A homogeneous high throughput nonradioactive method for measurement of functional activity of Gs-coupled receptors in membranes

J Biomol Screen. 2002 Feb;7(1):35-44. doi: 10.1177/108705710200700106.


A method is described for measuring the activity of G(s)-coupled receptors in a nonradioactive homogeneous membrane-based assay. This method has several major advantages over currently used methods for measuring functional activity of G(s)-coupled receptors. The assay is high throughput (>150,000 data points/day using a single reader). Dimethyl sulfoxide tolerance is high ( approximately 10%). Compared to complex cell-based assays, there is limited potential for nonspecific compound action. This resulted in low compound hit rates in robustness screening, where hit rates from a simulated screen were 1.0% (antagonist screen) and 0.1% (agonist screen). No continuous cell culture is required for the assay, reducing cell culture overheads and allowing the screen to run every day. Automation is simple and requires no temperature- or humidity-controlled incubation. No radioactivity is required. The method relies on measurement of cyclic AMP (cAMP) generation by fluorescence polarization assay using commercially available reagents. Membranes (1-2 microg protein per well, containing anti-cAMP antibody) are transferred to 384-well plates containing 1 microl test compound. For antagonist screens, agonist is added 15 min later. After 30 min incubation at room temperature, one further assay reagent (fluorescein-cAMP in a buffer containing detergent) is added. The signal may be read after 1 h and is stable for greater than 12 h. Typical Z' for the assay is approximately 0.5.

MeSH terms

  • Adrenergic beta-Agonists / pharmacology
  • Animals
  • Automation
  • Biotechnology / methods*
  • CHO Cells
  • Cell Membrane / metabolism
  • Colforsin / pharmacology
  • Cricetinae
  • Cyclic AMP / metabolism
  • Dose-Response Relationship, Drug
  • GTP-Binding Proteins / metabolism*
  • Inhibitory Concentration 50
  • Isoproterenol / pharmacology
  • Kinetics
  • Propranolol / pharmacology
  • Temperature
  • Time Factors
  • Vasoconstrictor Agents / pharmacology
  • Vasopressins / pharmacology


  • Adrenergic beta-Agonists
  • Vasoconstrictor Agents
  • Vasopressins
  • Colforsin
  • Propranolol
  • Cyclic AMP
  • GTP-Binding Proteins
  • Isoproterenol