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

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 ??-adrenergic receptor (??AR) system. When a chemical library containing ?1200 off-patent drugs was screened against cells expressing FAP-tagged ??ARs, all 33 known ??AR-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.

SUBMITTER: Wu Y

PROVIDER: S-EPMC3463215 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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

Wu Yang Y Tapia Phillip H PH Fisher Gregory W GW Simons Peter C PC Strouse J Jacob JJ Foutz Terry T Waggoner Alan S AS Jarvik Jonathan J Sklar Larry A LA

Molecular pharmacology 20120705 4

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 ...[more]

PMID: 22767611

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Project description:The androgen receptor (AR) is a steroid hormone receptor which regulates transcription of androgen-sensitive genes and is responsible for the development and maintenance of male secondary sexual characteristics. Chemicals that interfere with AR activity may lead to pathological conditions in androgen-sensitive tissues. A variety of reporter systems have been developed, driven by androgen-sensitive promoters, which screen for chemicals that modulate androgenic activity. We have developed a flexible, high-throughput AR transcriptional activation assay, designated the Multifunctional Androgen Receptor Screening (MARS) assay, to facilitate the identification of novel modulators of AR transcriptional activity using flow cytometry. Androgen-independent human prostate cancer-derived PC3 cells were transiently cotransfected with an expression vector for the wild-type human AR and an androgen-sensitive promoter regulating the expression of destabilized enhanced GFP (dsEGFP). The transfected cells were stimulated with established androgenic and antiandrogenic compounds and assessed for increased or decreased dsEGFP expression. To screen for antagonists of AR transcription, the AR agonist R1881 was coadministered at submaximal concentrations with potential AR antagonists. The assay was formatted for high-throughput screening using the HyperCyt flow cytometry system. Agents with established androgenic and antiandrogenic activity were used for validation of the MARS assay. AR agonists were found to potently induce dsEGFP. Furthermore, AR agonists induced dsEGFP expression in a dose-dependent manner. Alternatively, AR antagonists blocked dsEGFP expression when coadministered with low-dose R1881, which also occurred in a dose-dependent manner. Modulators of AR transcriptional activity can be successfully identified by the MARS assay, utilizing a rapid, flexible, sensitive, and high-throughput format. Dose-response curves can be successfully generated for these compounds, allowing for an assessment of potency. Because of its simplicity and high-throughput compatibility, the MARS assay and HyperCyt system combined with flow cytometric analysis represents a valuable and novel addition to the current repertoire of AR transcriptional activation screening assays.

Dataset Information

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

Publications

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

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