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Time-resolved fluorescence ligand binding for G protein–coupled receptors

Nature Protocols volume 8, pages 1307–1320 (2013)Cite this article

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Abstract

G protein–coupled receptors (GPCRs) and their ligands are traditionally characterized by radioligand-binding experiments. These experiments yield excellent quantitative data, but have low temporal and spatial resolution. In addition, the use of radioligands presents safety concerns. Here we provide a general procedure for an alternative approach with high temporal and spatial resolution, based on Tb+-labeled fluorescent receptor ligands and time-resolved fluorescence resonance energy transfer (TR-FRET). This protocol and its design are detailed here for the parathyroid hormone receptor, a class B GPCR, and its fluorescently labeled 34-amino acid peptide ligand, but it can be easily modified for other receptors and their appropriately labeled ligands. We discuss three protocol options that use Tb+-labeled fluorescent ligands: a time-resolved fluorescence separation option that works on native receptors but requires separation of bound and unbound ligand; a TR-FRET option using SNAP-tag-labeled receptors for high-throughput screening; and a TR-FRET option that uses fluorescently labeled antibodies directed against an epitope engineered into the Flag-labeled receptors' N terminus. These protocol options can be used as standard procedures with very high signal-to-background ratios in order to characterize ligands and their receptors in living cells and in cell membranes via straightforward plate-reader measurements.

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Figure 1: Illustration of time-resolved ligand-binding protocol formats for PTHRs.
Figure 2: Confocal imaging of [C13-d2]PTH′(1–34) on living cells.
Figure 3: Binding of Tb3+-cryptate-labeled PTH measured by TRFS protocols.
Figure 4: TR-FRET/SNAP.
Figure 5: TR-FRET/Ab.

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Acknowledgements

Research in the authors' laboratory is supported by the DFG (grant no. SFB 487 to M.J.L.) and the Bayerische Forschungsstiftung (fellowship to A.E.-N.).

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Authors and Affiliations

  1. Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany

    Alexander Emami-Nemini & Martin J Lohse

  2. Rudolf Virchow Center, Deutsche Forschungsgemeinschaft (DFG) Research Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany

    Alexander Emami-Nemini & Martin J Lohse

  3. Cisbio Bioassays, Parc Technologique Marcel Boiteux, Codolet, France

    Thomas Roux, Marion Leblay, Emmanuel Bourrier, Laurent Lamarque & Eric Trinquet

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  1. Alexander Emami-Nemini
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Contributions

A.E.-N., T.R. and M.L. performed the experiments and analyzed the data; L.L. and E.B. labeled PTHs; A.E.-N., E.T. and M.J.L. wrote the paper.

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Correspondence to Martin J Lohse.

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E.B., M.L., L.L., T.R. and E.T. are employees of Cisbio Bioassays.

Supplementary information

Supplementary Figure 1

cAMP accumulation by [C13-Tb3+]PTH'(1-34) and dissociation of [C35-d2]PTH'(1-34) from SNAP-PTHR. (a) cAMP accumulation by [C13-Tb3+]PTH'(1-34). [C13-Tb3+]PTH'(1-34) was tested in a cell-based cAMP radioimmuno assay. PTHR expressing HEK 293 ad cells generated similar cAMP amounts upon stimulation with 100 pM [C13-Tb3+]PTH'(1-34) or PTH'(1-34). Error bars represent SEM, n=3. (b) Dissociation of [C35-d2]PTH'(1-34) from SNAP-PTHR. SNAP-PTHR expressing HEK 293 ad cells were grown on 96-well plates, labeled with benzylguanine-Tb3+-cryptate and incubated with 5 nM [C35-d2]PTH'(1-34) for 4 h at 20 °C. Cells were washed three times with 100 μl Tag-lite medium within 3 min and 100 μl Tag-Lite labeling medium (assay buffer) or 100 μl assay buffer containing 1 μM PTH'(1-34) was added. Cells were incubated at 20 °C and TR-FRET ratios were measured at indicated time intervals using the Envision reader at 5 point scan mode. A representative experiment is shown. Error bars represent SEM of quadruplicates. (PDF 900 kb)

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Emami-Nemini, A., Roux, T., Leblay, M. et al. Time-resolved fluorescence ligand binding for G protein–coupled receptors. Nat Protoc 8, 1307–1320 (2013). https://doi.org/10.1038/nprot.2013.073

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