Discovery of Regulators of Receptor Internalization with High-Throughput Flow Cytometry

Wu, Yang; Tapia, Phillip H.; Fisher, Gregory W.; Simons, Peter C.; Strouse, J. Jacob; Foutz, Terry D.; Waggoner, Alan S.; Jarvik, Jonathan W.; Sklar, Larry A.

doi:10.1124/mol.112.079897

Cited by 23 publications

(35 citation statements)

References 52 publications

(50 reference statements)

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“…We and colleagues recently described a related singlecolor FAP-based assay for loss of receptor at the cell surface 7 that has been was applied to a number of screens against GPCRs using high-throughput flow cytometry, 9,10 including a screen of approximately 350,000 compounds against the human β2 adrenergic receptor. 11 In addition to bona fide agonists, the screen identified a number of compounds that reduce surface signal not by stimulating the receptor but by inhibiting fluorogen activation by the FAP.…”

Section: Resultsmentioning

confidence: 99%

Self-Checking Cell-Based Assays for GPCR Desensitization and Resensitization

et al. 2014

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Section: Resultsmentioning

confidence: 99%

Self-Checking Cell-Based Assays for GPCR Desensitization and Resensitization

et al. 2014

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“…5). Accordingly, a user may perform measurements of real-time signal exchange using different color channels; this has direct application for cellular trafficking studies such as the downregulation and re-sensitization of cell surface receptors (Fisher et al, 2014;Wu et al, 2012Wu et al, , 2014. On the other hand, fluorogen competition using similar affinity fluorogens, results in multi-color co-labeling instead of color-switch.…”

Section: Discussionmentioning

confidence: 99%

Breaking the color barrier – a multi-selective antibody reporter offers innovative strategies of fluorescence detection

Gallo

Jarvik

2017

Journal of Cell Science

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A novel bi-partite fluorescence platform exploits the high affinity and selectivity of antibody scaffolds to capture and activate small-molecule fluorogens. In this report, we investigated the property of multiselectivity activation by a single antibody against diverse cyanine family fluorogens. Our fluorescence screen identified three cellimpermeant fluorogens, each with unique emission spectra (blue, green and red) and nanomolar affinities. Most importantly, as a protein fusion tag to G-protein-coupled receptors, the antibody biosensor retained full activity -displaying bright fluorogen signals with minimal background on live cells. Because fluorogen-activating antibodies interact with their target ligands via non-covalent interactions, we were able to perform advanced multi-color detection strategies on live cells, previously difficult or impossible with conventional reporters. We found that by fine-tuning the concentrations of the different color fluorogen molecules in solution, a user may interchange the fluorescence signal (onset versus offset), execute real-time signal exchange via fluorogen competition, measure multi-channel fluorescence via colabeling, and assess real-time cell surface receptor traffic via pulsechase experiments. Thus, here we inform of an innovative reporter technology based on tri-color signal that allows user-defined fluorescence tuning in live-cell applications.

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“…Fluorogen activating protein (FAP) technology was first introduced in 2008 6 and has been successfully used to monitor receptor trafficking in live cells. [7][8][9][10] FAPs are a new class of biosensors that bind specifically to small-molecule fluorogens. The binding dramatically increases the quantum yield of these fluorogens from ~0 in solution to ~0.5, which corresponds to a fluorescence intensity enhancement of up to 160,000-fold.…”

Section: Introductionmentioning

confidence: 99%

Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen–Fluorogen Activating Protein Binding Pair

Stauffer

Stanfield

et al. 2016

SLAS Discovery

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A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to track receptor trafficking in live cells. Unlike the traditional fluorescent proteins (FPs), FAPs do not fluoresce unless bound to their specific small-molecule fluorogens, and thus FAP-based assays are highly sensitive. Application of the FAP-based assay for protein trafficking in high-throughput flow cytometry resulted in the discovery of a new class of compounds that interferes with the binding between fluorogens and FAP, thus blocking the fluorescence signal. These compounds are high-affinity, nonfluorescent analogs of fluorogens with little or no toxicity to the tested cells and no apparent interference with the normal function of FAP-tagged receptors. The most potent compound among these, N, 4-dimethyl-N-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide (ML342), has been investigated in detail. X-ray crystallographic analysis revealed that ML342 competes with the fluorogen, sulfonated thiazole orange coupled to diethylene glycol diamine (TO1-2p), for the same binding site on a FAP, AM2.2. Kinetic analysis shows that the FAP-fluorogen interaction is more complex than a homogeneous one-site binding process, with multiple conformational states of the fluorogen and/or the FAP, and possible dimerization of the FAP moiety involved in the process.

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Discovery of Regulators of Receptor Internalization with High-Throughput Flow Cytometry

Cited by 23 publications

References 52 publications

Self-Checking Cell-Based Assays for GPCR Desensitization and Resensitization

Self-Checking Cell-Based Assays for GPCR Desensitization and Resensitization

Breaking the color barrier – a multi-selective antibody reporter offers innovative strategies of fluorescence detection

Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen–Fluorogen Activating Protein Binding Pair

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