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