scholarly journals Cell-Free Assay of G-Protein-Coupled Receptors Using Fluorescence Polarization

2008 ◽  
Vol 13 (5) ◽  
pp. 424-429 ◽  
Author(s):  
Jessi Wildeson Jones ◽  
Tiffani A. Greene ◽  
Christine A. Grygon ◽  
Benjamin J. Doranz ◽  
Martha P. Brown
Keyword(s):  
G Protein ◽  
Fluorescence Polarization ◽  
G Protein Coupled Receptors ◽  
Peptide Ligand ◽  
Binding Assays ◽  
Free System ◽  
Molecular Binding ◽  
Soluble Cell ◽  
G Protein Coupled ◽  
Peptide Interaction

A recently developed nanotechnology, the Integral Molecular lipoparticle, provides an essentially soluble cell-free system in which G-protein-coupled receptors (GPCRs) in their native conformations are concentrated within virus-like particles. As a result, the lipoparticle provides a means to overcome 2 common obstacles to the development of homogeneous, nonradioactive GPCR ligand-binding assays: membrane protein solubilization and low receptor density. The work reported here describes the first application of this nanotechnology to a fluorescence polarization (FP) molecular binding assay format. The GPCR chosen for these studies was the well-studied chemokine receptor CXCR4 for which a peptide ligand (T-22) has been previously characterized. The EC50 determined for the CXCR4-T-22 peptide interaction via FP with CXCR4 lipoparticles (15 nM) is consistent with the IC50 determined for the unlabeled T-22 peptide via competitive binding (59 nM). ( Journal of Biomolecular Screening 2008:424-429)

scholarly journals Impact of a Red-Shifted Dye Label for High Throughput Fluorescence Polarization Assays of G Protein-Coupled Receptors

2000 ◽  
Vol 5 (5) ◽  
pp. 329-334 ◽  
Author(s):  
Peter Banks ◽  
Mylene Gosselin ◽  
Linda Prystay
Keyword(s):  
G Protein ◽  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
False Negative ◽  
G Protein Coupled Receptors ◽  
Negative Results ◽  
False Negative Results ◽  
Assay Precision ◽  
G Protein Coupled

High throughput screening fluorescence polarization assays using G protein-coupled receptors (GPCRs) as targets have been compared using fluorescein and BODIPY TMR-labeled peptides. The red-shifted BODIPY TMR dye exhibits improved assay performance relative to fluorescein due to improvement in both ligand affinity to the GPCRs and assay precision brought about by the higher intensity probe. Furthermore, the red-shifted dye demonstrates an insensitivity to the effects of the highly colored compound tartrazine, which can produce false-negative results for assays conducted with fluorescein as a label.

scholarly journals High Throughput Fluorescence Polarization: A Homogeneous Alternative to Radioligand Binding for Cell Surface Receptors

2000 ◽  
Vol 5 (2) ◽  
pp. 63-69 ◽  
Author(s):  
Michael Allen ◽  
Julian Reeves ◽  
Geoffrey Mellor
Keyword(s):  
Cell Surface ◽  
High Throughput ◽  
Fluorescence Polarization ◽  
Radioligand Binding ◽  
Low Cost ◽  
G Protein Coupled Receptors ◽  
Cell Surface Receptors ◽  
Binding Assays ◽  
Surface Receptors ◽  
G Protein Coupled

High throughput fluorescence polarization (FP) assays are described that offer a nonradioactive, homogeneous, and low-cost alternative to radioligand binding assays for cell surface receptors (G protein-coupled receptors and ligand-gated ion channels). FP assays were shown to work across a range of both peptide (vasopressin V1a and δ-opioid) and nonpeptide (β-adrenoceptor, 5-hydroxytryptamine3) receptors. Structure-activity relationships were investigated at β1-receptors and were found to be consistent with radioligand binding assays. FP was shown to tolerate up to 5% DMSO with no loss in sensitivity or signal window. From a random set of 1,280 compounds, 1.9% were found to significantly interfere with FP measurement. If fluorescent or quenching compounds were eliminated (3% of all compounds), less than 0.4% of compounds were found to interfere with FP measurement. Assays could be run in 384-well plates with little loss of signal window or sensitivity compared to 96-well plate assays. New advances in FP measurement have therefore enabled FP to offer a high throughput alternative to radioligand binding for cell surface receptors.

scholarly journals Comparative Analysis of Functional Assays for Characterization of Agonist Ligands at G Protein-Coupled Receptors

