High Throughput Screening Technologies for Direct Cyclic AMP Measurement

Gabriel, Daniela; Vernier, Magali; Pfeifer, Martin; Dasen, Boris; Tenaillon, Laurent; Bouhelal, Rochdi

doi:10.1089/15406580360545107

Cited by 128 publications

(68 citation statements)

References 21 publications

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“…Quantification of cAMP Levels-Protocol was conducted as previously described using the cAMP Glo luminescence based assay (Promega) (38). Briefly, 28 -30 h post-transfection, the cells were spun down (300 g, 3 min), resuspended at 1 ϫ 10 6 cells/ml in PBS solution supplemented with 0.02% glucose and 800 M ascorbic acid and aliquoted into 96-well round bottom opaque microplates.…”

Section: Methodsmentioning

confidence: 99%

Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins

Yao¹,

Malik

Griggs

et al. 2016

Journal of Biological Chemistry

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G protein ␣ subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of G␣ i . Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function.Heterotrimeric G proteins are key mediators of intracellular signaling pathways that control diverse cellular processes ranging from movement and division to differentiation and neuronal activity (1). G proteins consist of three subunits: G␣, G␤, and G␥. Bound with GDP, G␣ forms an inactive complex with its G␤␥ subunit partners. Interaction with activated receptor (GPCR) 3 promotes the exchange of GDP for GTP on G␣ and its separation from G␤␥. Both isolated G␣ and G␤␥ can then bind and activate or inhibit downstream effectors. GTP hydrolysis deactivates G␣, which subsequently reassociates with G␤␥ completing the cycle. This cycle is further regulated by two classes of additional proteins called regulators of G protein signaling. These function as either GTPase-activating proteins (which promote GTP hydrolysis) or GDP dissociation inhibitors (GDIs, which hinder exchange of GDP for GTP) (2). Important conformational transitions occurring at each stage of this regulated cycle have been characterized from extensive crystallographic studies. These include GDP, GTP analogue, G␤␥, GTPase-activating protein, GDI and most recently GPCR bound complex structures of G␣. However, the link between the observed conformations and the atomic level mechanisms involved in coupling receptor association, G protein activation, and effector interaction remain unclear.All G␣ proteins consist of a catalytic GTP binding Ras-like domain (termed RasD) and a heterotrimeric G protein specific helical domain (HD). Recent principal component analysis (PCA) of 53 available G␣ crystallographic structures identified three major conformationally distinct groups ( Fig. 1 and Ref. 3). These groups correspond to structures with bound GTP analogues, GDP, and GDI (red, green, and blue points in Fig. 1a, respectively). The major variation in the accumulated structures is the concerted displacements of three nucleotide-binding site loops termed the switch regions (SI, SII, and SIII), as well as a relatively small...

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Section: Methodsmentioning

confidence: 99%

Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins

Yao¹,

Malik

Griggs

et al. 2016

Journal of Biological Chemistry

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“…The LOCI technology has been compared with other assay types in different studies and has been described as a sensitive assay suited for high-throughput screening. 5,6 It has also been compared with existing eLISA assays 7,8 and found to be superior to eLISA with regard to speed, sensitivity, and hands-on time needed. It is a well-suited assay type for liquid handling robotics.…”

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confidence: 99%

Peptide Binding to HLA Class I Molecules: Homogenous, High-Throughput Screening, and Affinity Assays

Harndahl¹,

Justesen²,

Lamberth³

et al. 2009

SLAS Discovery

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The Human MHC Project aims at large-scale description of peptide-HLA binding to a wide range of HLA molecules covering all populations of the world and the accompanying generation of bioinformatics tools capable of predicting binding of any given peptide to any given HLA molecule. Here, the authors present a homogenous, proximity-based assay for detection of peptide binding to HLA class I molecules. It uses a conformation-dependent anti-HLA class I antibody, W6/32, as one tag and a biotinylated recombinant HLA class I molecule as the other tag, and a proximity-based signal is generated through the luminescent oxygen channeling immunoassay technology (abbreviated LOCI and commercialized as AlphaScreen). Compared with an enzyme-linked immunosorbent assay-based peptide-HLA class I binding assay, the LOCI assay yields virtually identical affinity measurements, although having a broader dynamic range, better signal-to-background ratios, and a higher capacity. They also describe an efficient approach to screen peptides for binding to HLA molecules. For the occasional user, this will serve as a robust, simple peptide-HLA binding assay. For the more dedicated user, it can easily be performed in a high-throughput screening mode using standard liquid handling robotics and 384-well plates. We have successfully applied this assay to more than 60 different HLA molecules, leading to more than 2 million measurements. (Journal of Biomolecular Screening 2009:173-180)

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“…the Ca 2+ -level or the cAMP concentration (Paredes et al, 2008;Hennen et al, 2013;Gabriel et al, 2003). In addition MODELLING AND MATHEMATICAL ANALYSIS OF SIGNALLING IN CHO CELLS 5 DMR has been introduced into the pharmaceutical field in order to specifically quantify the stimulation effect on the cytoskeleton (Fang et al, 2005;Schröder et al, 2011).…”

Section: Biological Background and Network Reconstructionmentioning

confidence: 99%

Modelling and mathematical analysis of the M$_{2}$ receptor-dependent joint signalling and secondary messenger network in CHO cells

Engelhardt

Holze

Elliott

et al. 2017

Mathematical Medicine and Biology: A Journal of the IMA

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The muscarinic M 2 receptor is a prominent member of the GPCR family and strongly involved in heart diseases. Recently published experimental work explored the cellular response to iperoxo-induced M 2 receptor stimulation in Chinese hamster ovary (CHO) cells. To better understand these responses, we modelled and analysed the muscarinic M 2 receptor-dependent signalling pathway combined with relevant secondary messenger molecules using mass action. In our literature-based joint signalling and secondary messenger model, all binding and phosphorylation events are explicitly taken into account in order to enable subsequent stoichiometric matrix analysis. We propose constraint flux sampling (CFS) as a method to characterize the expected shift of the steady state reaction flux distribution due to the known amount of cAMP production and PDE4 activation. CFS correctly predicts an experimentally observable ‡ Additional address: UCB Biosciences GmbH, Alfred-Nobel-Str. 10, 40789 Monheim, Germany influence on the cytoskeleton structure (marked by actin and tubulin) and in consequence a change of the optical density of cells. In a second step, we use CFS to simulate the effect of knock-out experiments within our biological system, and thus to rank the influence of individual molecules on the observed change of the optical cell density. In particular, we confirm the relevance of the protein RGS14, which is supported by current literature. A combination of CFS with Elementary Flux Mode analysis enabled us to determine the possible underlying mechanism. Our analysis suggests that mathematical tools developed for metabolic network analysis can also be applied to mixed secondary messenger and signalling models. This could be very helpful to perform model checking with little effort and to generate hypotheses for further research if parameters are not known.

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High Throughput Screening Technologies for Direct Cyclic AMP Measurement

Cited by 128 publications

References 21 publications

Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins

Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins

Peptide Binding to HLA Class I Molecules: Homogenous, High-Throughput Screening, and Affinity Assays

Modelling and mathematical analysis of the M$_{2}$ receptor-dependent joint signalling and secondary messenger network in CHO cells

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