A systems approach to investigate GPCR-mediated Ras signaling network in chemoattractant sensing.
Ontology highlight
ABSTRACT:
SUBMITTER: Xu X
PROVIDER: S-EPMC9250378 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Accurate detection of extracellular chemical gradients is essential for many cellular behaviors. Gradient sensing is challenging for small cells, which can experience little difference in ligand concentrations on the up-gradient and down-gradient sides of the cell. Nevertheless, the tiny cells of the yeast Saccharomyces cerevisiae reliably decode gradients of extracellular pheromones to find their mates. By imaging the behavior of polarity factors and pheromone receptors, we quantified the accuracy of initial polarization during mating encounters. We found that cells bias the orientation of initial polarity up-gradient, even though they have unevenly distributed receptors. Uneven receptor density means that the gradient of ligand-bound receptors does not accurately reflect the external pheromone gradient. Nevertheless, yeast cells appear to avoid being misled by responding to the fraction of occupied receptors rather than simply the concentration of ligand-bound receptors. Such ratiometric sensing also serves to amplify the gradient of active G protein. However, this process is quite error-prone, and initial errors are corrected during a subsequent indecisive phase in which polarity clusters exhibit erratic mobile behavior.
| S-EPMC6818790 | biostudies-literature
Project description:Abnormal feedback of serum calcium to parathyroid hormone (PTH) secretion is the hallmark of primary hyperparathyroidism (PHPT). Although the molecular pathogenesis of parathyroid neoplasia in PHPT has been linked to abnormal expression of genes involved in cell growth (e.g., cyclin D1, retinoblastoma, and β-catenin), the molecular basis of abnormal calcium sensing by calcium-sensing receptor (CaSR) and PTH hypersecretion in PHPT are incompletely understood. Through gene expression profiling, we discovered that an orphan adhesion G protein-coupled receptor (GPCR), GPR64/ADGRG2, is expressed in human normal parathyroid glands and is overexpressed in parathyroid tumors from patients with PHPT. Using immunohistochemistry, Western blotting, and coimmunoprecipitation, we found that GPR64 is expressed on the cell surface of parathyroid cells, is overexpressed in parathyroid tumors, and physically interacts with the CaSR. By using reporter gene assay and GPCR second messenger readouts we identified Gαs, 3',5'-cyclic adenosine monophosphate (cAMP), protein kinase A, and cAMP response element binding protein (CREB) as the signaling cascade downstream of GPR64. Furthermore, we found that an N-terminally truncated human GPR64 is constitutively active and a 15-amino acid-long peptide C-terminal to the GPCR proteolysis site (GPS) of GPR64 activates this receptor. Functional characterization of GPR64 demonstrated its ability to increase PTH release from human parathyroid cells at a range of calcium concentrations. We discovered that the truncated constitutively active, but not the full-length GPR64 physically interacts with CaSR and attenuates the CaSR-mediated intracellular Ca2+ signaling and cAMP suppression in HEK293 cells. Our results indicate that GPR64 may be a physiologic regulator of PTH release that is dysregulated in parathyroid tumors, and suggest a role for GPR64 in pathologic calcium sensing in PHPT. © 2016 American Society for Bone and Mineral Research.
| S-EPMC7211037 | biostudies-literature
Project description:Modern toxicological evaluations have evolved to consider toxicity as a perturbation of biological pathways or networks. As such, toxicity testing approaches are shifting from common end point evaluations to pathway based approaches, where the degree of perturbation of select biological pathways is monitored. These new approaches are greatly increasing the data available to toxicologists, but methods of analyses to determine the inter-relationships between potentially affected pathways are needed to fully understand the consequences of exposure. An approach to construct dose-response curves that use graph theory to describe network perturbations among three disparate mitogen-activated protein kinase (MAPK) pathways is presented. Mitochondrial stress was induced in human hepatocytes (HepG2) by exposing the cells to increasing doses of the complex I inhibitor, deguelin. The relative phosphorylation responses of proteins involved in the regulation of the stress response were measured. Graph theory was applied to the phosphorylation data to obtain parameters describing the network perturbations at each individual dose tested. The graph theory results depicted the dynamic nature of the relationship between p38, JNK, and ERK1/2 under conditions of mitochondrial stress and revealed shifts in the relationships between these MAPK pathways at low doses. The inter-relationship, or crosstalk, among these 3 traditionally linear MAPK cascades was further probed by coexposing cells to deguelin plus SB202190 (JNK and p38 inhibitor) or deguelin plus SB202474 (JNK inhibitor). The cells exposed to deguelin plus SB202474 resulted in significantly decreased viability, which could be visualized and attributed to the decrease of ERK1/2 network centrality. The approach presented here allows for the construction and visualization of dose-response curves that describe network perturbations induced by chemical stress, which provides an informative and sensitive means of assessing toxicological effects on biological systems.
