High-Throughput Screening of Interactions Between G ProteinCoupled Receptors and Ligands Using Confocal Optics Microscopy

  • Lenka Zemanová
  • Andreas Schenk
  • Martin J. Valler
  • G. Ulrich  Nienhaus
  • Ralf Heilker
Part of the Methods in Molecular Biology™ book series (MIMB, volume 305)


Interactions of extracellular ligands with proteins in the cellular plasma membrane are the starting point for various intracellular signaling cascades. In the pharmaceutical industry, particular attention has been paid to G protein-coupled receptors (GPCRs), which are involved in various disease processes. In so-called high-throughput screening (HTS) campaigns, large medicinal chemistry compound libraries were searched for bioactive molecules that would either induce or inhibit the activity of a specific disease-relevant GPCR. In the respective drug discovery assays, the test compound typically competes with the physiological ligand for a binding site on the receptor. The transmembrane receptor is prepared in the form of membrane fragments or, as described here, in so-called virus-like particles (VLiPs). As hundreds of thousands of test compounds must be analyzed, there is a strict need for low volume binding assays to save the expensive bioreagents, and to reduce the consumption of the test compounds. In this chapter, we describe the application of confocal optics microscopy to measure GPCR ligand interactions in low microliter assay volumes.

Key Words

Ligand receptor high-throughput screening fluorescence spectroscopy confocal optics miniaturization binding assay 


