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G protein-coupled receptors (GPCRs) orchestrate diverse physiological responses via signaling through G proteins, GPCR kinases (GRKs), and arrestins. While most G protein functions are well-established, the contributions of GRKs and arrestins remain incompletely understood. Here, we investigate the influence of β-arrestin-interacting GPCR domains (helix-bundle/C-terminus) on β-arrestin conformations and functions using refined biosensors and advanced cellular knockout systems. Focusing on prototypical class A (b2AR) and B (V2R) receptors and their chimeras (b2V2/V2b2), we show that most N-domain β-arrestin conformational changes are mediated by receptor C-terminus-interactions, while C-domain conformations respond to the helix-bundle or an individual combination of interaction interfaces. Moreover, we demonstrate that ERK1/2 signaling responses are governed by the GPCR helix-bundle, while β-arrestin co-internalization depends on the receptor C-terminus. However, receptor internalization is controlled via the overall GPCR configuration. Our findings elucidate how individual GPCR domains dictate downstream signaling events, shedding light on the structural basis of receptor-specific signaling. Class A and B GPCR show differential downstream regulation and functions. Here, the authors show how their C-termini largely mediate GRK-specific β-arrestin N-domain conformational changes and co-internalization, while GPCR helix-bundles govern pERK.

The MRGPRX family of receptors (MRGPRX1–4) is a family of mas-related G-protein-coupled receptors that have evolved relatively recently 1 . Of these, MRGPRX2 and MRGPRX4 are key physiological and pathological mediators of itch and related mast cell-mediated hypersensitivity reactions 2 – 5 . MRGPRX2 couples to both G i and G q in mast cells 6 . Here we describe agonist-stabilized structures of MRGPRX2 coupled to G i1 and G q 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 q . Together, these findings should accelerate the structure-guided discovery of therapeutic agents for pain, itch and mast cell-mediated hypersensitivity. Structural studies of the itch receptors MRGPRX2 and MRGPRX4 in complex with endogenous and synthetic ligands provide a basis for the development of therapeutic compounds for pain, itch and mast cell-mediated hypersensitivity.

The G-protein-coupled bile acid receptor (GPBAR) conveys the cross-membrane signalling of a vast variety of bile acids and is a signalling hub in the liver–bile acid–microbiota–metabolism axis 1 – 3 . Here we report the cryo-electron microscopy structures of GPBAR–G s complexes stabilized by either the high-affinity P395 4 or the semisynthesized bile acid derivative INT-777 1 , 3 at 3 Å resolution. These structures revealed a large oval pocket that contains several polar groups positioned to accommodate the amphipathic cholic core of bile acids, a fingerprint of key residues to recognize diverse bile acids in the orthosteric site, a putative second bile acid-binding site with allosteric properties and structural features that contribute to bias properties. Moreover, GPBAR undertakes an atypical mode of activation and G protein coupling that features a different set of key residues connecting the ligand-binding pocket to the G s -coupling site, and a specific interaction motif that is localized in intracellular loop 3. Overall, our study not only reveals unique structural features of GPBAR that are involved in bile acid recognition and allosteric effects, but also suggests the presence of distinct connecting mechanisms between the ligand-binding pocket and the G-protein-binding site in the G-protein-coupled receptor superfamily. Using cryo-electron microscopy, the authors report the structures of G-protein-coupled bile acid receptor–G s complexes and reveal the structural basis of bile acid recognition.

In the clades of animals that diverged from the bony fish, a group of Mas-related G-protein-coupled receptors (MRGPRs) evolved that have an active role in itch and allergic signals 1 , 2 . As an MRGPR, MRGPRX2 is known to sense basic secretagogues (agents that promote secretion) and is involved in itch signals and eliciting pseudoallergic reactions 3 – 6 . MRGPRX2 has been targeted by drug development efforts to prevent the side effects induced by certain drugs or to treat allergic diseases. Here we report a set of cryo-electron microscopy structures of the MRGPRX2–G i1 trimer in complex with polycationic compound 48/80 or with inflammatory peptides. The structures of the MRGPRX2–G i1 complex exhibited shallow, solvent-exposed ligand-binding pockets. We identified key common structural features of MRGPRX2 and describe a consensus motif for peptidic allergens. Beneath the ligand-binding pocket, the unusual kink formation at transmembrane domain 6 (TM6) and the replacement of the general toggle switch from Trp 6.48 to Gly 6.48 (superscript annotations as per Ballesteros–Weinstein nomenclature) suggest a distinct activation process. We characterized the interfaces of MRGPRX2 and the G i trimer, and mapped the residues associated with key single-nucleotide polymorphisms on both the ligand and G-protein interfaces of MRGPRX2. Collectively, our results provide a structural basis for the sensing of cationic allergens by MRGPRX2, potentially facilitating the rational design of therapies to prevent unwanted pseudoallergic reactions. Cryo-electron microscopy structures of the MRGPRX2–G i1 trimer in complex with polycationic compound 48/80 or inflammatory peptides provide insights into the sensing of cationic allergens by MRGPRX2, potentially facilitating the design of therapies to prevent unwanted pseudoallergic reactions.

G-protein-coupled receptors (GPCRs) are key cell-surface proteins that transduce external environmental cues into biochemical signals across the membrane. GPCRs are intrinsically allosteric proteins; they interact via spatially distinct yet conformationally linked domains with both endogenous and exogenous proteins, nutrients, metabolites, hormones, small molecules and biological agents. Here we explore recent high-resolution structural studies, which are beginning to unravel the atomic details of allosteric transitions that govern GPCR biology, as well as highlighting how the wide diversity of druggable allosteric sites across these receptors present opportunities for developing new classes of therapeutics. High-resolution structural studies of GPCRs have led to insights into the role of allostery in GPCR-mediated signal transduction.