| S-EPMC3939825 | biostudies-literature
Project description:Binding of arrestin to phosphorylated G-protein-coupled receptors (GPCRs) controls many aspects of cell signaling. The number and arrangement of phosphates may vary substantially for a given GPCR, and different phosphorylation patterns trigger different arrestin-mediated effects. Here, we determine how GPCR phosphorylation influences arrestin behavior by using atomic-level simulations and site-directed spectroscopy to reveal the effects of phosphorylation patterns on arrestin binding and conformation. We find that patterns favoring binding differ from those favoring activation-associated conformational change. Both binding and conformation depend more on arrangement of phosphates than on their total number, with phosphorylation at different positions sometimes exerting opposite effects. Phosphorylation patterns selectively favor a wide variety of arrestin conformations, differently affecting arrestin sites implicated in scaffolding distinct signaling proteins. We also reveal molecular mechanisms of these phenomena. Our work reveals the structural basis for the long-standing "barcode" hypothesis and has important implications for design of functionally selective GPCR-targeted drugs.
| S-EPMC7901245 | biostudies-literature
Project description:The activation of G-protein-coupled receptors (GPCRs) leads to the activation of mTORC2 in cell migration and metabolism. However, the mechanism that links GPCRs to mTORC2 remains unknown. Here, using Dictyostelium cells, we show that GPCR-mediated chemotactic stimulation induces hetero-oligomerization of phosphorylated GDP-bound Rho GTPase and GTP-bound Ras GTPase in directed cell migration. The Rho-Ras hetero-oligomers directly and specifically stimulate mTORC2 activity toward AKT in cells and after biochemical reconstitution using purified proteins in vitro. The Rho-Ras hetero-oligomers do not activate ERK/MAPK, another kinase that functions downstream of GPCRs and Ras. Human KRas4B functionally replace Dictyostelium Ras in mTORC2 activation. In contrast to GDP-Rho, GTP-Rho antagonizes mTORC2-AKT signaling by inhibiting the oligomerization of GDP-Rho with GTP-Ras. These data reveal that GPCR-stimulated hetero-oligomerization of Rho and Ras provides a critical regulatory step that controls mTORC2-AKT signaling.
| S-EPMC7716479 | biostudies-literature
Project description:Microorganisms sense environmental fluctuations in nutrients and light, coordinating their growth and development accordingly. Despite their critical roles in fungi, only a few G-protein coupled receptors (GPCRs) have been characterized. The Aspergillus nidulans genome encodes 86 putative GPCRs. Here, we characterise a carbon starvation-induced GPCR-mediated glucose sensing mechanism in A. nidulans. This includes two class V (gprH and gprI) and one class VII (gprM) GPCRs, which in response to glucose promote cAMP signalling, germination and hyphal growth, while negatively regulating sexual development in a light-dependent manner. We demonstrate that GprH regulates sexual development via influencing VeA activity, a key light-dependent regulator of fungal morphogenesis and secondary metabolism. We show that GprH and GprM are light-independent negative regulators of sterigmatocystin biosynthesis. Additionally, we reveal the epistatic interactions between the three GPCRs in regulating sexual development and sterigmatocystin production. In conclusion, GprH, GprM and GprI constitute a novel carbon starvation-induced glucose sensing mechanism that functions upstream of cAMP-PKA signalling to regulate fungal development and mycotoxin production.
| S-EPMC6812930 | biostudies-literature
Project description:Describing the architecture of robust, nonlinear cell signaling networks is essential to gain a predictive understanding of cellular behavior. The structure of the Drosophila Rho-signaling network, comprised of Rho-family GTPases, RhoGTP Exchange Factors (RhoGEFs), and RhoGTPase Activating Proteins (RhoGAPs), has been particularly difficult to infer due to the highly overlapping function and substrate specificity of network components. We developed a parameterized modeling approach to predict connectivity amongst components of the Rho-signaling network that was driven by hundreds of mRNA expression profiles derived from RNAi-mediated inhibition or overexpression of component genes. Our model incorporated rate kinetics, transcriptional feedback, and noise. We biochemically validated several novel predicted connections, and used this model to predict Rho-signaling response to particular conditions. While functional redundancy is a feature of all signaling systems that often prevents classical genetic methods from elucidating relationships between components, the methods described here provide the basis for describing any complex network architecture. Keywords: Genetic modification, RNAi-mediated gene inhibition 129 samples analyzed. Experiments were peformed in batches of 4 containing 1 control/reference sample (transfection of GFP alone) that was prepared in parallel with experimental samples. There are 30 reference samples. The majority of experiments were replicated 2-6 times.
