Homogeneous GTP binding assay employing QRET technology

Journal of Biomolecular Screening
Anita Rozwandowicz-JansenHarri Härmä

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)...Continue Reading

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Citations

Nov 22, 2011·Analytical Chemistry·Eija MartikkalaHarri Härmä
Apr 12, 2011·Analytical Biochemistry·Roope HuttunenHarri Härmä
Oct 31, 2015·Analytica Chimica Acta·Natalia Tong-OchoaHarri Härmä
May 24, 2018·Chemical Communications : Chem Comm·Sarah H Hewitt, Stephen J Butler
Feb 24, 2011·Journal of Biomolecular Screening·Eija MartikkalaHarri Härmä

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Methods Mentioned

BETA
nucleotide exchange
QRET
saturation binding
fluorescence filtration

Software Mentioned

GraphPad Prism

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