Abstract
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.
Footnotes
This research was supported by the National Institutes of Health National Institute of Mental Health [Grants 1U54-MH084690-02, 5U54-MH084690-03, 1R03-MH093192]; and the National Institutes of Health National Center for Research Resources [Grant 5U54-RR022241].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- GPCR
- G protein-coupled receptor
- FAP
- fluorogen-activating protein
- β2AR
- β2-adrenergic receptor
- αAR
- α-adrenergic receptor
- MCF
- median channel fluorescence
- ISO
- isoproterenol
- HCS
- high-content screening
- HTS
- high-throughput screening
- HTFC
- high- throughput flow cytometry
- PCL
- Prestwick Chemical Library
- TO1-2p
- sulfonated thiazole orange coupled to diethylene glycol diamine
- ICI 118,551
- 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol
- RV
- response value
- DHA
- dihydroalprenolol
- GFP
- green fluorescent protein
- DMSO
- dimethylsulfoxide
- HPSM
- HEPES-potassium-sodium-magnesium
- ALP
- alprenolol
- PRO
- propranolol
- LEVON
- levonordefrin
- ANI
- anisomycin
- NAF
- naftopidil
- DOM
- domperidone
- PIZ
- pizotifen
- BUC
- bucindolol
- CAV
- carvedilol
- CYC
- cycloheximide
- AC
- adenylate cyclase
- ERK
- extracellular signal-regulated kinase
- MAPK
- mitogen-activated protein kinase.
- Received May 8, 2012.
- Accepted July 5, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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