Characterizing the Pharmacology of G Protein-Coupled Receptors in Transfected Cell Lines

  • Kathryn A. Seely
  • Paul L. PratherEmail author
Part of the Neuromethods book series (NM, volume 60)


A remarkable potential exists for current and future development of therapeutic drugs acting at GPCRs. As one of the initial steps in GPCR drug development, in vitro assays are required to characterize the pharmacology of new ligands acting at distinct GPCRs. This is routinely accomplished by first employing cellular models to establish high affinity, selectivity, and efficacy of a test compound for a specific GPCR involved in a disease process of interest. However, several limitations are encountered when native cell lines or isolated tissues expressing low levels of endogenous GCPRs are employed for receptor characterization. To overcome many of these issues, cells are routinely transfected with cDNA of a desired GPCR to create cell lines stably expressing a sufficient receptor density to allow for adequate pharmacological studies. Although several commercial suppliers offer stably transfected cell lines expressing various GPCRs, as well as kits to examine several signal transduction pathways regulated by GPCRs, the cost and specialized equipment required to conduct these essential studies are often out of reach for many laboratories. Therefore, the purpose of this chapter is to provide a brief, simple, economical, and straightforward guide for the production of a cell line stably expressing a GPCR of interest and the methods required to characterize the basic pharmacology of ligands acting at that receptor. Specifically, methods will describe how to stably transfect cells and to conduct receptor binding studies for determination of receptor density and ligand affinity. Finally, methods will be presented to subsequently characterize the functional signaling of the expressed GPCR, from G-protein activation to regulation of two distinct intracellular effectors.

Key words

Transfection Saturation binding Competition binding GTPγS binding cAMP ERK-MAPK Affinity Efficacy Signal transduction 


  1. 1.
    Eglen RM, Reisine T (2009) New insights into GPCR function: implications for HTS. Meth Mol Biol 552:1–13.CrossRefGoogle Scholar
  2. 2.
    Lagerstrom MC, Schioth HB (2008) Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov 7:339–357.PubMedCrossRefGoogle Scholar
  3. 3.
    Howlett AC (1995) Pharmacology of cannabinoid receptors. Ann Rev Pharmacol Toxicol 35:607–634.CrossRefGoogle Scholar
  4. 4.
    Herkenham M, Lynn AB, Little MD et al (1990) Cannabinoid receptor localization in brain. Proc Nat Acad Sci USA 87:1932–1936.PubMedCrossRefGoogle Scholar
  5. 5.
    Galiegue S, Mary S, Marchand J et al (1995) Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. Eur J Biochem 232:54–61.PubMedCrossRefGoogle Scholar
  6. 6.
    Conti S, Costa B, Colleoni M et al (2002) Anti-inflammatory action of endocannabinoid palmitoylethanolamide and the synthetic cannabinoid nabilone in a model of acute inflammation in the rat. Br J Pharmacol 135:181–187.PubMedCrossRefGoogle Scholar
  7. 7.
    Shoemaker JL, Joseph BK, Ruckle MB et al (2005) The endocannabinoid noladin ether acts as a full agonist at human CB2 cannabinoid receptors. J Pharmacol Exp Ther 314:868–875.PubMedCrossRefGoogle Scholar
  8. 8.
    Shoemaker JL, Ruckle MB, Mayeux PR et al (2005) Agonist-directed trafficking of response by endocannabinoids acting at CB2 receptors. J Pharmacol Exp Ther 315:828–838.PubMedCrossRefGoogle Scholar
  9. 9.
    Graham FL, van der Eb AJ (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456–467.PubMedCrossRefGoogle Scholar
  10. 10.
    Taghian DG, Nickoloff JA (1995) Electrotransformation of Chinese hamster ovary cells. Methods Mol Biol 48:115–121.PubMedGoogle Scholar
  11. 11.
    Martin NA, Prather PL (2001) Interaction of co-expressed mu- and delta-opioid receptors in transfected rat pituitary GH3 cells. Mol Pharmacol 59:774–783.PubMedGoogle Scholar
  12. 12.
    Martin NA, Terruso MT, Prather PL (2001) Agonist Activity of the delta-antagonists TIPP and TIPP-psi in cellular models expressing endogenous or transfected delta-opioid receptors. J Pharmacol Exp Ther 298:240–248.PubMedGoogle Scholar
  13. 13.
    Cheng Y, Prusoff W (1973) Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (IC50) of an enzymatic reaction. Biochem Pharmacol 22:3099–3108.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Pharmacology and ToxicologyUniversity of Arkansas for Medical SciencesLittle RockUSA

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