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

Impairment of G protein-coupled seven-transmembrane (7 TM) receptor functio n has been implicated in a variety of different pathologic conditions, sugg esting that the discovery of specific antagonists may lead to the developme nt of successful therapeutic agents. The effect of different agents on rece ptor-ligand interaction is often measured directly in a receptor binding as say; however, this assay format can be time consuming and does not detect a gents that interact at sites distal to the native ligand binding site. Cycl ic adenosine monophospate (cAMP) represents a ubiquitous second messenger g enerated in response to ligand binding to many 7 TM receptors. The present study describes the practical adaptation of scintillation proximity methodo logy, using FlashPlate(TM) (NEN Life Sciences, Boston, MA) technology to ev aluate cAMP production. The bioassay is based on competition between endoge nously produced cAMP and exogenously added radiolabeled [I-125]-CAMp. Cycli c AMP capture is mediated through an anti-cAMP antibody onto a microplate w ell surface. Removal of unbound radioligand is not necessary because only l igand 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 scintillati on proximity technology allows accurate and specific evaluation of G protei n-coupled receptor function as measured by cAMP production and is suitable for high throughput screening.