Real‐Time Detection of Protein Trafficking with High‐Throughput Flow Cytometry (HTFC) and Fluorogen‐Activating Protein (FAP) Base Biosensor

Abstract

We combined 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 allows drug discovery for trafficking receptors, such as G protein–coupled receptors, receptor tyrosine kinases, and ion channels, which were previously not suitable for high‐throughput screening by flow cytometry. The system has been validated using the β2‐adrenergic receptor (β2AR) system and extended to other GPCRs. When a chemical library containing ∼1200 off‐patent drugs was screened against cells expressing FAP‐tagged β2AR, all known β2AR active ligands in the library were successfully identified, together with a few compounds that were later confirmed to regulate receptor internalization in a nontraditional manner. The unexpected discovery of new ligands by this approach indicates the potential of using this protocol for GPCR de‐orphanization. In addition, screens of multiplexed targets promise improved efficiency with minor protocol modification. Curr. Protoc. Cytom. 67:9.43.1‐9.43.11. © 2014 by John Wiley & Sons, Inc.

PI3K class II α regulates δ-opioid receptor export from thetrans-Golgi network

“…The PC12 cells expressing PI3K C2A shRNA were transfected with a fusion construct expressing a previously characterized fluorogen-activated peptide (FAP) N-terminally tagged to δR (FAP-δR) and imaged live via fluorescence confocal microscopy. The FAP-δR was chosen for its far-red emission spectra, which results from a malachite green (MG)–based fluorogen ( Szent-Gyorgyi et al , 2008 , 2013 ; Wu et al , 2014 ; Pratt et al , 2015 ). In control cells not expressing the PI3K C2A shRNA, FAP-tagged δR was localized to the cell surface; however, in the cells stably expressing the PI3K C2A shRNA, a prominent Golgi-localized pool of δR was observed ( Figure 5C ).…”

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

“…With appropriate fluorogens and experimental design, the system has been used to successfully identify molecules that regulate receptor internalization pathways in both a canonical and a noncanonical fashion. 7,9,21 However, potential modulators of FAP-fluorogen binding have not been reported. These modulators directly interfere with the binding between fluorogen and the FAP tag rather than regulating trafficking of the receptor fused to the FAP tag ( Fig.…”

A versatile optical tool for studying synaptic GABAA receptor trafficking

“…This paper lays the groundwork for future investigations of GABA A R trafficking with the γ2 pH FAP sensor. Importantly, this technique can be further extended toward pharmacology-focused efforts in high-throughput screenings (Fisher et al, 2014;Snyder et al, 2015), assay development based on flow cytometry (Saunders et al, 2012;Wu et al, 2014) or high-resolution 96-well plate assays (Larsen et al, 2016) and for in vivo protein labeling (Liu et al, 2016;Zhang et al, 2015). In summary, our γ2 pH FAP is the first protein-FAP conjugate characterized in primary neurons, providing a unique tool to monitor multistage GABA A R trafficking in living cells with relevance both for basic science research and applied pharmacology.…”

“…Moreover, high-affinity MG-FAP binding means that a stable fluorescent module that allows for measurements of receptors undergoing internalization and recycling is formed (Pratt et al, 2015;Szent-Gyorgyi et al, 2013;Yan et al, 2015). Measuring drug-induced changes in FAP-tagged receptor trafficking has proven largely successful (Fisher et al, 2010(Fisher et al, , 2014Grover et al, 2012;Pratt et al, 2015;Snyder et al, 2015;Wu et al, 2014) placing this technique at the forefront of pharmacological screening.…”