Elsevier

Analytical Biochemistry

Volume 397, Issue 2, 15 February 2010, Pages 202-207
Analytical Biochemistry

Europium-labeled GTP as a general nonradioactive substitute for [35S]GTPγS in high-throughput G protein studies

https://doi.org/10.1016/j.ab.2009.10.028Get rights and content

Abstract

[35S]GTPγS, the nonhydrolyzable radioactive GTP analog, has been a powerful tool in G protein studies and has set the standards in this field of research. However, its radioactive nature imposes clear limitations to its use in regular laboratory practice and in high-throughput experimentation. The europium-labeled GTP analog (Eu-GTP) has been used as an alternative in the analysis of G protein activation by G protein-coupled receptors in cellular membrane preparations. Here we expand the usage of Eu-GTP and show that it can be applied in other types of assays where [35S]GTPγS has been previously utilized. We demonstrate the applicability of the modified Eu-GTP binding technology to analysis of heterotrimeric and monomeric G proteins of natural and recombinant sources, from different organisms, in assays with soluble proteins and membrane-containing assays of a high-throughput format. The deci-nanomolar KD of Eu-GTP for the tested G proteins is similar to that of other fluorescent-modified GTP analogs, while the sensitivity achieved in time-resolved fluorescence analysis of Eu-GTP exceeds that of the radioactive measurements. Overall, the results of our modified Eu-GTP binding assay present Eu-GTP as a general nonradioactive alternative for G protein studies, especially attractive in high-throughput experiments.

Section snippets

Materials

Drosophila melanogaster rab5 cDNA (clone GH24702) and pGEX-dCdc42-WT (barcode 1259) were from the Drosophila Genomics Resource Center. Recombinant GST-tagged human Ras1 (Cat. No. 553325), anti-Gβγ antibodies (Cat. No. 371821), and imidazole were from Merck. Complete protease inhibitor cocktail was from Roche. GDP, GTPγS, Thesit, lysozyme, mastoparan, and serotonin were from Sigma. PMSF and isopropyl-1-thio-d-galactopyranoside (IPTG) were from Roth. Oxotremorine-M was from Tocris Bioscience.

[35S]GTPγS and Eu-GTP record similar GPCR activation in natural membranes

To begin our analysis, we wanted to directly compare Eu-GTP and [35S]GTPγS for their capacities to monitor activation of GPCRs in natural membranes. We purified porcine brain membranes (see Materials and methods) and stimulated them with two agonists, serotonin and oxotremorine-M. These agents activate various metabotropic serotonin receptors (5-HT1a, 5HT2c, etc.) and muscarinic receptors, respectively, which in turn activate G proteins of the Gq and Gi/o groups [21], [27]. First, the

Discussion

In the present article we demonstrate the utility of the Europium-labeled GTP analog, Eu-GTP, as a general substitute of the conventionally used radioactive [35S]GTPγS in various formats of analysis of GPCR signaling and G protein activity. Eu-GTP is found to be applicable in membrane-containing assays, as well as in solution-based assays to study heterotrimeric G proteins, purified Gα-subunits, and monomeric G proteins of different types and organisms. We show that Eu-GTP can be used in the

Acknowledgments

We thank Silke Buestorf for technical assistance, Joerg Hartig for help with radioactive experiments, and Hans Werner Hofer for advice. This work was funded by TR-SFB11 (Deutsche Forschungsgemeinschaft) to V.L.K.

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These authors contributed equally to this work.

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