G-protein-coupled receptors (GPCRs) remain major drug targets, despite our incomplete understanding of how they signal through 16 non-visual G-protein signal transducers (collectively named the ...transducerome) to exert their actions. To address this gap, we have developed an open-source suite of 14 optimized bioluminescence resonance energy transfer (BRET) Gαβγ biosensors (named TRUPATH) to interrogate the transducerome with single pathway resolution in cells. Generated through exhaustive protein engineering and empirical testing, the TRUPATH suite of Gαβγ biosensors includes the first Gα15 and GαGustducin probes. In head-to-head studies, TRUPATH biosensors outperformed first-generation sensors at multiple GPCRs and in different cell lines. Benchmarking studies with TRUPATH biosensors recapitulated previously documented signaling bias and revealed new coupling preferences for prototypic and understudied GPCRs with potential in vivo relevance. To enable a greater understanding of GPCR molecular pharmacology by the scientific community, we have made TRUPATH biosensors easily accessible as a kit through Addgene.
DREADDs are chemogenetic tools widely used to remotely control cellular signaling, neuronal activity, and behavior. Here we used a structure-based approach to develop a new Gi-coupled DREADD using ...the kappa-opioid receptor as a template (KORD) that is activated by the pharmacologically inert ligand salvinorin B (SALB). Activation of virally expressed KORD in several neuronal contexts robustly attenuated neuronal activity and modified behaviors. Additionally, co-expression of the KORD and the Gq-coupled M3-DREADD within the same neuronal population facilitated the sequential and bidirectional remote control of behavior. The availability of DREADDs activated by different ligands provides enhanced opportunities for investigating diverse physiological systems using multiplexed chemogenetic actuators.
•Structure-guided approach for κ-opioid receptor (KOR)-DREADD (KORD) design•KORD is selectively activated by salvinorin B, and not by endogenous opioids•KORD robustly silenced multiple neuronal subtypes•Inhibitory KORD combined with excitatory hM3Dq for multiplexed behavioral control
The κ-opioid receptor (KOR) was used as a template to generate a novel inhibitory DREADD (KORD), which is activated by salvinorin B and insensitive to endogenous opioid peptides. Sequential activation of the inhibitory KOR-DREADD and an excitatory M3-DREADD facilitated the bidirectional, multiplexed modulation of behavior.
Recent studies show that GPCRs rapidly interconvert between multiple states although our ability to interrogate, monitor and visualize them is limited by a relative lack of suitable tools. We ...previously reported two nanobodies (Nb39 and Nb6) that stabilize distinct ligand- and efficacy-delimited conformations of the kappa opioid receptor. Here, we demonstrate via X-ray crystallography a nanobody-targeted allosteric binding site by which Nb6 stabilizes a ligand-dependent inactive state. As Nb39 stabilizes an active-like state, we show how these two state-dependent nanobodies can provide real-time reporting of ligand stabilized states in cells in situ. Significantly, we demonstrate that chimeric GPCRs can be created with engineered nanobody binding sites to report ligand-stabilized states. Our results provide both insights regarding potential mechanisms for allosterically modulating KOR with nanobodies and a tool for reporting the real-time, in situ dynamic range of GPCR activity.
The MRGPRX family of receptors (MRGPRX1-4) is a family of mas-related G-protein-coupled receptors that have evolved relatively recently
. Of these, MRGPRX2 and MRGPRX4 are key physiological and ...pathological mediators of itch and related mast cell-mediated hypersensitivity reactions
. MRGPRX2 couples to both G
and G
in mast cells
. Here we describe agonist-stabilized structures of MRGPRX2 coupled to G
and G
in ternary complexes with the endogenous peptide cortistatin-14 and with a synthetic agonist probe, respectively, and the development of potent antagonist probes for MRGPRX2. We also describe a specific MRGPRX4 agonist and the structure of this agonist in a complex with MRGPRX4 and G
. Together, these findings should accelerate the structure-guided discovery of therapeutic agents for pain, itch and mast cell-mediated hypersensitivity.
The κ-opioid receptor (KOR) has emerged as an attractive drug target for pain management without addiction, and biased signaling through particular pathways of KOR may be key to maintaining this ...benefit while minimizing side-effect liabilities. As for most G protein-coupled receptors (GPCRs), however, the molecular mechanisms of ligand-specific signaling at KOR have remained unclear. To better understand the molecular determinants of KOR signaling bias, we apply structure determination, atomic-level molecular dynamics (MD) simulations, and functional assays. We determine a crystal structure of KOR bound to the G protein-biased agonist nalfurafine, the first approved KOR-targeting drug. We also identify an arrestin-biased KOR agonist, WMS-X600. Using MD simulations of KOR bound to nalfurafine, WMS-X600, and a balanced agonist U50,488, we identify three active-state receptor conformations, including one that appears to favor arrestin signaling over G protein signaling and another that appears to favor G protein signaling over arrestin signaling. These results, combined with mutagenesis validation, provide a molecular explanation of how agonists achieve biased signaling at KOR.
Elucidating how the brain's serotonergic network mediates diverse behavioral actions over both relatively short (minutes-hours) and long period of time (days-weeks) remains a major challenge for ...neuroscience. Our relative ignorance is largely due to the lack of technologies with robustness, reversibility, and spatio-temporal control. Recently, we have demonstrated that our chemogenetic approach (eg, Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)) provides a reliable and robust tool for controlling genetically defined neural populations. Here we show how short- and long-term activation of dorsal raphe nucleus (DRN) serotonergic neurons induces robust behavioral responses. We found that both short- and long-term activation of DRN serotonergic neurons induce antidepressant-like behavioral responses. However, only short-term activation induces anxiogenic-like behaviors. In parallel, these behavioral phenotypes were associated with a metabolic map of whole brain network activity via a recently developed non-invasive imaging technology DREAMM (DREADD Associated Metabolic Mapping). Our findings reveal a previously unappreciated brain network elicited by selective activation of DRN serotonin neurons and illuminate potential therapeutic and adverse effects of drugs targeting DRN neurons.
Chemogenetic tools such as designer receptors exclusively activated by designer drugs (DREADDs) are routinely used to modulate neuronal and non-neuronal signaling and activity in a relatively ...noninvasive manner. The first generation of DREADDs were templated from the human muscarinic acetylcholine receptor family and are relatively insensitive to the endogenous agonist acetylcholine but instead are activated by clozapine-N-oxide (CNO). Despite the undisputed success of CNO as an activator of muscarinic DREADDs, it has been known for some time that CNO is subject to a low rate of metabolic conversion to clozapine, raising the need for alternative chemical actuators of muscarinic-based DREADDs. Here we show that DREADD agonist 21 (C21) (11-(1-piperazinyl)-5H-dibenzob,e1,4diazepine) is a potent and selective agonist at both excitatory (hM3Dq) and inhibitory (hM4Di) DREADDs and has excellent bioavailability, pharmacokinetic properties, and brain penetrability. We also show that C21-induced activation of hM3Dq and hM4Di in vivo can modulate bidirectional feeding in defined circuits in mice. These results indicate that C21 represents an alternative to CNO for in vivo studies where metabolic conversion of CNO to clozapine is a concern.
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