Open AccessProgress in Methodology
Improved Reporter Gene Assays Used to Identify Ligands Acting on Orphan Seven‐Transmembrane Receptors
Author(s) -
Kotarsky Knut,
Nilsson Niclas E.,
Olde Björn,
Owman Christer
Publication year - 2003
Publication title -
pharmacology & toxicology
Language(s) - English
Resource type - Journals
eISSN - 1600-0773
pISSN - 0901-9928
DOI - 10.1111/j.1600-0773.2003.pto930601.x
Subject(s) - reporter gene , receptor , biology , computational biology , bioreporter , clone (java method) , orphan receptor , high throughput screening , gene , microbiology and biotechnology , cell culture , biochemistry , genetics , gene expression , transcription factor
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 (FFA 1 R) and GPR43 (FFA 2 R). The identification was accomplished using a reverse pharmacology approach.