High‐throughput flow cytometry compatible biosensor based on fluorogen activating protein technology

Abstract

Monitoring the trafficking of multiple proteins simultaneously in live cells is of great interest because many receptor proteins are found to function together with others in the same cell. However, existing fluorescent labeling techniques have restricted the mechanistic study of functional receptor pairs. We have expanded a hybrid system combining fluorogen activating protein (FAP) technology and high-throughput flow cytometry to a new type of biosensor that is robust, sensitive, and versatile. This provides the opportunity to study multiple trafficking proteins in the same cell. Human beta2 adrenergic receptor (β2AR) fused with FAP AM2.2 and murine C-C chemokines receptor type 5 fused with FAP MG13 was chosen for our model system. The function of the receptor and the binding between MG13 and fluorogen MG-2p have been characterized by flow cytometry and confocal microscopy assays. The binding of fluorogen and the FAP pair is highly specific, while both FAP-tagged fusion proteins function similarly to their wild type counterparts. The system has successfully served as a counter screen assay to eliminate false positive compounds identified in a screen against NIH Molecular Libraries Small Molecule Repository targeting regulators of the human β2AR.

Induction of HIV-Blocking Anti-CCR5 IgA in Peyers's Patches without Histopathological Alterations

“…Antisera to ECL1 N, ECL2 N, and Nter1 N also induced CCR5 receptor downregulation over short and long incubation times (150 min and 48 h), as measured using FAP technology (23,24). All sera were tested at a 1:100 dilution.…”

“…CCR5 internalization was assayed using U937 cells stably expressing mouse CCR5 fused at its N terminus to the fluorogen-activating protein (FAP) HL4-MG and expressing a control receptor, B2AR, fused at its N terminus to a different fluorogen-activating protein, HL1.0.1-T01, as described previously (23,24). Cells were grown at 37°C, 5% CO 2 in RPMI 1640 plus 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin-streptomycin.…”

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

“…The fluorogen activating protein system has demonstrated its utility as a protein reporter system since 2008 [6][7][8][9][10][11]20 by taking advantage of the significant fluorescent quantum yield increase upon FAP-fluorogen binding to track receptor trafficking in real time. 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.…”

“…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.…”

Dark dyes–bright complexes: fluorogenic protein labeling

“…Using dye addition and flow cytometric analysis, labeling is complete within <30 sec, and results are in quantitative agreement with surface-selective antibody labeling approaches for measurement of G-protein coupled receptor (GPCR) desensitization. This approach substantially reduces the variability seen with antibody labeling approaches, and potentially opens the door to very high-throughput analysis of cell surface abundance of proteins in high throughput flow cytometry[32–34]. …”