Progress in methodology improved reporter gene assays used to identify ligands acting on orphan seven-transmembrane receptors.
Author
Summary, in English
Seven-transmembrane G-protein-coupled receptors play a central role in physiology by facilitating cell communication through recognition of a wide range of ligands. Even more important, they represent important drug targets. Unfortunately, for many of these receptors the endogenous ligands, and hence their functions, remain to be identified. These receptors are referred to as "orphan" receptors. A pre-requisite for the identification of ligands activating orphan receptors is powerful assay systems. Until now, reporter gene assays have not been in common use in this process. Here, we summarize our development of improved reporter gene assays. We optimized reporter gene assays in respect of (i) the promoter region of the construct, (ii) the reporter enzyme used, (iii) and the assay procedure. Furthermore, an unique fluorescence-based clone selection step was introduced, allowing rapid selection of the most sensitive reporter cell clones when establishing stable reporter cell lines. Mathematical formulae are provided to enable a simple and reliable comparison between different cell lines, when tested with a compound of interest. The resulting reporter cell lines responded in a very sensitive way to the stimulation of various test receptors. The reporter system was termed HighTRACE® (high-throughput reporter assay with clone election). Its high assay quality makes it suitable as a primary screening tool. Ligands for two recently unknown 7TM receptors were identified using the HighTRACE® system i.e., two cell surface free fatty acid receptors, GPR40 (FFA1R) and GPR43 (FFA2R). The identification was accomplished using a reverse pharmacology approach.
Department/s
- Mucosal Immunology
- Department of Experimental Medical Science
- Drug Target Discovery
Publishing year
2003
Language
English
Pages
249-258
Publication/Series
Pharmacology and Toxicology
Volume
93
Issue
6
Full text
- Available as PDF - 158 kB
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Links
Document type
Journal article review
Publisher
Wiley-Blackwell
Topic
- Basic Medicine
- Neurosciences
Status
Published
Research group
- Mucosal Immunology
- Drug Target Discovery
ISBN/ISSN/Other
- ISSN: 1600-0773