Design and Use of Fluorescent Ligands to Study Ligand–Receptor Interactions in Single Living Cells

  • Stephen J. BriddonEmail author
  • Barrie Kellam
  • Stephen J. Hill
Part of the Methods in Molecular Biology book series (MIMB, volume 746)


The interaction of ligands with G protein-coupled receptors (GPCRs) has been traditionally studied using radiolabelled variants of receptor ligands. More recently, increased knowledge about the way in which GPCRs exist in a highly organised membrane environment has led to an interest in investigating receptor–ligand interactions in single cells. In addition, substantial improvements in imaging technology and an increase in the expense of radioactive waste disposal have resulted in an expansion in the use of fluorescent technologies. One major requirement for these methods is a suitable fluorescent ligand for the receptor of interest. The design of fluorescent ligands for GPCRs is complex, and has to take into account their pharmacological, photophysical, and also physicochemical properties. Here, we describe some basic considerations in the design of fluorescent GPCR ligands, including choice of pharmacological template, linker, and fluorophore. In addition, we describe basic protocols for determining the photophysical properties of the ligand and determining the cellular localisation of their interaction with the target receptors. Finally, we provide a basic protocol for using fluorescent GPCR ligands to quantify the number and diffusion of receptor–ligand complexes in small areas of the cell membrane.

Key words

G protein-coupled receptors Fluorescent ligand Fluorophore Confocal microscopy Fluorescence correlation spectroscopy Single cell Ligand binding 


