Concept: G protein
A highly crystallizable T4 lysozyme (T4L) was fused to the N-terminus of the β(2) adrenergic receptor (β(2)AR), a G-protein coupled receptor (GPCR) for catecholamines. We demonstrate that the N-terminal fused T4L is sufficiently rigid relative to the receptor to facilitate crystallogenesis without thermostabilizing mutations or the use of a stabilizing antibody, G protein, or protein fused to the 3rd intracellular loop. This approach adds to the protein engineering strategies that enable crystallographic studies of GPCRs alone or in complex with a signaling partner.
Regulator of G protein Signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates heterotrimeric G protein and H-Ras signaling pathways. RGS14 possesses an RGS domain that binds active Gαi/o-GTP subunits to promote GTP hydrolysis, and a G protein regulatory (GPR) motif that selectively binds inactive Gαi1/3-GDP subunits to form a stable heterodimer at cellular membranes. RGS14 also contains two tandem Ras/Rap-binding domains (RBDs) that bind H-Ras. Here we show that RGS14 preferentially binds activated H-Ras-GTP in live cells to enhance H-Ras cellular actions, and that this interaction is regulated by inactive Gαi1-GDP and GPCRs. Using bioluminescence resonance energy transfer (BRET) in live cells, we show that RGS14-Luciferase and active H-Ras(G/V)-Venus exhibit a robust BRET signal at the plasma membrane that is markedly enhanced in the presence of inactive Gαi1-GDP, but not active Gαi1-GTP. Active H-Ras(G/V) interacts with a native RGS14:Gαi1 complex in brain lysates, and co-expression of RGS14 and Gαi1 in PC12 cells greatly enhances H-Ras(G/V) stimulatory effects on neurite outgrowth. Stimulation of the Gαi-linked α2A-adrenergic receptor induces a conformational change in the Gαi1:RGS14:H-Ras(G/V) complex, which may allow subsequent regulation of the complex by other binding partners. Together, these findings indicate that inactive Gαi1-GDP enhances theaffinity of RGS14 for H-Ras-GTP in live cells, resulting in a ternary signaling complex that is further regulated by GPCRs.
Beta-arrestins regulate G protein-coupled receptor signaling as competitive inhibitors and protein adaptors. Low molecular weight biased ligands that bind receptors and discriminate between the G protein dependent arm and beta-arrestin, clathrin-associated arm of receptor signaling are considered therapeutically valuable as a result of this distinctive pharmacological behavior. Other than receptor agonists, compounds that activate beta-arrestins are not available. We show that within minutes of exposure to the cationic triphenylmethane dyes malachite green and brilliant green, tissue culture cells recruit beta-arrestins to clathrin scaffolds in a receptor-activation independent manner. In the presence of these compounds G protein signaling is inhibited, ERK and GSK3β signaling are preserved, and the recruitment of the beta2-adaptin, AP2 adaptor complex to clathrin as well as transferrin internalization are reduced. Moreover, malachite green binds beta-arrestin2-GFP coated immunotrap beads relative to GFP only coated beads. Triphenylmethane dyes are FDA approved for topical use on newborns as components of triple-dye preparations, and are not approved but used effectively as aqueous antibiotics in fish husbandry. As possible carcinogens their chronic ingestion in food preparations, particularly through farmed fish, is discouraged in the US and Europe. Our results indicate triphenylmethane dyes as a result of novel pharmacology may have additional roles as beta-arrestin/clathrin pathway signaling modulators in both pharmacology research and clinical therapy.
