AM2.2-β2AR/MG13-CCR5 dual transfected cells were characterized by flow cytometry and confocal microscopy analyses. The binding between fluorogens and their associated FAPs and the biological function of the receptors were tested individually. The interaction between the cell membrane impermeable fluorogen TO1-2p and AM2.2 has been characterized elsewhere (9
). TO1-2p binds to cell surface expressing FAP AM2.2 with ~2nM affinity and a t1/2
of less than 5 secs, where t1/2
represents the time for 50% occupancy of the cell surface fluorogen binding sites. Both kinetic and equilibrium binding between cell membrane impermeable fluorogen MG-2p and FAP MG13 ar plotted in . As illustrated, the binding between MG-2p and its FAP MG13 is also highly specific with essentially no non-specific binding. At room temperature (25°C), the addition of 100 nM MG-2p resulted in an immediate fluorescent signal increase in MG13-CCR5 transfected cells with t1/2
~100 s (line a in ). Signals from wild type U937 cells remain unchanged (line b in ). The equilibrium binding curve plotted in revealed that the EC50
between MG-2p and surface displayed MG13 is ~150 nM (16
). In combination with the TO1-2p/AM2.2 binding results reported earlier, our data suggest that the fluorogen-FAP reporter system is robust with a fast on-rate, adequately high affinity, and low background fluorescence.
Figure 1 Flow cytometric characterization of MG-2p and surface displayed MG13-CCR5 in AM2.2-β2AR/MG13-CCR5 dual transfected cells. (A) Kinetic binding of MG-2p to cell surface MG-13. The median channel fluorescence (MCF) in FL4 channel was plotted over (more ...)
In order to validate the biological function of the FAP-receptor fusion protein, we designed a set of high-throughput flow cytometry compatible experiments and validated the results with confocal microscopy.
Dual transfected cells were treated with nine different concentrations of β2AR agonist isoproterenol (ISO) (17
) or CCR5 agonist RANTES (18
) alone as controls, or with a mixture of the two drugs. shows the average fluorescent signal from both FL1 (green filled circles, left Y axis, represents the signal from β2AR) and FL4 (red filled squares, right Y axis, represents the signal from CCR5) channels from the labeled wells. The first quartile of the plate (wells A1 to A12) contains cells were treated with increasing concentration of RANTES (128 pM to 38 nM); the 2nd
quartile (C1 to C12) contains cells treated with 10 nM to 30 μM ISO; cells in the 3rd
quartile were treated with increasing concentrations of both RANTES and ISO; cells in the last quartile were simultaneously treated with increasing concentrations of ISO and decreasing concentrations of RANTES. The experiment was done in triplicate. For clarity, only one representative set of data from each quartile is plotted in the graph, and the average for all four sets of experiments is presented in . The fluorescent signal from both receptors decreased in a dose-dependent manner when exposed to the receptor-specific ligand, and no cross-talk was observed between these two GPCRs. The measured EC50
was very similar regardless of the set up of these experiments, and yielded 0.45 ± 0.15 μM for ISO induced β2AR internalization and 11 ± 2 nM for RANTES induced CCR5 internalization. The amount of off-target drugs does not appear to affect the behavior of the other receptor. These values are in close agreement with reported affinities for ISO-β2AR binding (17
) or RANTES-CCR5 binding (high and low affinity 0.022 and 18 nM (19
Figure 2 Functional characterization of AM2.2-β2AR/MG13-CCR5 dual transfected cells. (A) Mean channel fluorescent signal from selected wells of a 96 well plate. The FL1 signal from AM2.2-β2AR is plotted on the left Y axis and the FL4 signal from (more ...)
