Detection and Quantification of β2AR Internalization in Living Cells Using FAP-Based Biosensor Technology

Fisher, Gregory W.; Adler, Sally A.; Fuhrman, Margaret H.; Waggoner, Alan S.; Bruchez, Marcel P.; Jarvik, Jonathan W.

doi:10.1177/1087057110370892

Cited by 61 publications

(87 citation statements)

References 15 publications

(26 reference statements)

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“…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.…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

Section: Methodsmentioning

confidence: 99%

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

Pastori

Diomede

Venuti

et al. 2014

J Virol

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The chemokine receptor CCR5 is essential for HIV infection and is thus a potential target for vaccine development. However, because CCR5 is a host protein, generation of anti-CCR5 antibodies requires the breaking of immune tolerance and thus carries the risk of autoimmune responses. In this study, performed in mice, we compared 3 different immunogens representing surface domains of murine CCR5, 4 different adjuvants, and 13 different immunization protocols, with the goal of eliciting HIV-blocking activity without inducing autoimmune dysfunction. In all cases the CCR5 sequences were presented as fusions to the Flock House virus (FHV) capsid precursor protein. We found that systemic immunization and mucosal boosting elicited CCR5-specific antibodies and achieved consistent priming in Peyer's patches, where most cells showed a phenotype corresponding to activated B cells and secreted high levels of IgA, representing up to one-third of the total HIV-blocking activity. Histopathological analysis revealed mild to moderate chronic inflammation in some tissues but failed in reporting signs of autoimmune dysfunction associated with immunizations. Antisera against immunogens representing the N terminus and extracellular loops 1 and 2 (Nter1 and ECL1 and ECL2) of CCR5 were generated. All showed specific anti-HIV activity, which was stronger in the anti-ECL1 and -ECL2 sera than in the anti-Nter sera. ECL1 and ECL2 antisera induced nearly complete long-lasting CCR5 downregulation of the receptor, and especially, their IgG-depleted fractions prevented HIV infection in neutralization and transcytosis assays. In conclusion, the ECL1 and ECL2 domains could offer a promising path to achieve significant anti-HIV activity in vivo. IMPORTANCEThe study was the first to adopt a systematic strategy to compare the immunogenicities of all extracellular domains of the CCR5 molecule and to set optimal conditions leading to generation of specific antibodies in the mouse model. There were several relevant findings, which could be translated into human trials. (i) Prime (systemic) and boost (mucosal) immunization is the best protocol to induce anti-self antibodies with the expected properties. (ii) Aluminum is the best adjuvant in mice and thus can be easily used in nonhuman primates (NHP) and humans. (iii) The Flock House virus (FHV) system represents a valid delivery system, as the structure is well known and is not pathogenic for humans, and it is possible to introduce constrained regions able to elicit antibodies that recognize conformational epitopes. (iv) The best CCR5 vaccine candidate should include either extracellular loop 1 or 2 (ECL1 or ECL2), but not N terminus domains.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Pastori

Diomede

Venuti

et al. 2014

J Virol

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“…Table S1) were somewhat higher than have been reported for other cell-based assays, as we observed previously for FAPtagged β2AR. 7 This may be a consequence of the fact that the EC 50 s that we obtain represent half-maximal values for receptor internalization or surface retention, not halfmaximal signaling per se. It is important to note, however, that the rank order of response to the three different agonists reflects the well-established rank order of selectivity for each of the three adrenergic receptors against which they were tested.…”

Section: Resultsmentioning

confidence: 99%

“…All receptors used in this study were tagged at their extracellular N-termini with FAP HL1.0.1-TO1 as previously described 7 and were expressed in U937 cells (a nonadherent human lymphoma line) and HEK 293 cells (an adherent human embryonic kidney cell line). Two membraneimpermeant fluorogens specific to HL1.0.1-TO1 were used (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…7 Here we describe a related new assay system that concomitantly measures receptor loss from the surface and receptor accumulation inside the cell. This system represents an improvement over previous systems for two primary reasons: (1) it is intrinsically self-checking, since in the absence of artifact, receptor loss and receptor accumulation will be reciprocally related, and (2) it provides a straightforward means to measure receptor resensitization as well as desensitization.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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

Tapia

Jarvik

et al. 2014

CP Cytometry

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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.

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Detection and Quantification of β2AR Internalization in Living Cells Using FAP-Based Biosensor Technology

Cited by 61 publications

References 15 publications

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

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

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

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

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