G protein-coupled receptors (GPCRs) can initiate intracellular signalling cascades by coupling to an array of heterotrimeric G proteins and arrestin adaptor proteins. Understanding the contribution of each of these coupling options to GPCR signalling has been hampered by a paucity of tools to selectively perturb receptor function. Here we employ CRISPR/Cas9 genome editing to eliminate selected G proteins (Gαq and Gα11) or arrestin2 and arrestin3 from HEK293 cells, together with the elimination of receptor phosphorylation sites, to define the relative contribution of G proteins, arrestins and receptor phosphorylation to the signalling outcomes of the free fatty acid receptor 4 (FFA4). A lack of FFA4-mediated elevation of intracellular [Ca2+] in Gαq/Gα11-null cells and agonist-mediated receptor internalization in arrestin2/3-null cells confirmed previously reported canonical signalling features of this receptor, thereby validating the genome-edited HEK293 cells. FFA4-mediated ERK1/2 activation was totally dependent on Gq/11 but intriguingly was substantially enhanced for FFA4 receptors lacking sites of regulated phosphorylation. This was not due to a simple lack of desensitization of Gq/11 signalling because the Gq/11-dependent calcium response was desensitized by both receptor phosphorylation and arrestin-dependent mechanisms whilst a substantially enhanced ERK1/2-response was only observed for receptors lacking phosphorylation sites and not in arrestin2/3-null cells. In conclusion, we validate CRISPR/Cas9 engineered HEK293 cells lacking Gq/11 or arrestin2/3 as systems for GPCR signalling research and employ these cells to reveal a previously unappreciated interplay of signalling pathways where receptor phosphorylation can impact on ERK1/2 signalling through a mechanism that is likely independent of arrestins.
In response to environments that cause cellular stress, animals engage in sleep behavior that facilitates recovery from the stress. In Caenorhabditis elegans, stress-induced sleep(SIS) is regulated by cytokine activation of the ALA neuron, which releases FLP-13 neuropeptides characterized by an amidated arginine-phenylalanine (RFamide) C-terminus motif. By performing an unbiased genetic screen for mutants that impair the somnogenic effects of FLP-13 neuropeptides, we identified the gene dmsr-1, which encodes a G-protein coupled receptor similar to an insect RFamide receptor. DMSR-1 is activated by FLP-13 peptides in cell culture, is required for SIS in vivo, is expressed non-synaptically in several wake-promoting neurons, and likely couples to a Gi/o heterotrimeric G-protein. Our data expand our understanding of how a single neuroendocrine cell coordinates an organism-wide behavioral response, and suggest that similar signaling principles may function in other organisms to regulate sleep during sickness.
Mapping physiological G protein-coupled receptor signaling pathways reveals a role for receptor phosphorylation in airway contraction
- Proceedings of the National Academy of Sciences of the United States of America
- Published almost 3 years ago
G protein-coupled receptors (GPCRs) are known to initiate a plethora of signaling pathways in vitro. However, it is unclear which of these pathways are engaged to mediate physiological responses. Here, we examine the distinct roles of Gq/11-dependent signaling and receptor phosphorylation-dependent signaling in bronchial airway contraction and lung function regulated through the M3-muscarinic acetylcholine receptor (M3-mAChR). By using a genetically engineered mouse expressing a G protein-biased M3-mAChR mutant, we reveal the first evidence, to our knowledge, of a role for M3-mAChR phosphorylation in bronchial smooth muscle contraction in health and in a disease state with relevance to human asthma. Furthermore, this mouse model can be used to distinguish the physiological responses that are regulated by M3-mAChR phosphorylation (which include control of lung function) from those responses that are downstream of G protein signaling. In this way, we present an approach by which to predict the physiological/therapeutic outcome of M3-mAChR-biased ligands with important implications for drug discovery.
G-protein-coupled receptors (GPCRs) are critically regulated by β-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the β2 adrenergic receptor (β2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of β-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-β-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human β2AR-β-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between β2AR and β-arrestin 1 using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and chemical crosslinking. Electron microscopy two-dimensional averages and three-dimensional reconstructions reveal bimodal binding of β-arrestin 1 to the β2AR, involving two separate sets of interactions, one with the phosphorylated carboxy terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of crosslinked residues suggest engagement of the finger loop of β-arrestin 1 with the seven-transmembrane core of the receptor. In contrast, focal areas of raised HDX levels indicate regions of increased dynamics in both the N and C domains of β-arrestin 1 when coupled to the β2AR. A molecular model of the β2AR-β-arrestin signalling complex was made by docking activated β-arrestin 1 and β2AR crystal structures into the electron microscopy map densities with constraints provided by HDX-MS and crosslinking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented here provides a framework for better understanding the basis of GPCR regulation by arrestins.