To further confirm the action of the receptors we performed confocal microscopy to monitor receptor trafficking in live cells. As shown in , both β2AR (green) and CCR5 (red) are visualized on the plasma membrane of untreated cells; β2AR was internalized after 40 min treatment with 10 μM ISO () while CCR5 remain on the surface; when cells were treated with 100 nM RANTES, CCR5 was detected inside the cells and β2AR remained on the cell surface (). However, both receptors were observed in the cytoplasmic compartment of the cells when treated with a mixture of the two drugs (). It is worthwhile noting that it required 60 min incubation with 10 μM ISO for ~90% of surface β2AR to internalize (9
), whereas 30 min incubation is sufficient to induce maximal internalization of CCR5 in the presence of RANTES. This observation is consistent with the rapid binding of RANTES to CCR5 and the fast internalization rate of CCR5 reported in literature (20
). Together, these confirm that the MG13 tag does not interfere with the normal function of CCR5.
The function of cell surface expressed FAP-β2AR has been previously characterized (9
). We further investigated the function of MG13-CCR5 by comparing receptor internalization induced by 3 different human chemokines: CCL3 (MIP-1α), CCL4 (MIP-1β) in addition to RANTES (CCL5), dose-dependent receptor internalization data is presented in . As illustrated, the three human chemokines induced murine CCR5 internalization with EC50
between 7 nM for RANTES to 70 nM for MIP-1α. The reported affinity of human chemokines binding to human CCR5 is between 0.2 to 10 nM, with MIP-1α < MIP-1β < RANTES (20
). However, Mack et al
reported that the affinity between human chemokines and murine CCR5 is an order of magnitude lower measured by a CCR5 down-regulation assay in murine natural killer cells (22
). These results are in overall agreement with our observations, indicating that the transfected murine FAP-CCR5 functions normally compared to its wild-type counterpart.
Figure 3 Comparison of dose-response curves of murine CCR5 internalization induced by human chemokines CCL3 (blue diamonds), CCL4 (purple triangles), and CCL5 (red open circles). The measured EC50 values are approximately 70 nM (CCL3), 20 nM (CCL4), and 7 nM (CCL5). (more ...)
The feasibility of high-throughput flow cytometry with the dual transfected cells was validated by screening against the Prestwick Chemical Library (PCL) that contains ~1,200 off-patent drugs, including 10 active β2AR agonists but no chemokine agonists. Fourteen molecules, including all 10 β2AR agonists, induced β2AR internalization in a dose-response manner. The measured EC50
of these molecules inducing β2AR internalization in dual transfected cells are in close agreement with that obtained from U937 cells transfected with β2AR alone (9
). On the other hand, none of the PCL compounds appeared to induce CCR5 internalization. These results were not surprising because the PCL does not contain any chemokine-like molecules.
The characterized cell line was then used in a high-throughput flow cytometry based approach to validate the activity of ~1,100 molecules previously identified from a 340,000 compound library as reported in PubChem (PubChem AID: 588765). These molecules were cherry picked because they were “active” in an HTS assay for β2AR agonists (PubChem AIDs: 504448 (project summary) and 504454 (primary screen)). Since there is no ligand shared between adrenoceptor β2AR and chemokine receptor CCR5 reported in literature, a compound that simultaneously reduces the fluorescence signal from both β2AR and CCR5 is most likely to be a false positive hit.
shows the analyzed results from a complete 384 well plate. Signal from FL1 channel (β2AR) is plotted on the left Y axis, and signal from FL8 channel (CCR5) is plotted on the right Y axis. Each dot in the graph represents the average channel fluorescence of cells from a single well, where green half circles and red half diamonds represent the FL1 signal from AM2.2-β2AR and FL4 signal from MG13-CCR5, respectively. Open dark green and dark red circles represent FL1 and FL4 signal from our negative control (NCntrl) wells when cells only received DMSO. Open diamonds represent FL1 and FL4 signal from positive control (PCntrl) wells where cells were treated with 10 μM ISO and 13 nM RANTES. In this high-throughput assay, we evaluated the activity of each compound by calculating its response value (RV):
Where MCF represents mean channel fluorescence. RV indicates the level of fluorescent signal depletion, and an RV of 0 indicates that the compound behaves like our negative control DMSO which has no affect on the surface receptor. An RV of 100 indicates that the compound reduced cell surface fluorescence to the same level as our positive controls, ISO and RANTES. In the approach described, we defined an active compound for β2AR with an RV of > 42, and an active compound for CCR5 when the RV is > 50. Red dotted lines in the figure show RVs of 0, 50 and 100 for the CCR5 signal.
