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Progress in methodology improved reporter gene assays used to identify ligands acting on orphan seven-transmembrane receptors.

Kotarsky, Knut LU ; Nilsson, Niclas LU ; Olde, Björn LU and Owman, Christer LU (2003) In Pharmacology and Toxicology 93(6). p.249-258
Abstract
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Pharmacology and Toxicology
volume
93
issue
6
pages
249 - 258
publisher
Wiley-Blackwell
external identifiers
  • wos:000188030400001
  • pmid:14675457
  • scopus:0346041542
ISSN
1600-0773
DOI
10.1111/j.1600-0773.2003.pto930601.x
language
English
LU publication?
yes
id
76f3d72b-5b0b-4f4f-a070-497613d5e628 (old id 119614)
date added to LUP
2007-07-16 13:06:01
date last changed
2017-07-30 03:48:57
@article{76f3d72b-5b0b-4f4f-a070-497613d5e628,
  abstract     = {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.},
  author       = {Kotarsky, Knut and Nilsson, Niclas and Olde, Björn and Owman, Christer},
  issn         = {1600-0773},
  language     = {eng},
  number       = {6},
  pages        = {249--258},
  publisher    = {Wiley-Blackwell},
  series       = {Pharmacology and Toxicology},
  title        = {Progress in methodology improved reporter gene assays used to identify ligands acting on orphan seven-transmembrane receptors.},
  url          = {http://dx.doi.org/10.1111/j.1600-0773.2003.pto930601.x},
  volume       = {93},
  year         = {2003},
}