  1. 1.
    Middleton, R.J., and Kellam B. (2005) Fluorophore-tagged GPCR ligands. Curr. Op. Chem. Biol. 9, 517–525.CrossRefGoogle Scholar
  2. 2.
    Daly, C.J. and McGrath, J.C. (2003) Fluorescent ligands, antibodies, and proteins for the study of receptors. Pharmacol. Ther. 100, 101–118.PubMedCrossRefGoogle Scholar
  3. 3.
    Leopoldo, M., Lacivita, E., Berardi, F., and Perrone, R. (2009) Developments in fluorescent probes for receptor research. Drug. Discov. Today 14, 706–712.PubMedCrossRefGoogle Scholar
  4. 4.
    Ostrom, R.S, and Insel, P.A. (2004) The evolving role of lipid rafts and caveolae in G protein-coupled receptor signalling: implication for molecular pharmacology. Br. J. Pharmacol. 143, 235–245.PubMedCrossRefGoogle Scholar
  5. 5.
    Insel, P.A., Head, B.P., Ostrom, R.S., Patel, H.H., Swaney, J.S., Tang, C.M., Roth, D.M. (2005) Caveolae and lipid rafts: G-protein-coupled receptor signalling microdomains in cardiac myocytes. Ann. N.Y. Acad. Sci. 1047, 166–172.CrossRefGoogle Scholar
  6. 6.
    Hern, J.A., Baig, A.H., Mashanov, G.I., Birdsall, B., Corrie, J.E., Lazareno, S., Molloy, J.E., and Birdsall, N.J. (2010) Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules. Proc. Natl. Acad. Sci. USA 107, 2693–2698.PubMedCrossRefGoogle Scholar
  7. 7.
    Miquel, M.R., Segura, V., Ali, Z., D’Ocon, M.P., McGrath, J.C., and Daly, C.J. (2005) 3-D image analysis of fluorescent drug binding. Mol. Imaging 4, 40–52.PubMedGoogle Scholar
  8. 8.
    Briddon, S.J., Cordeaux, Y., Middleton, R.J., Weinstein, J, George, M.W., Kellam, B., and Hill, S.J. (2004) Quantitative analysis of the formation and diffusion of A(1)-adenosine receptor-antagonist complexes in single living cells Proc. Natl. Acad. Sci. USA 101, 4673–4678.CrossRefGoogle Scholar
  9. 9.
    Daly, C.J., Ross, R., Whyte, J., Henstridge, C., Irving, A., and McGrath, J. (2010) Fluorescent ligand binding reveals heterogeneous distribution of adrenoceptors and ‘cannabinoid-like’ receptors in small arteries. Br. J. Pharmacol. In Press, DOI: 10.1111/j.1476-5381.2009.00608.xGoogle Scholar
  10. 10.
    Jones, J.W., Greene, T.A., Grygon, C.A., Doranz, B.J., and Brown, M.P. (2008) Cell-free assay of G-protein-coupled receptors using fluorescence polarization. J. Biomol. Screen 13, 424–429.PubMedCrossRefGoogle Scholar
  11. 11.
    Harikumar, K.G., Pinon, D.L. and Miller, L.J. (2006) Fluorescence characteristics of hydrophobic partial agonist probes of the cholecystokinin receptor. Biosci. Rep. 26, 89–100.PubMedCrossRefGoogle Scholar
  12. 12.
    Harikumar, K.G., Hosohata, K., Pinon, D.I., and Miller, L.J. (2006) Use of probes with fluorescent indicator distributed throughout the pharmacophore to examine the peptide agonist-binding environment of the family B G protein-coupled secretin receptor. J. Biol. Chem. 281, 2543–2550.PubMedCrossRefGoogle Scholar
  13. 13.
    Ilien, B., Franchet, C., Bernard, P., Morisset, S., Well, C.O., Bourguignon, J.J., Hibert, M., and Galzi, J.L. (2003) Fluorescence resonance energy transfer to probe human M1 muscarinic receptor structure and drug binding properties. J. Neurochem. 85, 768–778.PubMedCrossRefGoogle Scholar
  14. 14.
    Tahtaoui, C., Guillier, F., Klotz, P., Galzi, J.L., Hibert, M. and Ilien, B (2005) On the use of nonfluorescent dye labelled ligands in FRET-based receptor binding studies. J. Med. Chem. 48, 7847–7859.PubMedCrossRefGoogle Scholar
  15. 15.
    Middleton, R.J., Briddon, S.J., Cordeaux, Y., Yates, A.S., Dale, C.L., George, M.W., Baker, J.G., Hill, S.J., and Kellam, B. (2007) New fluorescent adenosine A1-receptor agonists which allow quantification of ligand-receptor interactions in microdomains of single living cells. J. Med. Chem. 50, 782–793.PubMedCrossRefGoogle Scholar
  16. 16.
    Briddon, S.J. and Hill, S.J. (2007) Pharmacology under the microscope: the use of fluorescence correlation spectroscopy to determine the properties of ligand receptor complexes. Trends Pharmacol. Sci. 28, 637–645.PubMedCrossRefGoogle Scholar
  17. 17.
    Cordeaux Y, Briddon SJ, Alexander SPH, Kellam B & Hill SJ (2008) Agonist-occupied A3 adenosine receptors exist within heterogeneous complexes in membrane microdomains of individual living cells. FASEB J. 22, 850–860.PubMedCrossRefGoogle Scholar
  18. 18.
    Hegener O, Prenner L, Runkel F, Baader SL, Kappler J & Haberlein H (2004) Dynamics of beta2-adrenergic receptor-ligand complexes on living cells. Biochemistry 43, 6190–6199.PubMedCrossRefGoogle Scholar
  19. 19.
    Ziemek, R., Brennauer, A., Schneider, E., Cabrele, C., Beck-Sickinger, A.G., Bernhardt, G., and Buschauer, A. (2006) Fluorescence- and luminescence -based methods for the determination of affinity and activity of neuropeptide Y2 receptor. Eur. J. Pharmacol. 551, 10–18.PubMedCrossRefGoogle Scholar
  20. 20.
    Schwille P (2001) Fluorescence correlation spectroscopy and its potential for intracellular applications Cell Biochem. Biophys. 34, 383–408.Google Scholar
  21. 21.
    Kim S, Heinze K & Schwille P (2007) Fluorescence correlation spectroscopy in living cells. Nat. Methods 4, 963–973.PubMedCrossRefGoogle Scholar
  22. 22.
    Baker, J.G., Middleton, R.J., Adams, L., Briddon, S.J., Kellam, B, and Hill, S.J. (2010) Pharmacological characteristics of different fluorescent ligands at the human adenosine-A1 receptor. Br. J. Pharmacol 159, 772–786.Google Scholar
  23. 23.
    Wohland T, Friedrich K, Hovius R & Vogel H (1999) Study of ligand-receptor interactions by fluorescence correlation spectroscopy with different fluorophores: evidence that the homopentameric 5-hydroxytryptamine type 3As receptor binds only one ligand. Biochemistry 38, 8671–8681.PubMedCrossRefGoogle Scholar
  24. 24.
    Cornelius, P., Lee, E., Lin, W., Wang, R., Werner, W., Brown, J.A., Stuhmeier, F., Boyd, J.G., and McClure, K. (2009) Design, synthesis and pharmacology of fluorescently labeled analogs of serotonin: application to screening of the 5-HT2C receptor. J. Biomol. Screen. 14, 360–370.PubMedCrossRefGoogle Scholar
  25. 25.
    Briddon, S.J., Hern, J.A., and Hill, S.J. (2009) Use of fluorescence correlation spectroscopy to study GPCRs. In G-protein coupled receptors – Essential Methods (Poyner, D. and Wheatley, M., ed.), Wiley-Blackwell, 169–191.Google Scholar
  26. 26.
    Pramanik A & Rigler R (2001) FCS-analysis of ligand-receptor interactions in living cells. In Fluorescence Correlation Spectroscopy: Theory and Applications, (Elson, E. & Rigler, R., ed.) Springer, Heidelberg. 101–129.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Stephen J. Briddon
    • 1
    Email author
  • Barrie Kellam
  • Stephen J. Hill
  1. 1.Institute of Cell Signalling, School of Biomedical SciencesUniversity of NottinghamNottinghamUK

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