2003 ◽  
Vol 8 (5) ◽  
pp. 500-510 ◽  
Author(s):  
Anke Niedernberg ◽  
Sorin Tunaru ◽  
Andree Blaukat ◽  
Bruce Harris ◽  
Evi Kostenis
Keyword(s):  
G Protein ◽  
Partial Agonist ◽  
A Priori ◽  
G Protein Coupled Receptors ◽  
Binding Assays ◽  
Functional Assays ◽  
Gtpγs Binding ◽  
Sphingosine 1 Phosphate ◽  
Intracellular Ca ◽  
G Protein Coupled

A variety of functional assays are available for agonist or antagonist screening of G protein-coupled receptors (GPCRs), but it is a priori not predictable which assay is the most suitable to identify agonists or antagonists of GPCRs with therapeutic value in humans. More specifically, it is not known how a given set of GPCR agonists compares in different functional assays with respect to potency and efficacy and whether the level of the signaling cascade that is analyzed has any impact on the detection of agonistic responses. To address this question, the authors used the recently cloned human S1P5 receptor as a model and compared a set of 3 lipid ligands (sphingosine 1-phosphate [S1P], dihydro sphingosine 1-phosphate [dhS1P], and sphingosine) in 5 different functional assays: GTPγS binding, inhibition of adenylyl cyclase activity, mobilization of intracellular Ca2+ via the FLIPR and aequorin technology, and MAP kinase (ERK1/2) activation. S1P induced agonistic responses in all except the ERK1/2 assays with EC50 values varying by a factor of 10. Whereas dhS1P was identified as a partial agonist in the GTPγS assay, it behaved as a full agonist in all other settings. Sphingosine displayed partial agonistic activity exclusively in GTPγS binding assays. The findings suggest that assays in a given cellular background may vary significantly with respect to suitability for agonist finding and that ligands producing a response may not readily be detectable in all agonist assays. ( Journal of Biomolecular Screening 2003:500-510)

scholarly journals Considerations for Using Fluorescence Polarization in the Screening of G Protein-Coupled Receptors

2002 ◽  
Vol 7 (2) ◽  
pp. 111-117 ◽  
Author(s):  
Peter Banks ◽  
Michael Harvey
Keyword(s):  
Performance Assessment ◽  
G Protein ◽  
Fluorescence Polarization ◽  
Detection Method ◽  
G Protein Coupled Receptors ◽  
Fluorescence Techniques ◽  
Assay Performance ◽  
Detection Volume ◽  
G Protein Coupled

Fluorescence polarization must be classified as a unique detection method relative to other intensity-based methods because both bound and unbound tracer is measured. Other fluorescence techniques require either physical removal of unbound tracer or a means for distinguishing only bound tracer. The presence of unbound tracer in the detection volume has profound consequences on how assay performance is gauged. The intent here is to provide tools for accurate assay performance assessment and to discuss some of the practical considerations necessary for understanding the advantages and limitations of the technology. An emphasis is placed on applications using G protein-coupled receptors.

NMR studies of CCK-8/CCK1 complex support membrane-associated pathway for ligand-receptor interaction

2002 ◽  
Vol 80 (5) ◽  
pp. 383-387 ◽  
Author(s):  
Craig Giragossian ◽  
Maria Pellegrini ◽  
Dale F Mierke
Keyword(s):  
G Protein ◽  
Structural Features ◽  
G Protein Coupled Receptors ◽  
Peptide Ligands ◽  
Receptor Interaction ◽  
Peptide Ligand ◽  
Receptor Subtype ◽  
Nmr Studies ◽  
Receptor Complexes ◽  
G Protein Coupled

The interaction of peptide ligands with their associated G-protein-coupled receptors has been examined by a number of different experimental approaches over the years. We have been developing an approach utilizing high-resolution NMR to determine the structural features of the peptide ligand, well-designed fragments of the receptor, and the ligand–receptor complexes formed upon titration of the peptide hormone. The results from these investigations provide evidence for a membrane-associated pathway for the initial interaction of peptide ligands with the receptor. Here, our results from the investigation of the interaction of CCK-8 with the CCK1 receptor are described. Our spectroscopic results clearly show that both CCK-8 and the regions of CCK1 with which it interacts are closely associated with the zwitterionic interface of the lipids utilized in our solution spectroscopic studies.Key words: G-protein-coupled receptors, NMR structural characterization, cholecystokinin, CCK-8, cholecystokinin receptor, subtype 1, CCK1, peptide hormones.

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