Detects compound activity against known and/or orphan GPCR targets in a high content screening environment

The Transfluor® Assay is a cell-based fluorescence assay used to screen for G-protein coupled receptor (GPCR) ligands and other potential drugs that regulate GPCRs by deactivating or desensitizing a common pathway. By attaching a fluorescent label to beta-arrestin, the location of the receptor-arrestin complex may be monitored during receptor activation. Since desensitization only occurs with an activated receptor, monitoring beta-arrestin translocation and subsequent receptor recycling provides a method to detect the activation of any GPCR.

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Studying this mechanism enables researchers to identify the impact of GPCR ligands on a variety of physiological processes including behavior, mood, inflammation, nervous system, and olfactory responses. In addition, researchers can gain insight into the complexities of receptor degradation and recycling for a given target.


  • Validated with over 100 GPCRs, the Transfluor assay provides the assurance to work across all GPCR classes (Class I, II, III), regardless of interacting G-protein (Gs, Gi/o and Gq/11).
  • Single read-out is compatible with all GPCR subtypes, including Gi, Gs, Go, Gq, eliminating the need for multiple GPCR assays
  • No GPCR labeling or tagging avoids custom molecular biology that may alter your target’s response.
  • Universal format requires no prior knowledge of interacting G-protein, enabling orphan GPCR screening.
  • GFP-based technology does not require additional substrates to monitor beta-arrestin translocation.
  • MetaXpress® Application Module automates analysis of beta-arrestin movement to the cell membrane (pits) and endocytic vesicles.
  • Ligand Independent Translocation (LITe™) Assay is an agonist-independent assay used to verify the translocation of beta-arrestin-GFP in orphan GPCRs.
With the Transfluor technology, arrestin-GFP is dispersed throughout the cytosol in unstimulated cells. Upon ligand binding with the GPCR, the arrestin-GFP quickly translocates from the cytosol to the cell membrane and then into clathrin-coated pits at the membrane. The receptor then internalizes into endocytic vesicles prior to reprocessing and returns to the cell membrane. Pits and vesicles can be imaged and quantified by commercially-available image analysis instrumentation, such as ImageXpress® Micro or ImageXpress® Ultra System, forming the basis for a universal, high-throughput, high-content assay for all GPCRs (known and orphan).

Orphan GPCR Assay

In contrast to current methods of screening GPCRs, the Transfluor technology is based on the mechanism for termination of GPCR signaling, known as receptor desensitization. This mechanism is shared by virtually all GPCRs and is activated by ligand binding. Transfluor technology requires no prior knowledge of the interacting G-protein. This important feature of the Transfluor technology makes it ideal for screening orphan GPCRs (oGPCR).

A proprietary technique to assist in validating orphan GPCR screens, called LITe™ (Ligand Independent Translocation), is an agonist-independent assay used to verify the translocation of ß arrestin-GFP in orphan GPCRs.

Transfluor References

Garippa RJ, Hoffman AF, Gradl G, Kirsch A.
High-throughput confocal microscopy for beta-arrestin-green fluorescent protein translocation G protein-coupled receptor assays using the Evotec Opera.
Methods Enzymol. 2006;414:99-120.

Hudson CC, Oakley RH, Sjaastad MD, Loomis CR.
High-content screening of known G protein-coupled receptors by arrestin translocation.
Methods Enzymol. 2006;414:63-78.

Oakley RH, Hudson CC, Sjaastad MD, Loomis CR.
The ligand-independent translocation assay: an enabling technology for screening orphan G protein-coupled receptors by arrestin recruitment.
Methods Enzymol. 2006;414:50-63.

Haasen D, Wolff M, Valler MJ, Heilker R.
Comparison of G-protein coupled receptor desensitization-related beta-arrestin redistribution using confocal and non-confocal imaging.
Comb Chem High Throughput Screen. 2006 Jan;9(1):37-47.

Ghosh RN, DeBiasio R, Hudson CC, Ramer ER, Cowan CL, Oakley RH.
Quantitative cell-based high-content screening for vasopressin receptor agonists using transfluor technology.
J Biomol Screen. 2005 Aug;10(5):476-84.

