Discovery of regulators of receptor internalization with high-throughput flow cytometry

Mol Pharmacol. 2012 Oct;82(4):645-57. doi: 10.1124/mol.112.079897. Epub 2012 Jul 5.


We developed a platform combining fluorogen-activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform facilitates drug discovery for trafficking receptors such as G protein-coupled receptors and was validated with the β₂-adrenergic receptor (β₂AR) system. When a chemical library containing ∼1200 off-patent drugs was screened against cells expressing FAP-tagged β₂ARs, all 33 known β₂AR-active ligands in the library were successfully identified, together with a number of compounds that might regulate receptor internalization in a nontraditional manner. Results indicated that the platform identified ligands of target proteins regardless of the associated signaling pathway; therefore, this approach presents opportunities to search for biased receptor modulators and is suitable for screening of multiplexed targets for improved efficiency. The results revealed that ligands may be biased with respect to the rate or duration of receptor internalization and that receptor internalization may be independent of activation of the mitogen-activated protein kinase pathway.

Publication types

  • Research Support, N.I.H., Extramural
  • Validation Study

MeSH terms

  • Adrenergic beta-2 Receptor Agonists / pharmacology
  • Adrenergic beta-2 Receptor Antagonists / pharmacology
  • Binding, Competitive
  • Flow Cytometry / methods
  • Green Fluorescent Proteins / genetics
  • High-Throughput Screening Assays / methods*
  • Humans
  • Ligands
  • Protein Transport
  • Receptors, Adrenergic, beta-2 / genetics
  • Receptors, Adrenergic, beta-2 / metabolism*
  • Receptors, G-Protein-Coupled / agonists
  • Receptors, G-Protein-Coupled / antagonists & inhibitors
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Small Molecule Libraries / pharmacology*
  • Time Factors
  • Transfection
  • U937 Cells


  • Adrenergic beta-2 Receptor Agonists
  • Adrenergic beta-2 Receptor Antagonists
  • Ligands
  • Receptors, Adrenergic, beta-2
  • Receptors, G-Protein-Coupled
  • Recombinant Fusion Proteins
  • Small Molecule Libraries
  • Green Fluorescent Proteins