2012-12-31 | E-GEOD-18307 | biostudies-arrayexpress
Project description:The GTP-binding protein-coupled receptors (GPCRs) play important roles in physiology and neuronal signaling. More than a thousand genes, excluding the olfactory receptors, have been identified that encode these integral membrane proteins. Their pharmacological and functional properties make them fascinating targets for drug development, since various disease states can be treated and overcome by pharmacologically addressing these receptors and/or their downstream interacting partners. The activation of the GPCRs typically causes transient changes in the intracellular second messenger concentrations as well as in membrane conductance. In contrast to ion channel-mediated electrical signaling which results in spontaneous cellular responses, the GPCR-mediated metabotropic signals operate at a different time scale. Here we have studied the kinetics of two common GPCR-induced signaling pathways: (a) Ca2+ release from intracellular stores and (b) cyclic adenosine monophosphate (cAMP) production. The latter was monitored via the activation of cyclic nucleotide-gated (CNG) ion channels causing Ca2+ influx into the cell. Genetically modified and stably transfected cell lines were established and used in stopped-flow experiments to uncover the individual steps of the reaction cascades. Using two homologous biogenic amine receptors, either coupling to Go/q or Gs proteins, allowed us to determine the time between receptor activation and signal output. With ~350 ms, the release of Ca2+ from intracellular stores was much faster than cAMP-mediated Ca2+ entry through CNG channels (~6 s). The measurements with caged compounds suggest that this difference is due to turnover numbers of the GPCR downstream effectors rather than the different reaction cascades, per se.
| S-EPMC9369311 | biostudies-literature
Project description:The perturbation of signal-transduction molecules elicits genomic-expression effects that are typically neither restricted to a small set of genes nor uniform. Instead there are broad, varied, and complex changes in expression across the genome. These observations suggest that signal transduction is not mediated by isolated pathways of information flow to distinct groups of genes in the genome. Rather, multiple entangled paths of information flow influence overlapping sets of genes. Using the Ras-cAMP pathway in Saccharomyces cerevisiae as a model system, we perturbed key pathway elements and collected genomic-expression data. Singular value decomposition was applied to separate the genome-wide transcriptional response into weighted expression components exhibited by overlapping groups of genes. Molecular interaction data were integrated to connect gene groups to perturbed signaling elements. The resulting series of linked subnetworks maps multiple putative pathways of information flow through a dense signaling network, and provides a set of testable hypotheses for complex gene-expression effects across the genome.
| S-EPMC1457029 | biostudies-literature
Project description:Silicosis is a major health issue among workers exposed to crystalline silica. Genetic susceptibility has been implicated in silicosis. The present research demonstrates key regulatory targets and propagated network of gene/miRNA/transcription factor (TF) with interactions responsible for silicosis by integrating publicly available microarray data using a systems biology approach. Array quality is assessed with the Quality Metrics package of Bioconductor, limma package, and the network is constructed using Cytoscape. We observed and enlist 235 differentially expressed genes (DEGs) having up-regulation expression (85 nos) and down-regulation expression (150 nos.) in silicosis; and 24 TFs for the regulation of these DEGs entangled with thousands of miRNAs. Functional enrichment analysis of the DEGs enlighten that, the maximum number of DEGs are responsible for biological process viz, Rab proteins signal transduction (11 nos.) and Cellular Senescence (20 nos.), whereas IL-17 signaling pathway (16 nos.) and Signalling by Nuclear Receptors (14 nos.) etc. are Biological Pathway involving more DEGs. From the identified 1100 high target microRNA (miRNA)s involved in silicosis, 1055 miRNAs are found to relate with down-regulated genes and 847 miRNAs with up-regulated genes. The CDK19 gene (Up-regulated) is associated with 617 miRNAs whereas down-regulated gene ARID5B is regulated by as high as 747 high target miRNAs. In Prediction of Small-molecule signatures, maximum scoring small-molecule combinations for the DEGs have shown that CGP-60774 (with 20 combinations), alvocidib (with 15 combinations) and with AZD-7762 (24 combinations) with few other drugs having the high probability of success.
| S-EPMC7809153 | biostudies-literature