Ozawa K, Hudson CC, Wille KR, Karaki S, Oakley RH.
Development and validation of algorithms for measuring G-protein coupled receptor activation in cells using the LSC-based imaging cytometer platform.
Cytometry A. 2005 May;65(1):69-76.

Oakley RH, Hudson CC, Cruickshank RD, Meyers DM, Payne RE Jr, Rhem SM, Loomis CR.
The cellular distribution of fluorescently labeled arrestins provides a robust, sensitive, and universal assay for screening G protein-coupled receptors.
Assay Drug Dev Technol. 2002 Nov;1(1 Pt 1):21-30.

This patented technology, licensed from Duke University, uses cell lines genetically engineered to express both the labeled beta-arrestin and the GPCR of interest and can be used to screen for natural or synthetic ligands, including agonists or antagonists. The Transfluor Assay requires no prior knowledge of the interacting G-protein, making it ideally suited for screening orphan GPCRs (oGPCR).

Figure 1.ß2AR-expressing cells were stimulated with isoproterenol. Left: control, center: pits, right: vesicles.
Top: Transfluor assay imaged with ImageXpress. Bottom: Transfluor assay imaged with Discovery-1.

Figure 2.Segmentation and analysis run with the GPCR Cycling Application Module for MetaXpress.
Dose response to isoproterenol. Top: pits, bottom: vesicles. Error bars show standard error.

Partial GPCR Listing

The Transfluor technology monitors receptor activity by detecting movement of beta-arrestin-GFP in the cell.
A partial listing of GPCRs that have been shown to translocate beta-arrestin-GFP is shown below.
Gs Gi/o Gq/11
  • A2a adenosine
  • A2b adenosine
  • ß1-adrenergic
  • ß2-adrenergic
  • CRF1 corticotropin releasing factor
  • D1 dopamine
  • D5 dopamine
  • FSH follicle-stimulating hormone
  • Glucagon
  • LH luteinizing hormone
  • PTH1 parathyroid hormone
  • E2 prostaglandin
  • E4 prostaglandin
  • Secretin
  • VIP1 vasoactive intestinal peptide
  • V2 vasopressin
  • alpha 2a-adrenergic
  • alpha 2b-adrenergic
  • alpha 2c-adrenergic
  • A1 adenosine
  • A3 adenosine
  • Apelin
  • C5a anaphylatoxin
  • CCR5 chemokinee
  • CXCR1 chemokine
  • CXCR2 chemokine
  • CXCR4 chemokine
  • D2 dopamine
  • D3 dopamine
  • D4 dopamine
  • Edg1 endothelial diff. gene
  • Edg2 endothelial diff. gene
  • Edg3 endothelial diff. gene
  • Edg5 endothelial diff. gene
  • 5HT1A hydroxytryptamine
  • delta-opioid
  • mu-opioid
  • MCH1 melanin conc. hormone
  • M2Ach muscarinic acetylcholine
  • E3 prostaglandin
  • N-formyl peptide
  • Neuropeptide FF
  • Somatostatin
  • alpha 1b-adrenergic
  • AT1A angiotensin II
  • CCK-A cholecystokinin
  • CCK-B cholecystokinin
  • Cytomegalovirus US28
  • ETA endothelin
  • GnRH (type2) gonadotropin releasing hormone
  • 5HT2A hydroxytryptamine
  • 5HT2C hydroxytryptamine
  • m1ACh muscarinic acetylcholine
  • mGluR1 metabotropic glutamate
  • NK1 neurokinin
  • NK3 neurokinin
  • NT1 neurotensin
  • Orexin-1
  • Oxytocin
  • PAR2 proteinase-activated
  • Platelet-activating factor
  • TRHR-1 thyrotropin releasing hormone
  • TRHR-2 thyrotropin releasing hormone
Other Receptor Listing
  • 12 Drosophila GPCRs
  • Fz4 frizzled receptor
  • TßRIII transforming growth factor-ß