Figure 4 Analyzed screening data from a complete 384 well plate collected with CyanADP flow cytometer connected to Hypercyt™ autosampler. The signal from AM2.2-β2AR is plotted on the left Y axis, and the signal from MG13-CCR5 is plotted on the (more ...)
As illustrated in the figure, many compounds appeared to significantly reduce the fluorescent signal from β2AR, but only a handful of the compounds had a significant impact on the CCR5 signal. The three wells that have a RV of 50 or higher for the CCR5 signal all show reduced fluorescent signal from β2AR (marked with red ellipses). From all 1,100 compounds screened by this assay, only 9 of them simultaneously reduced the fluorescent signal from β2AR and CCR5. Due to the low possibility of ligand cross talk between β2AR and CCR5, all 9 compounds were considered false positives and removed from our list of potential β2AR agonists. It is noteworthy that there is observed chemical similarity in this small group of compounds that affected both receptors, such as two of the compounds identified in the plate shown in , marked with red circles (see inserts in ). The only difference between these two compounds is an ortho −Cl−
vs a para-CH3 endgroup. This not only demonstrates the successful application of the two color/dual receptor/single cell system in a high-throughput flow cytometry format, but again validates the biological relevance of the assay.
These experiments indicate that the two FAP-tagged receptors functioned in a remarkably similar fashion to their natural counterparts. The system can be used to quantitatively measure the amount of both β2AR and CCR5 ligands, and serve as a model system for FAP-based biosensors. The concept can easily be adapted to other receptor pairs of interest such as GPCR/epidermal growth factor receptor pair with crosstalk activity, the virus entry receptor/coreceptor pair CD4/CCR5, receptors that form hetero-dimers such as human serotonin 5-HT2A
receptor and dopamine D2
R receptor, and the chemokine receptor complexes CCR5/CXCR4, CXCR5/CXCR4, CXCR3/CXCR7 etc (4
). The system could also be adapted to track the behavior of a receptor pair due to drug synergy effects to understand the mechanism of action.
In conclusion, after the successful development of a FAP-based high-throughput flow cytometry platform with a single receptor, we extended our analysis to a two color/dual receptor single cell system. The system has been validated by confocal microscopy imaging and flow cytometry. Both fluorogens bound to a specific FAP with no detectable cross-reaction. The FAP tag that fused to the N-terminus of the receptors does not interfere with the normal function of the receptor, and the EC50
values of ligand induced receptor internalization measured by the system are consistent regardless of the amount of non-associated ligand in solution. We adapted the dual transfected cell system in a high-throughput flow cytometry assay to eliminate compounds that reduced the fluorescent signal from both β2AR and CCR5, and to verify compounds that have an impact on the β2AR signal, only. Out of the ~1,200 compounds screened, 9 compounds significantly decreased the fluorescent signal from both receptors, including 2 that share similar chemical structure. These results suggested the two color/dual receptor/single cell system reported herein is biologically pertinent, sensitive and high-throughput ready. In addition to the obvious advantage of increasing the throughput of drug screens, this system opens the door for simultaneously monitoring the trafficking and interaction of multiple receptors in the same cell. In addition to the TO1-2p-AM2.2 and MG-2p-MG13 fluorogen-FAP binding pairs discussed here, other fluorogen-FAP binding pairs with different excitation/emission profiles are also under investigation, such as fluorogen dimethylindole red and its specific FAP named K7 (23
). The unique feature of low fluorescent background of fluorogens and FAP alone and high yield after binding also increase the possibility of studying the behavior of low expression receptors.