A Chimeric Reporter Gene Allowing for Clone Selection and High-Throughput Screening of Reporter Cell Lines Expressing G-Protein-Coupled Receptors

2001 ◽  
Vol 288 (2) ◽  
pp. 209-215 ◽  
Author(s):  
Knut Kotarsky ◽  
Christer Owman ◽  
Björn Olde
Keyword(s):  
G Protein ◽  
Cell Lines ◽  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
G Protein Coupled Receptors ◽  
Reporter Cell ◽  
Clone Selection ◽  
G Protein Coupled

scholarly journals Multiplexing Fluo-4 NW and a GeneBLAzer® Transcriptional Assay for High-Throughput Screening of G-Protein-Coupled Receptors

2006 ◽  
Vol 11 (6) ◽  
pp. 644-651 ◽  
Author(s):  
Bonnie J. Hanson
Keyword(s):  
Cell Line ◽  
G Protein ◽  
Cell Lines ◽  
High Throughput ◽  
Jurkat Cell ◽  
High Throughput Screening ◽  
G Protein Coupled Receptors ◽  
Beta Lactamase ◽  
Assay Format ◽  
G Protein Coupled

Activation of G-protein-coupled receptors (GPCRs) leads to a cascade of signaling events, including calcium mobilization and downstream transcriptional activation of various proteins. Two commonly used methods of high-throughput screening for GPCRs include calcium-sensitive dyes, such as Fluo-4 NW, and reporter gene assays, such as beta-lactamase. To determine whether the advantages of each assay format could be combined by multiplexing, Jurkat and CHO-K1 cell lines over-expressing the M1 muscarinic receptor and beta-lactamase under control of an NFAT response element were tested in a multiplexed format. The Jurkat cell line was further screened with a subset of the LOPAC1280 library. The multiplexing assay was compatible with both the CHO-K1 and Jurkat cell lines. For the screen, there was 100% correlation of on-target hits in the multiplexed format, and several false positives with each assay format were identified. Therefore, not only can the assays be multiplexed, but by multiplexing, the false positives associated with each assay format also could be easily identified. In addition to enhanced reliability, this method saves time and money because only half the amount of compounds, cells, and consumables are needed to screen a cell line in a multiplexed mode versus separate screening by both methods.

Development of an intact cell reporter gene β-lactamase assay for G protein-coupled receptors for high-throughput screening

2003 ◽  
Vol 314 (1) ◽  
pp. 16-29 ◽  
Author(s):  
Priya Kunapuli ◽  
Richard Ransom ◽  
Kathy L Murphy ◽  
Doug Pettibone ◽  
Julie Kerby ◽  
...  
Keyword(s):  
G Protein ◽  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Intact Cell ◽  
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)

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