High throughput quantitation of cAMP production mediated by activation of seven transmembrane domain receptors

被引:41
作者
Kariv, I [1 ]
Stevens, ME [1 ]
Behrens, DL [1 ]
Oldenburg, KR [1 ]
机构
[1] DuPont Pharmaceut Co, Expt Stn E400 5412, Wilmington, DE 19880 USA
关键词
D O I
10.1177/108705719900400105
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Impairment of G protein-coupled seven-transmembrane (7 TM) receptor function has been implicated in a variety of different pathologic conditions, suggesting that the discovery of specific antagonists may lead to the development of successful therapeutic agents. The effect of different agents on receptor-ligand interaction is often measured directly in a receptor binding assay; however, this assay format can be time consuming and does not detect agents that interact at sites distal to the native ligand binding site. Cyclic adenosine monophospate (cAMP) represents a ubiquitous second messenger generated in response to ligand binding to many 7 TM receptors. The present study describes the practical adaptation of scintillation proximity methodology, using FlashPlate(TM) (NEN Life Sciences, Boston, MA) technology to evaluate cAMP production. The bioassay is based on competition between endogenously produced cAMP and exogenously added radiolabeled [I-125]-CAMp. Cyclic AMP capture is mediated through an anti-cAMP antibody onto a microplate well surface. Removal of unbound radioligand is not necessary because only ligand within less than or equal to 20 mu m of the plate surface is detected due to the proximity effect. The data indicate that the use of scintillation proximity technology allows accurate and specific evaluation of G protein-coupled receptor function as measured by cAMP production and is suitable for high throughput screening.
引用
收藏
页码:27 / 32
页数:6
相关论文
共 11 条
  • [1] [Anonymous], 1994, The G-Protein linked Receptor Fact Book
  • [2] Modes of peptide binding in G protein-coupled receptors
    Berthold, M
    Bartfai, T
    [J]. NEUROCHEMICAL RESEARCH, 1997, 22 (08) : 1023 - 1031
  • [3] Scintillation proximity assay: A versatile high-throughput screening technology
    Cook, ND
    [J]. DRUG DISCOVERY TODAY, 1996, 1 (07) : 287 - 294
  • [4] GUAN XM, 1995, MOL PHARMACOL, V48, P492
  • [5] Molecular approaches to receptors as targets for drug discovery
    Herz, JM
    Thomsen, WJ
    Yarbrough, GG
    [J]. JOURNAL OF RECEPTOR AND SIGNAL TRANSDUCTION RESEARCH, 1997, 17 (05): : 671 - 776
  • [6] Dopamine receptors and brain function
    Jaber, M
    Robinson, SW
    Missale, C
    Caron, MG
    [J]. NEUROPHARMACOLOGY, 1996, 35 (11) : 1503 - 1519
  • [7] CRH and the immune system
    Karalis, K
    Louis, JM
    Bae, D
    Hilderbrand, H
    Majzoub, JA
    [J]. JOURNAL OF NEUROIMMUNOLOGY, 1997, 72 (02) : 131 - 136
  • [8] KOCH WS, 1996, LIFE SCI, V58, P741
  • [9] SEQUENCE ALIGNMENT OF THE G-PROTEIN COUPLED RECEPTOR SUPERFAMILY
    PROBST, WC
    SNYDER, LA
    SCHUSTER, DI
    BROSIUS, J
    SEALFON, SC
    [J]. DNA AND CELL BIOLOGY, 1992, 11 (01) : 1 - 20
  • [10] WATSON J, 1996, FLASH NEWS