High Throughput Screening of G-Protein Coupled Receptors via Flow Cytometry

2003 ◽  
Vol 6 (4) ◽  
pp. 389-397 ◽  
Author(s):  
A. Waller ◽  
P. Simons ◽  
E. Prossnitz ◽  
B. Edwards ◽  
L. Sklar
Keyword(s):  
Flow Cytometry ◽  
G Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
G Protein Coupled Receptors ◽  
G Protein Coupled

scholarly journals A Homogeneous Enzyme Fragment Complementation-Based β-Arrestin Translocation Assay for High-Throughput Screening of G-Protein-Coupled Receptors

2008 ◽  
Vol 13 (8) ◽  
pp. 737-747 ◽  
Author(s):  
Xiaoning Zhao ◽  
Adrie Jones ◽  
Keith R. Olson ◽  
Kun Peng ◽  
Tom Wehrman ◽  
...  
Keyword(s):  
G Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Somatostatin Receptor ◽  
Gene Families ◽  
G Protein Coupled Receptors ◽  
Functional Assay ◽  
Gpcr Activation ◽  
Fragment Complementation ◽  
G Protein Coupled

G-protein-coupled receptors (GPCRs) represent one of the largest gene families in the human genome and have long been regarded as valuable targets for small-molecule drugs. The authors describe a new functional assay that directly monitors GPCR activation. It is based on the interaction between β-arrestin and ligand-activated GPCRs and uses enzyme fragment complementation technology. In this format, a GPCR of interest is fused to a small (~4 kDa), optimized α fragment peptide (termed ProLink™) derived from β-galactosidase, and β-arrestin is fused to an N-terminal deletion mutant of β-galactosidase (termed the enzyme acceptor [EA]). Upon activation of the receptor, the β-arrestin-EA fusion protein binds the activated GPCR. This interaction drives enzyme fragment complementation, resulting in an active β-galactosidase enzyme, and thus GPCR activation can be determined by quantifying β-galactosidase activity. In this report, the authors demonstrate the utility of this technology to monitor GPCR activation and validate the approach using a Gαi-coupled GPCR, somatostatin receptor 2. Potential application to high-throughput screens in both agonist and antagonist screening modes is exemplified. ( Journal of Biomolecular Screening 2008:737-747)

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 Miniaturization of Whole Live Cell-Based GPCR Assays Using Microdispensing and Detection Systems

2004 ◽  
Vol 9 (3) ◽  
pp. 186-195 ◽  
Author(s):  
Oleg Kornienko ◽  
Raul Lacson ◽  
Priya Kunapuli ◽  
Jonathan Schneeweis ◽  
Ira Hoffman ◽  
...  
Keyword(s):  
G Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
G Protein Coupled Receptors ◽  
Assay Development ◽  
Functional Responses ◽  
Detection Systems ◽  
G Protein Coupled ◽  
Receptor Agonists And Antagonists ◽  
Assay Volume

Cell-based β-lactamase reporter gene assays designed to measure the functional responses of G-protein-coupled receptors (GPCRs) were miniaturized to less than 2 μL total assay volume in a 3456-well microplate. Studies were done to evaluate both receptor agonists and antagonists. The pharmacology of agonists and antagonists for target GPCRs originally developed in a 96-well format was recapitulated in a 3456-well microplate format without compromising data quality or EC50/IC50 precision. These assays were employed in high-throughput screening campaigns, allowing the testing of more than 150,000 compounds in 8 h. The instrumentation used and practical aspects of the assay development are discussed.

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