title: Homogeneous GTP binding assay employing QRET technology.
abstract: Functional cell signaling assays have become important tools for measuring ligand-induced receptor activation in cell-based biomolecular screening. Guanosine-5'-triphosphate (GTP) is a generic signaling marker responsible for the first intracellular signaling event of the G-protein-coupled receptors (GPCRs). [(35)S]GTPgammaS binding assay is the classical well-established method for measuring agonist-induced G-protein activation requiring a separation of free and bound fractions prior to measurement. Here a novel, separation-free, time-resolved fluorescence GTP binding assay has been developed based on a non-fluorescence resonance energy transfer (FRET) single-label approach and quenching of a nonbound europium-labeled, nonhydrolyzable GTP analog (Eu-GTP). The quenching resonance energy transfer (QRET) method relies on the use of Eu-GTP, providing a time-resolved fluorescent detection as an alternative to the radiolabel [(35)S]GTPgammaS assay. Upon activation of recombinant human alpha(2A)-adrenoceptors (alpha(2A)-AR) expressed in Chinese hamster ovary cells, guanosine-5'-diphosphate is released from the alpha-subunit of Gi-proteins, enabling the subsequent binding of Eu-GTP. Activation of alpha(2A)-AR with 5 different alpha(2)-AR agonists was measured quantitatively using the developed QRET GTP assay and compared to [(35)S]GTPgammaS and heterogeneous Eu-GTP filtration assays. Equal potencies and efficacy rank orders were observed in all 3 assays but with a lower signal-to-background ratio and increased assay variation in the QRET assay compared to the Eu-GTP filtration and the nonhomogeneous [(35)S]GTPgammaS binding assays.
authors.affiliation: Laboratory of Biophysics, Institute of Biomedicine, University of Turku, Finland. aniroz@utu.fi
authors.forename: Anita
authors.initials: A
authors.lastname: Rozwandowicz-Jansen
authors.forename: Jonne
authors.initials: J
authors.lastname: Laurila
authors.forename: Eija
authors.initials: E
authors.lastname: Martikkala
authors.forename: Heini
authors.initials: H
authors.lastname: Frang
authors.forename: Ilkka
authors.initials: I
authors.lastname: Hemmilä
authors.forename: Mika
authors.initials: M
authors.lastname: Scheinin
authors.forename: Pekka
authors.initials: P
authors.lastname: Hänninen
authors.forename: Harri
authors.initials: H
authors.lastname: Härmä
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004735:Energy Transfer; D005453:Fluorescence; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D013997:Time Factors
chemical_list: D008565:Membrane Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate