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Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay
Published on: November 11, 2018
Homogeneous GTP binding assay employing QRET technology
Anita Rozwandowicz-Jansen1, Jonne Laurila, Eija Martikkala
1Laboratory of Biophysics, Institute of Biomedicine, University of Turku, Finland. aniroz@utu.fi
Journal of Biomolecular Screening
|January 28, 2010
Summary
A novel fluorescence assay using europium-labeled Guanosine-5'-triphosphate (Eu-GTP) enables time-resolved measurement of G-protein-coupled receptor activation. This separation-free method offers an alternative to traditional radiolabeled assays but shows a lower signal-to-background ratio.
Area of Science:
- Biochemistry
- Pharmacology
- Cell Signaling
Background:
- G-protein-coupled receptors (GPCRs) are crucial in cell signaling, with Guanosine-5 '-triphosphate (GTP) binding assays commonly used to measure their activation.
- The traditional [(35)S]GTPgammaS assay requires separation steps, limiting its efficiency.
- Novel, separation-free assays are needed for high-throughput screening of GPCR activity.
Purpose of the Study:
- To develop and validate a novel, separation-free, time-resolved fluorescence assay for measuring GTP binding to activated GPCRs.
- To compare the performance of the new assay against established methods using Guanosine-5 '-triphosphate.
Main Methods:
- Development of a Quenching Resonance Energy Transfer (QRET) assay utilizing a non-fluorescent europium-labeled, nonhydrolyzable GTP analog (Eu-GTP).
- Assay measures Eu-GTP binding to Gi-proteins upon activation of recombinant human alpha(2A)-adrenoceptors (alpha(2A)-AR) expressed in Chinese hamster ovary cells.
- Comparison with the [(35)S]GTPgammaS binding assay and a heterogeneous Eu-GTP filtration assay.
Main Results:
- The QRET assay successfully measured alpha(2A)-AR activation by five different agonists.
- Potency and efficacy rank orders were consistent across the QRET, [(35)S]GTPgammaS, and Eu-GTP filtration assays.
- The QRET assay exhibited a lower signal-to-background ratio and increased variability compared to the other two methods.
Conclusions:
- The novel QRET assay provides a viable, separation-free, time-resolved fluorescence alternative for measuring GTP binding and GPCR activation.
- While demonstrating comparable results in potency and efficacy, the QRET assay requires optimization to improve signal-to-background and reduce assay variation for broader application.
