Improved reporter gene assays used to identify ligands acting on orphan seven-transmembrane receptors

被引:53
作者
Kotarsky, K [1 ]
Nilsson, NE [1 ]
Olde, B [1 ]
Owman, C [1 ]
机构
[1] Wallenberg Neurosci Ctr, Dept Physiol Sci, Div Mol Neurobiol, S-22184 Lund, Sweden
来源
PHARMACOLOGY & TOXICOLOGY | 2003年 / 93卷 / 06期
关键词
D O I
10.1111/j.1600-0773.2003.pto930601.x
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
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(R) (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(R) 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.
引用
收藏
页码:249 / 258
页数:10
相关论文
共 94 条
  • [1] Molecular cloning of the human Edg2 protein and its identification as a functional cellular receptor for lysophosphatidic acid
    An, SZ
    Dickens, MA
    Bleu, T
    Hallmark, OG
    Goetzl, EJ
    [J]. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1997, 231 (03) : 619 - 622
  • [2] Rhodopsin crystal: new template yielding realistic models of G-protein-coupled receptors?
    Archer, E
    Maigret, B
    Escrieut, C
    Pradayrol, L
    Fourmy, D
    [J]. TRENDS IN PHARMACOLOGICAL SCIENCES, 2003, 24 (01) : 36 - 40
  • [3] Identification of melanin concentrating hormone (MCH) as the natural ligand for the orphan somatostatin-like receptor 1 (SLC-1)
    Bächner, D
    Kreienkamp, HJ
    Weise, C
    Buck, F
    Richter, D
    [J]. FEBS LETTERS, 1999, 457 (03) : 522 - 524
  • [4] A beta-arrestin green fluorescent protein biosensor for detecting G protein-coupled receptor activation
    Barak, LS
    Ferguson, SSG
    Zhang, J
    Caron, MG
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1997, 272 (44) : 27497 - 27500
  • [5] Neurobiology of the Caenorhabditis elegans genome
    Bargmann, CI
    [J]. SCIENCE, 1998, 282 (5396) : 2028 - 2033
  • [6] Molecular tinkering of G protein-coupled receptors: an evolutionary success
    Bockaert, J
    Pin, JP
    [J]. EMBO JOURNAL, 1999, 18 (07) : 1723 - 1729
  • [7] Characterization of the cloned guinea pig leukotriene B4 receptor:: comparison to its human orthologue
    Boie, Y
    Stocco, R
    Sawyer, N
    Greig, GM
    Kargman, S
    Slipetz, DM
    O'Neill, GP
    Shimizu, T
    Yokomizo, T
    Metters, KM
    Abramovitz, M
    [J]. EUROPEAN JOURNAL OF PHARMACOLOGY, 1999, 380 (2-3) : 203 - 213
  • [8] Induction of NFAT-mediated transcription by G(q)-coupled receptors in lymphoid and non-lymphoid cells
    Boss, V
    Talpade, DJ
    Murphy, TJ
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1996, 271 (18) : 10429 - 10432
  • [9] The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids
    Briscoe, CP
    Tadayyon, M
    Andrews, JL
    Benson, WG
    Chambers, JK
    Eilert, MM
    Ellis, C
    Elshourbagy, NA
    Goetz, AS
    Minnick, DT
    Murdock, PR
    Sauls, HR
    Shabon, U
    Spinage, LD
    Strum, JC
    Szekeres, PG
    Tan, KB
    Way, JM
    Ignar, DM
    Wilson, S
    Muir, AI
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 2003, 278 (13) : 11303 - 11311
  • [10] The orphan G protein-coupled receptors GPR41 and GPR43 are activated by propionate and other short chain carboxylic acids
    Brown, AJ
    Goldsworthy, SM
    Barnes, AA
    Eilert, MM
    Tcheang, L
    Daniels, D
    Muir, AI
    Wigglesworth, MJ
    Kinghorn, I
    Fraser, NJ
    Pike, NB
    Strum, JC
    Steplewski, KM
    Murdock, PR
    Holder, JC
    Marshall, FH
    Szekeres, PG
    Wilson, S
    Ignar, DM
    Foord, SM
    Wise, A
    Dowell, SJ
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 2003, 278 (13) : 11312 - 11319