title: Multiparametric homogeneous method for identification of ligand binding to G protein-coupled receptors: receptor-ligand binding and β-arrestin assay.
abstract: Two homogeneous assay systems have been combined to provide a new cell-based functional assay. The assay can be used to identify ligand binding to β(2)-adrenergic receptors, but also the downstream response can be determined in the same assay. Both the quenching resonance energy transfer (QRET) and the DiscoveRx PathHunter assay formats allow the use of intact cells. The homogeneous QRET technique is a single-label approach based on nonspecific quenching of the time-resolved luminescence, enabling agonist and antagonist receptor binding measurements. The commercial PathHunter assay is in turn based on enzyme fragment complementation, which can be detected on the basis of chemiluminescence signal. In the PathHunter technology the enzyme complementation is recorded immediately downstream of agonist-induced receptor activation. The new multiparametric detection technology combines these two assay methods enabling the identification of agonist, and antagonist binding to the receptor, and the agonist-induced response. Using the QRET and the PathHunter methods a panel of β(2)-adrenergic receptor ligands (epinephrine, terbutaline, metaproterenol, salmeterol, propranolol, alprenolol, bisoprolol, ICI 118,551, and bucindolol) was tested to prove the assay performance. The signal-to-background ratio for tested ligands ranged from 5 to 11 and from 6 to 18 with QRET and PathHunter, respectively. Combined homogeneous assay technique can provide an informative method for screening purposes and an efficient way to monitor receptor-ligand interaction, thus separating agonist from antagonist.
authors: Laboratory of Biophysics, Department of Cell Biology and Anatomy, Institute of Biomedicine, University of Turku, Turku, Finland. khkopr@utu.fi
authors: Kari
authors: K
authors: Kopra
authors: Markus
authors: M
authors: Kainulainen
authors: Piia
authors: P
authors: Mikkonen
authors: Anita
authors: A
authors: Rozwandowicz-Jansen
authors: Pekka
authors: P
authors: Hänninen
authors: Harri
authors: H
authors: Härmä
mesh_terms: D058666:Adrenergic beta-2 Receptor Agonists / Q000737:chemistry / Q000378:metabolism; D058672:Adrenergic beta-2 Receptor Antagonists / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D019390:Arrestins / Q000032:analysis* / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000378:metabolism*; D000071557:beta-Arrestins
chemical_list: D058666:Adrenergic beta-2 Receptor Agonists; D058672:Adrenergic beta-2 Receptor Antagonists; D019390:Arrestins; D008024:Ligands; D018343:Receptors, Adrenergic, beta-2; D000071557:beta-Arrestins