  1. 1.
    Nathans J. and Hogness D. S. (1983) Isolation, sequence analysis, and intron-exon arrangement of the gene encoding bovine rhodopsin. Cell 34, 807–814.PubMedCrossRefGoogle Scholar
  2. 2.
    Dixon R. A., Kobilka B. K., Strader D. J., Benovic J. L., Dohlman H. G., Frielle T., Bolanowski M. A., Bennett C. D., Rands E., and Diehl R. E. (1986) Cloning of the gene and cDNA for mammalian beta-adrenergic receptor and homology with rhodopsin. Nature 321, 75–79.PubMedCrossRefGoogle Scholar
  3. 3.
    Ji T. H., Grossmann M., and Ji I. (1998) G protein-coupled receptors. I. Diversity of receptor-ligand interactions. J. Biol. Chem. 273, 17,299–17,302.PubMedCrossRefGoogle Scholar
  4. 4.
    Alouani S. (2000) Scintillation proximity binding assay. Methods Mol. Biol. 138, 135–141.PubMedGoogle Scholar
  5. 5.
    Ramm P. (1999) Imaging systems in assay screening. Drug Discov. Today 4, 401–410.PubMedCrossRefGoogle Scholar
  6. 6.
    Sorg G., Schubert H. D., Buttner F. H., and Heilker R. (2002) Automated high throughput screening for serine kinase inhibitors using a LEADseeker scintillation proximity assay in the 1536-well format. J. Biomol. Screen. 7, 11–19.PubMedGoogle Scholar
  7. 7.
    Sorg G., Schubert H. D., Buttner F. H., Valler M. J., and Heilker R. (2002) Comparison of photomultiplier tube and charge coupled device-based scintillation counting. Life Science News 11, 1–3.Google Scholar
  8. 8.
    Jessop R. A. (1998) Imaging proximity assays. Proc. SPIE 3259, 228–233.CrossRefGoogle Scholar
  9. 9.
    Banks P. and Harvey M. (2002) Considerations for using fluorescence polarization in the screening of g protein-coupled receptors. J. Biomol. Screen. 7, 111–117.PubMedCrossRefGoogle Scholar
  10. 10.
    Harris A., Cox S., Burns D., and Norey C. (2003) Miniaturization of fluorescence polarization receptor-binding assays using CyDye-labeled ligands. J. Biomol. Screen. 8, 410–420.PubMedCrossRefGoogle Scholar
  11. 11.
    Auer M., Moore K. J., Meyer-Almes F. J., Guenther R., Pope A. J., and Stoeckli K. (1998) Fluorescence correlation spectroscopy: lead discovery by miniaturized HTS. Drug Discov. Today 3, 457–465.CrossRefGoogle Scholar
  12. 12.
    Zemanova L., Schenk A., Valler M. J., Nienhaus G. U., and Heilker R. (2003) Confocal optics microscopy for biochemical and cellular high-throughput screening. Drug Discov. Today 8, 1085–1093.PubMedCrossRefGoogle Scholar
  13. 13.
    Ehrenberg M. and Rigler R. (1974) Rotational brownian motion and fluorescence intensity fluctuations. Chem. Phys. 4, 390–401.CrossRefGoogle Scholar
  14. 14.
    Magde D., Elson E. L., and Webb W. W. (1972) Thermodynamic fluctuations in a reacting system—measurement by fluorescence correlation spectroscopy. Phys. Rev. Lett. 29, 705–708.CrossRefGoogle Scholar
  15. 15.
    Magde D., Elson E. L., and Webb W. W. (1974) Fluorescence correlation spectroscopy. II. An experimental realization. Biopolymers 13, 29–61.PubMedCrossRefGoogle Scholar
  16. 16.
    Rigler R. (1995) Fluorescence correlations, single molecule detection and large number screening. Applications in biotechnology. J. Biotechnol. 41, 177–186.PubMedCrossRefGoogle Scholar
  17. 17.
    Thompson N. L. (1991) Fluorescence correlation spectroscopy, in Topics in Fluorescence Spectroscopy, Vol. 1 (Lakowicz J. R., ed.), Plenum Press, New York, pp. 337–378.Google Scholar
  18. 18.
    Chen Y., Muller J. D., So P. T., and Gratton E. (1999) The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys. J. 77, 553–567.PubMedCrossRefGoogle Scholar
  19. 19.
    Kask P., Palo K., Ullmann D., and Gall K. (1999) Fluorescence-intensity distribution analysis and its application in biomolecular detection technology. Proc. Natl. Acad. Sci. USA 96, 13,756–13,761.PubMedCrossRefGoogle Scholar
  20. 20.
    Schwille P., Meyer-Almes F. J., and Rigler R. (1997) Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution. Biophys. J. 72, 1878–1886.PubMedCrossRefGoogle Scholar
  21. 21.
    Winkler T., Kettling U., Koltermann A., and Eigen M. (1999) Confocal fluorescence coincidence analysis: an approach to ultra high-throughput screening. Proc. Natl. Acad. Sci. USA 96, 1375–1378.PubMedCrossRefGoogle Scholar
  22. 22.
    Kask P., Palo K., Fay N., Brand L., Mets U., Ullmann D., Jungmann J., Pschorr J., and Gall K. (2000) Two-dimensional fluorescence intensity distribution analysis: theory and applications. Biophys. J. 78, 1703–1713.PubMedCrossRefGoogle Scholar
  23. 23.
    Palo K., Brand L., Eggeling C., Jager S., Kask P., and Gall K. (2002) Fluorescence intensity and lifetime distribution analysis: toward higher accuracy in fluorescence fluctuation spectroscopy. Biophys. J. 83, 605–618.PubMedCrossRefGoogle Scholar
  24. 24.
    Klumpp M., Scheel A., Lopez-Calle E., Busch M., Murray K. J., and Pope A. J. (2001) Ligand binding to transmembrane receptors on intact cells or membrane vesicles measured in a homogeneous 1-microliter assay format. J. Biomol. Screen. 6, 159–170.PubMedCrossRefGoogle Scholar
  25. 25.
    Rudiger M., Haupts U., Moore K. J., and Pope A. J. (2001) Single-molecule detection technologies in miniaturized high throughput screening: binding assays for G protein-coupled receptors using fluorescence intensity distribution analysis and fluorescence anisotropy. J. Biomol. Screen. 6, 29–37.PubMedGoogle Scholar
  26. 26.
    Scheel A. A., Funsch B., Busch M., Gradl G., Pschorr J., and Lohse M. J. (2001) Receptor-ligand interactions studied with homogeneous fluorescence-based assays suitable for miniaturized screening. J. Biomol. Screen. 6, 11–18.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2005

Authors and Affiliations

  • Lenka Zemanová
    • 1
  • Andreas Schenk
    • 2
  • Martin J. Valler
    • 3
  • G. Ulrich  Nienhaus
    • 1
  • Ralf Heilker
    • 3
  1. 1.Department of BiophysicsUniversity of UlmUlmGermany
  2. 2.Tecan Austria GmbHGrödigAustria
  3. 3.Department of Integrated Lead DiscoveryBoehringer Ingelheim Pharma GmbH & Co KGBiberachGermany

Personalised recommendations