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

Fisher, Gregory W.; Fuhrman, Margaret H.; Adler, Sally A.; Szent-Györgyi, Christopher; Waggoner, Alan S.; Jarvik, Jonathan W.

doi:10.1177/1087057114534299

Cited by 26 publications

(36 citation statements)

References 9 publications

(16 reference statements)

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“…The molecules can also serve as a control for the newly available, two-fluorogen self-checking assays to monitor receptor trafficking. 26 In addition, the new probe could be used to make more precise measurements of the fraction of fluorescent signal that is due to background fluorescence. This more accurate determination of the net prestimulation and poststimulation signal should lead to higher Z′ factors in screening, thus permitting the identification of weak (but genuine) agonists and antagonists that would otherwise be missed.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…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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“…The noncovalent dye‐binding nature of FAP technology is a feature that can allow dye exchanges, as shown in the AM2‐2 TO1‐2p and TO1‐2p‐Cy5 paradigm and could potentially permit bound dye washouts . The current developed dL5** MG dyes have subnanomolar K d s, taking hours for dye dissociation.…”

Section: Fluorogen Activating Protein Platformmentioning

confidence: 99%

“…The second dye only labels receptors that are remaining at the plasma membrane, and internalized proteins are protected from displacement. Because all dyes are cell‐excluded, this labeling approach is selective for surface trafficking and does not label proteins contained within biosynthetic compartments . C, Drug is added to cells followed by cell‐impermeable dye for a surface FAP‐POI measurement to quantify cell surface POI trafficking changes.…”

Section: Cell Surface Protein Trafficking Measurements: Receptorsmentioning

confidence: 99%

Fluorogen activating protein toolset for protein trafficking measurements

Perkins

Bruchez

2020

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Assessing protein trafficking in live cells is a core strength of fluorogen activating protein technology. The rapid and specific labeling of a tagged protein of interest, intracellular or surface, with a variety of fluorogenic dye derivatives creates a toolset suitable for scalable, dynamic multi‐color fluorescence experiments and direct quantitative trafficking‐related measurements in single cells and cell populations, using fluorometry, flow cytometry and microscopy.

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

Cited by 26 publications

References 9 publications

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

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

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

Fluorogen activating protein toolset for protein trafficking measurements

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