In-vitro selection-based identification of novel G-protein coupled receptors activating peptides
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PROVIDER: PRJEB65864 | ENA |
REPOSITORIES: ENA
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Project description:This SuperSeries is composed of the following subset Series: GSE25886: Analysis of mRNA levels of GPCR and GPCR-related signaling proteins in 3 mouse cell lines: AtT20, BV2 and N18 GSE25894: Analysis of mRNA levels of GPCR and GPCR-related signaling proteins in the HEK293 cell line Refer to individual Series
2011-02-01 | E-GEOD-25901 | biostudies-arrayexpress
Project description:G protein-coupled receptors (GPCRs) are typically characterized by their seven transmembrane (7TM) architecture, and interaction with two universal signal-transducers namely, the heterotrimeric G-proteins and β-arrestins (βarrs). Synthetic ligands and receptor mutants have been designed to elicit transducer-coupling preferences and distinct downstream signaling outcomes for many GPCRs. This raises the question if some naturally-occurring 7TMRs may selectively engage one of these two signal-transducers, even in response to their endogenous agonists. Although there are scattered hints in the literature that some 7TMRs lack G-protein coupling but interact with βarrs, an in-depth understanding of their transducer-coupling preference, GRK-engagement, downstream signaling and structural mechanism remains elusive. Here, we use an array of cellular, biochemical and structural approaches to comprehensively characterize two non-canonical 7TMRs namely, the human decoy D6 receptor (D6R) and the human complement C5a receptor (C5aR2), in parallel with their canonical GPCR counterparts, CCR2 and C5aR1, respectively. We discover that D6R and C5aR2 couple exclusively to βarrs, exhibit distinct GRK-preference, and activate non-canonical downstream signaling partners. We also observe that βarrs, in complex with these receptors, adopt distinct conformations compared to their canonical GPCR counterparts despite being activated by a common natural agonist. Our study therefore establishes D6R and C5aR2 as bona-fide arrestin-coupled receptors (ACRs), and provides important insights into their regulation by GRKs and downstream signaling with direct implications for biased agonism.
2021-11-03 | PXD027887 | Pride
Project description:G-protein coupled receptors (GPCRs) are pivotal in regulating T cell responses in steady state and inflammation. GPCR expression was intensively studied in bulk cDNA of T cell populations, but limited data are available with respect to expression in individual cells. We here present an analysis of a selected set of 125 different GPCRs expressed on distinct single T cells under naive conditions and during experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis. We found that neuroinflammation induces characteristic changes in GPCR heterogeneity and patterning, and we identified functionally relevant subgroups with specific GPCR expression profiles among spinal cord-infiltrating CD4 T cells. In spinal cord-infiltrating T helper 17 (Th17) cells, for example, expression of receptors such as Cxcr3, Cxcr4, P2ry10, or S1pr1 was associated with reduced pathogenicity, and we show that these correlations also exists on the protein level. Using CXCR4 and S1P1 as examples, we demonstrate that pharmacological targeting of these receptors is able to modulate Th17 pathogenicity in vitro and in vivo. Taken together, GPCR single-cell expression analysis provides a basis for the development of new strategies for pharmacological modulation of pathogenic immune cell subtypes.
2017-07-15 | GSE99778 | GEO
Project description:G protein-coupled receptors (GPCRs) regulate many aspects of physiology and represent actionable targets for drug discovery. Activation of specific signaling pathways downstream of G-protein coupled receptors (GPCRs) or targeting the receptors at selected cellular locations has the potential to provide therapeutic actions with fewer side effects. However, the understanding of the molecular mechanisms underlying GPCR function is limited in the dynamic cellular environment, hampering drug discovery efforts towards selective ligands. Proximity biotin labeling based on an engineered ascorbic acid peroxidase (APEX) combined with quantitative mass spectrometry is a powerful method to delineate these mechanisms given its capacity to simultaneously capture proximal protein interaction networks and the cellular location of the receptor. However, a major challenge is to extract the various information from these complex datasets. Here, we describe a computational framework for proximity labeling datasets which predicts ligand-dependent subcellular location of GPCRs and quantitatively deconvolutes the effect of receptor location and proximal interactors. We applied this approach to the mu-opioid receptor and not only monitored distinct effects of ligands on receptor trafficking, but also discovered two novel regulators, EYA4 and KCTD12, which modulate MOR-driven G protein-dependent signaling.
