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)

scholarly journals Development of a Generic Dual-Reporter Gene Assay for Screening G-Protein-Coupled Receptors

2005 ◽  
Vol 10 (5) ◽  
pp. 437-446 ◽  
Author(s):  
Toby C. Kent ◽  
Kevin S. J. Thompson ◽  
Louise H. Naylor
Keyword(s):  
G Protein ◽  
Reporter Gene ◽  
High Throughput Screening ◽  
Reporter Gene Assay ◽  
G Protein Coupled Receptors ◽  
Rapid Expansion ◽  
Compound Libraries ◽  
Dual Reporter ◽  
Gene Assay ◽  
G Protein Coupled

Multiple assay formats have been developed for the pharmacological characterization of G-protein-coupled receptors (GPCRs) and for screening orphan receptors. However, the increased pace of target identification and the rapid expansion of compound libraries present the need to develop novel assay formats capable of screeningmultipleGPCRs simultaneously. To address this need, the authors have developed a generic dual-reporter gene assay that can detect ligand activity at 2 GPCRs within the same assay. Two stableHEK293 cell lineswere generated expressing either a firefly ( Photinus) luciferase gene under the control ofmultiple cAMP-response elements (CREs) or a Renillaluciferase gene under the control ofmultiple 12-Otetradecanoylphorbol-13-acetate (TPA)-responsive elements (TREs). Coseeded reporter cells were used to assess ligandbinding activity at bothGβ s-and Gβ q-coupled receptors. By selectively coexpressing receptors with a chimeric G-protein, agonist activitywas assessed atGβ i/o-coupled receptors in combinationwith eitherGβ s-or Gβ q-coupled receptors. The dual-reporter gene assaywas shown to be capable of simultaneously performing duplexed screens for a variety of agonist and/or antagonist combinations. The data generated from the duplexed reporter assays were pharmacologically relevant, and Zβ factor analysis indicated the suitability of both agonist and antagonist screens for use in high-throughput screening.

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