Classically, G-protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by β-arrestin (β-arr). However, some GPCRs continue to signal through G protein from internalized compartments, mediated by a GPCR–G protein–β-arr ‘megaplex’. Nevertheless, the molecular architecture of the megaplex remains unknown. Here, we present its cryo-electron microscopy structure, which shows simultaneous engagement of human G protein and bovine β-arr to the core and phosphorylated tail, respectively, of a single active human chimeric β2-adrenergic receptor with the C-terminal tail of the arginine vasopressin type 2 receptor (β2V2R). All three components adopt their canonical active conformations, suggesting that a single megaplex GPCR is capable of simultaneously activating G protein and β-arr. Our findings provide a structural basis for GPCR-mediated sustained internalized G protein signaling.

G-protein-coupled receptors (GPCRs) relay numerous extracellular signals by triggering intracellular signaling through coupling with G proteins and arrestins. Recent breakthroughs in the structural determination of GPCRs and GPCR–transducer complexes represent important steps toward deciphering GPCR signal transduction at a molecular level. A full understanding of the molecular basis of GPCR-mediated signaling requires elucidation of the dynamics of receptors and their transducer complexes as well as their energy landscapes and conformational transition rates. Here, we summarize current insights into the structural plasticity of GPCR–G-protein and GPCR–arrestin complexes that underlies the regulation of the receptor’s intracellular signaling profile.

G-protein-coupled receptors (GPCRs) are membrane proteins that modulate physiology across human tissues in response to extracellular signals. GPCR-mediated signalling can differ because of changes in the sequence 1 , 2 or expression 3 of the receptors, leading to signalling bias when comparing diverse physiological systems 4 . An underexplored source of such bias is the generation of functionally diverse GPCR isoforms with different patterns of expression across different tissues. Here we integrate data from human tissue-level transcriptomes, GPCR sequences and structures, proteomics, single-cell transcriptomics, population-wide genetic association studies and pharmacological experiments. We show how a single GPCR gene can diversify into several isoforms with distinct signalling properties, and how unique isoform combinations expressed in different tissues can generate distinct signalling states. Depending on their structural changes and expression patterns, some of the detected isoforms may influence cellular responses to drugs and represent new targets for developing drugs with improved tissue selectivity. Our findings highlight the need to move from a canonical to a context-specific view of GPCR signalling that considers how combinatorial expression of isoforms in a particular cell type, tissue or organism collectively influences receptor signalling and drug responses. Transcriptomics, proteomics, single-cell RNA sequencing, population-wide genetic association studies and structure–function analyses provide a picture of how the differential expression of G-protein-coupled receptor isoforms can diversify signalling in different tissues.

At least 120 non-olfactory G-protein-coupled receptors in the human genome are ‘orphans’ for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs. Yeast-based screening identifies the benzodiazepine drug lorazepam as a non-selective positive allosteric modulator of the G-protein-coupled receptor (GPCR) GPR68; homology modelling and molecular docking of 3.1 million molecules found a new compound, ‘ogerin’, as a potent GPR68 modulator, which suppressed recall in fear conditioning in wild-type mice, and the general method of combining physical and structure-based screening may lead to the discovery of selective ligands for other GPCRs. Finding ligands for GPCR orphans At least 120 non-olfactory G-protein-coupled receptors (GPCRs) in the human genome are 'orphans', meaning that their endogenous ligands are not known. Bryan Roth and colleagues use yeast-based screening to identify the benzodiazepine drug lorazepam as a non-selective positive allosteric modulator of GPR68, a proton receptor with no known small-molecule modulators. Homology modelling and molecular docking of 3.1 million molecules identified a new compound 'ogerin', as a potent GPR68 modulator. Ogerin suppressed recall in fear conditioning in wild-type mice. The procedures used in this work, combining physical and structure-based screening, may serve as a general method for identifying selective ligands for other GPCRs.

G protein-coupled receptors (GPCRs) generally accommodate specific ligands in the orthosteric-binding pockets. Ligand binding triggers a receptor allosteric conformational change that leads to the activation of intracellular transducers, G proteins and β-arrestins. Because these signals often induce adverse effects, the selective activation mechanism for each transducer must be elucidated. Thus, many orthosteric-biased agonists have been developed, and intracellular-biased agonists have recently attracted broad interest. These agonists bind within the receptor intracellular cavity and preferentially tune the specific signalling pathway over other signalling pathways, without allosteric rearrangement of the receptor from the extracellular side 1 – 3 . However, only antagonist-bound structures are currently available 1 , 4 – 6 , and there is no evidence to support that biased agonist binding occurs within the intracellular cavity. This limits the comprehension of intracellular-biased agonism and potential drug development. Here we report the cryogenic electron microscopy structure of a complex of G s and the human parathyroid hormone type 1 receptor (PTH1R) bound to a PTH1R agonist, PCO371. PCO371 binds within an intracellular pocket of PTH1R and directly interacts with G s . The PCO371-binding mode rearranges the intracellular region towards the active conformation without extracellularly induced allosteric signal propagation. PCO371 stabilizes the significantly outward-bent conformation of transmembrane helix 6, which facilitates binding to G proteins rather than β-arrestins. Furthermore, PCO371 binds within the highly conserved intracellular pocket, activating 7 out of the 15 class B1 GPCRs. Our study identifies a new and conserved intracellular agonist-binding pocket and provides evidence of a biased signalling mechanism that targets the receptor–transducer interface. A new intracellular agonist-binding pocket is identified that is common to many G protein-coupled receptors, which will have implications for the development of biased compounds that target this large group of receptors.