2024-03-30 | PXD031415 | Pride
Project description:Cas9:sgRNA in vitro selection libraries
| PRJNA212837 | ENA
Project description:G-protein coupled receptors (GPCRs) regulate various aspects of cellular behavior and represent actionable targets for drug discovery. Activation of specific signaling pathways downstream of G-protein coupled receptors (GPCRs) or targeting the receptors at selected cellular locations has the potential to provide therapeutic actions with fewer side effects. However, the understanding of the molecular mechanisms underlying GPCR activation is limited, hampering drug discovery efforts towards selective ligands. Proximity biotin labeling based on an engineered ascorbic acid peroxidase (APEX) combined with quantitative mass spectrometry is a powerful method to delineate these mechanisms given its capacity to simultaneously capture proximal protein interaction networks and the cellular location of the receptor. However, a major challenge is to extract the various information from these complex datasets. Here, we describe a computational framework for proximity labeling datasets which predicts ligand-dependent subcellular location of GPCRs and quantitatively deconvolutes the effect of receptor location and proximal interactors. We applied this approach to the mu-opioid receptor and not only monitored distinct effects of ligands on receptor trafficking, but also discovered two novel regulators, EYA4 and KCTD12, which modulate MOR-driven G protein-dependent signaling.
2024-03-31 | PXD031393 | panorama
Project description:Type 1 Diabetes is still an incurable disease characterized by autoimmune destruction of insulin-producing beta cells within the islet of Langerhans in the pancreas. Currently, there are no methods to monitor beta-cell mass in humans or deliver therapeutics specifically to beta cells. Here we performed Cluster Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments and toggle SELEX experiments to identify RNA aptamers specific for human islets. In the cluster SELEX, we started from a random library of RNA nucleotides composed of a 40 nucleotide long variable region flanked by two constant regions. We performed eight selection cycles using hand-picked islets and islet-depleted acinar tissue from 4 cadaveric human donors as positive and negative selectors. In the toggle SELEX, we conducted eight cycles of selection using islets and acinar tissue from mice, followed by two cycles of selection using human tissues. The polyclonal libraries from the two selection strategies showed a convergent evolution of ligands and increased specificity for human islets.
2022-02-23 | GSE197262 | GEO
Project description:Biased GPCR agonists are orthosteric ligands that possess pathway-selective efficacy, activating or inhibiting only a subset of the signaling repertoire of their cognate receptors. In vitro, D-Trp12,Tyr34-bPTH(7-34) (PTH-{beta}arr), a biased agonist for the type 1 parathyroid hormone receptor, antagonizes receptor-G protein coupling but activates arrestin-dependent signaling. In vivo, both PTH-{beta}arr and the conventional agonist PTH(1-34) stimulate anabolic bone formation. To understand how two PTH1R ligands with markedly different in vitro efficacy could elicit similar in vivo responses, we analyzed transcriptional profiles from calvarial bone of mice treated for 8 weeks with vehicle, PTH-{beta}arr or PTH(1-34). Treatment of wild type mice with PTH-{beta}arr primarily affected pathways that promote expansion of the osteoblast pool, notably cell cycle regulation, cell survival and migration. These responses were absent in beta-arrestin2 null mice, identifying them as downstream targets of beta-arrestin2-mediated signaling. In contrast, PTH(1-34) primarily affected pathways classically associated with enhanced bone formation, including collagen synthesis and matrix mineralization. PTH(1-34) actions were less dependent on beta-arrestin2, as might be expected of a ligand capable of G protein activation. These results illustrate the uniqueness of biased agonism in vivo and demonstrate that functional selectivity can be exploited to change the quality of GPCR efficacy.
2013-03-07 | GSE35181 | GEO
Project description:G-protein-coupled receptors (GPCRs) are membrane proteins that enable cells to sense molecular signals and to respond in a broad range of biological processes according to the circumstances of cells. Recent studies elucidated that short-medium chain fatty acids can stimulate GPCRs to activate signaling pathways for adipocyte differentiation. As for differentiation of Porcine Subcutaneous Pre-Adipocyte (PSPA), it has been demonstrated that octanoate supplementation in differentiation medium is essential for growth arrest which triggers differentiation initiation of PSPA followed by lipid synthesis and accumulation to develop into fully matured adipocyte. Previously we detected 10 candidate GPCR genes considered to affect adipocyte differentiation of PSPA. Transfection of small interfering RNA (siRNA) for the candidate genes indicated that knockdown of three genes (A, B and C) obviously decreased the differentiation of PSPA. Therefore we investigated gene expression profiles of PSPA transfected with siRNA for each of three GPCR genes using a porcine custom microarray. Our result indicated that larger numbers of down-regulated genes were detected more than up-regulated genes by the siRNA transfection. Gene ontology analysis elucidated that genes up-regulated by knockdown of Gene A, B and C were categorized in biological processes of skeletal system development, mitosis, and cell adhesion, respectively. Interestingly, knockdown of all three genes commonly down-regulated genes categorized in PPAR signaling pathway and immune response. Differentiation of PSPA might be attributable to the alteration of gene expression profiles regulated by GPCRs identified in this study.
2019-05-31 | GSE109067 | GEO
Project description:Cellular responses depend on the interactions of extracellular ligands, such as nutrients, growth factors, or drugs, with specific cell-surface receptors. The sensitivity of these interactions to non-physiological conditions, however, makes them challenging to study using in vitro assays. Here we present HATRIC, a technology that successfully identifies target receptors for ligands ranging from small molecules to intact viruses at physiological pH from as few as 1 million cells.
2017-07-07 | MSV000081228 | MassIVE