiding unwanted clinical side effects. This review highlights the modulation of serotonin 2A, 2C, and four receptors by β-arrestins, which may represent a fulcrum for biasing receptor responsiveness in vivo.

Hallucinogens mediate many of their psychoactive effects by activating serotonin 2A receptors (5-HT(2A)R). Although serotonin is the cognate endogenous neurotransmitter and is not considered hallucinogenic, metabolites of serotonin also have high affinity at 5-HT(2A)R and can induce hallucinations in humans. Here we report that serotonin differs from the psychoactive N-methyltryptamines by its ability to engage a β-arrestin2-mediated signaling cascade in the frontal cortex. Serotonin and 5-hydroxy-L-tryptophan (5-HTP) induce a head-twitch response in wild-type (WT) mice that is a behavioral proxy for 5-HT(2A)R activation. The response in β-arrestin2 knock-out (βarr2-KO) mice is greatly attenuated until the doses are elevated, at which point, βarr2-KO mice display a head-twitch response that can exceed that of WT mice. Direct administration of N-methyltryptamines also produces a greater response in βarr2-KO mice. Moreover, the inhibition of N-methyltransferase blocks 5-HTP-induced head twitches in βarr2-KO mice, indicating that N-methyltryptamines, rather than serotonin, primarily mediate this response. Biochemical studies demonstrate that serotonin stimulates Akt phosphorylation in the frontal cortex and in primary cortical neurons through the activation of a β-arrestin2/phosphoinositide 3-kinase/Src/Akt cascade, whereas N-methyltryptamines do not. Furthermore, disruption of any of the components of this cascade prevents 5-HTP-induced, but not N-methyltryptamine-induced, head twitches. We propose that there is a bifurcation of 5-HT(2A)R signaling that is neurotransmitter and β-arrestin2 dependent. This demonstration of agonist-directed 5-HT(2A)R signaling in vivo may significantly impact drug discovery efforts for the treatment of disorders wherein hallucinations are part of the etiology, such as schizophrenia, or manifest as side effects of treatment, such as depression.

Visual and auditory hallucinations accompany certain neuropsychiatric disorders, such as schizophrenia, and they also can be induced by the use or abuse of certain drugs. The heptahelical serotonin 2A receptors (5-HT2ARs) are molecular targets for drug-induced hallucinations. However, the cellular mechanisms by which the 5-HT2AR mediates these effects are not well understood. Drugs acting at the 5-HT2AR can trigger diverse signaling pathways that may be directed by the chemical properties of the drug. beta-arrestins are intracellular proteins that bind to heptahelical receptors and represent a point where such divergences in ligand-directed functional signaling could occur. Here we compare the endogenous agonist, serotonin, to a synthetic 5-HT2AR hallucinogenic agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI), in mice lacking beta-arrestin-2, as well as in cells lacking beta-arrestins. In mice, we find that serotonin induces a head twitch response by a beta-arrestin-2-dependent mechanism. However, DOI invokes the behavior independent of beta-arrestin-2. The two structurally distinct agonists elicit different signal transduction and trafficking patterns upon activation of 5-HT2AR, which hinge on the presence of beta-arrestins. Our study suggests that the 5-HT2AR-beta-arrestin interaction may be particularly important in receptor function in response to endogenous serotonin levels, which could have major implications in drug development for treating neuropsychiatric disorders such as depression and schizophrenia.

Opiate drugs, such as morphine, are renowned for their analgesic properties. To date, opioid narcotics represent the largest and most potent class of pain relievers available to treat both acute and chronic pain conditions. The use of opiates, however, is severely limited by several adverse side effects. Upon chronic use, opiates can produce tolerance, physical dependence and addiction. Although these conditions warrant consideration, there are acute effects that present more immediate concerns when choosing opiate narcotics for pain therapy. One of the most prevalent side effects, which continues for as long as the opiate is used for pain control, is constipation. This can impact patient compliance, as it is often one of the top reasons why patients discontinue opiate treatment. The challenge, therefore, is to develop pain therapies that preserve potent analgesia while preventing constipation.

Seven transmembrane receptors were originally named and characterized based on their ability to couple to heterotrimeric G proteins. The assortment of coupling partners for G protein-coupled receptors has subsequently expanded to include other effectors (most notably the βarrestins). This diversity of partners available to the receptor has prompted the pursuit of ligands that selectively activate only a subset of the available partners. A biased or functionally selective ligand may be able to distinguish between different active states of the receptor, and this would result in the preferential activation of one signaling cascade more than another. Although application of the "standard" operational model for analyzing ligand bias is useful and suitable in most cases, there are limitations that arise when the biased agonist fails to induce a significant response in one of the assays being compared. In this article, we describe a quantitative method for measuring ligand bias that is particularly useful for such cases of extreme bias. Using simulations and experimental evidence from several κ opioid receptor agonists, we illustrate a "competitive" model for quantitating the degree and direction of bias. By comparing the results obtained from the competitive model with the standard model, we demonstrate that the competitive model expands the potential for evaluating the bias of very partial agonists. We conclude the competitive model provides a useful mechanism for analyzing the bias of partial agonists that exhibit extreme bias.