The kappa opioid receptor (KOR) is involved in mediating pruritus; agonists targeting this receptor have been used to treat chronic intractable itch. Conversely, antagonists induce an inch response at the site of injection. As a G protein-coupled receptor (GPCR), the KOR has potential for signaling via G proteins and βarrestins, however, it is not clear which of these pathways are involved in the KOR modulation of itch. In this study asked whether the actions of KOR in pruritus involve βarrestins by using βarrestin2 knockout (βarr2-KO) mice as well as a recently described biased KOR agonist that biases receptor signaling toward G protein pathways over βarrestin2 recruitment. We find that the KOR antagonists nor-binaltorphimine (NorBNI) and 5'-guanidinonaltrindole (5'GNTI) induce acute pruritus in C57BL/6J mice, with reduced effects in KOR-KO mice. βarr2-KO mice display less of a response to KOR antagonist-induced itch compared to wild types, however no genotype differences are observed from chloroquine phosphate (CP)-induced itch, suggesting that the antagonists may utilize a KOR-βarrestin2 dependent mechanism. The KOR agonist U50,488H was equally effective in both WT and βarr2-KO mice in suppressing CP-induced itch. Furthermore, the G protein biased agonist, Isoquinolinone 2.1 was as effective as U50,488H in suppressing the itch response induced by KOR antagonist NorBNI or CP in C57BL/6J mice. Together these data suggest that the antipruritic effects of KOR agonists may not require βarrestins.
G protein-coupled receptors (GPCRs) are the largest class of receptors in the human genome and some of the most common drug targets. It is now well established that GPCRs can signal through multiple transducers, including heterotrimeric G proteins, GPCR kinases and β-arrestins. While these signalling pathways can be activated or blocked by ‘balanced’ agonists or antagonists, they can also be selectively activated in a ‘biased’ response. Biased responses can be induced by biased ligands, biased receptors or system bias, any of which can result in preferential signalling through G proteins or β-arrestins. At many GPCRs, signalling events mediated by G proteins and β-arrestins have been shown to have distinct biochemical and physiological actions from one another, and an accurate evaluation of biased signalling from pharmacology through physiology is crucial for preclinical drug development. Recent structural studies have provided snapshots of GPCR-transducer complexes, which should aid in the structure-based design of novel biased therapies. Our understanding of GPCRs has evolved from that of two-state, on-and-off switches to that of multistate allosteric microprocessors, in which biased ligands transmit distinct structural information that is processed into distinct biological outputs. The development of biased ligands as therapeutics heralds an era of increased drug efficacy with reduced drug side effects.
G-protein coupled receptors (GPCRs), a major gatekeeper of extracellular signals on plasma membrane, are unarguably one of the most important therapeutic targets. Given the recent discoveries of allosteric modulations, an allosteric wiring diagram of intramolecular signal transductions would be of great use to glean the mechanism of receptor regulation. Here, by evaluating betweenness centrality (CB ) of each residue, we calculate maps of information flow in GPCRs and identify key residues for signal transductions and their pathways. Compared with preexisting approaches, the allosteric hotspots that our CB -based analysis detects for A2 A adenosine receptor (A2 A AR) and bovine rhodopsin are better correlated with biochemical data. In particular, our analysis outperforms other methods in locating the rotameric microswitches, which are generally deemed critical for mediating orthosteric signaling in class A GPCRs. For A2 A AR, the inter-residue cross-correlation map, calculated using equilibrium structural ensemble from molecular dynamics simulations, reveals that strong signals of long-range transmembrane communications exist only in the agonist-bound state. A seemingly subtle variation in structure, found in different GPCR subtypes or imparted by agonist bindings or a point mutation at an allosteric site, can lead to a drastic difference in the map of signaling pathways and protein activity. The signaling map of GPCRs provides valuable insights into allosteric modulations as well as reliable identifications of orthosteric signaling pathways. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.