The intramolecular biosensors were designed to harbor the tet-racysteine tag positioned at various locations within the intracellular surface of the receptor in addition to a C-terminally fused Renilla luciferase. More precisely, the CCPGCC tag was inserted in two positions within the second intracellular loop, three within the third, and three within the carboxyl terminus domain of the receptor. For ease of cloning, compatible restriction sites were introduced by polymerase chain reaction (PCR) at the 5′ and 3′ ends of the receptor to facilitate its insertion into its corresponding mammalian expression vector. Briefly, HA-tagged hβ₂AR (20) in a pcDNA3.1(−) backbone vector was used as a template and amplified by PCR using the BamHI-β₂AR forward and the β₂AR-EcoRI-BamHI reverse primers. The resulting PCR product was cloned into an accepting vector; pIRESpuro3-signal peptide-HA-RlucII using BamHI. We screened for correct orientation using PstI. The introduction of the CCPGCC motif was accomplished by overlapping PCR where the wild-type receptor was flanked by the appropriate primers (Table 1) in order to introduce the desired TC tag within the coding sequence (11). In the first round, fragment one was generated using NheI-XhoI-forward primer and the appropriate FlAsH internal reverse primer. Fragment 2 was generated using the appropriate FlAsH internal forward primer and β₂AR-EcoRI-BamHI reverse primer. Both fragments were then combined in equal portions and used as templates for the second round of PCR using NheI-XhoI-Kozak-β₂AR forward and β₂AR-EcoRI-BamHI reverse primer. This product was cloned into pIRESpuro3-SP-HA-RlucII backbone using NheI and EcoRI. All constructs were confirmed by bidirectional sequencing (Génome Québec).

HEK 293 SL cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 5% vol/vol fetal bovine serum and 1% w/v penicillin–streptomycin from Wisent. The cells were maintained in a controlled environment, 37°C in a humidified atmosphere at 95% air and 5% CO₂.

HEK 293 SL cells were plated at a density of 2.0 × 10⁵ cells per well in clear 6-well plates (Thermo Scientific, 140675) prior to transfection. On the following day, cells were transfected with 1 µg of each of the eight β₂AR FlAsH biosensors along with pcDNA3.1(−) for a total of 1.5 µg per well using Lipofectamine 2000 (Invit-rogen) following the manufacturer’s instructions. Alternatively, 1 µg of AT1R-ICL3-p3-RlucII or AT1R-C-tail-p1-RlucII and 500 ng of the FP-ICL3-p4-RlucII biosensor was also used.

The day following transfection, cells were detached with 0.25% Trypsin–EDTA (Wisent) and 2.0 × 10⁴ cells were re-plated onto a poly-l-ornithine (Sigma-Aldrich) treated clear bottom black 96-well plate (Thermo Scientific, 165305). The next day, the cells were washed once with phosphate-buffered saline (PBS) and fixed with 2% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature. Successively, the cells were blocked with a 1% bovine serum albumin (Fisher Scientific) PBS solution for 1 h at room temperature to prevent non-specific interactions of the antibodies. Cells were then incubated with a monoclonal mouse anti-HA primary antibody for 1 h (BioLegend, 1:200, previously Covance). Afterward, the cells were washed three times with PBS and an Alexa fluor-488 goat anti-mouse IgG secondary antibody (Life Technologies, 1:1,000) was used to label cells. To confirm the ability of recombinant receptors to localize to the cell surface, the Operetta High Content Imaging system (Perkin Elmer) with a 20× WD objective was used. The excitation filter was set at 475/15 nm and its corresponding emission filter at 525/25 which permitted to capture the signal produced by Alexa fluor-488.

HEK 293 SL cells were transfected with 1 µg of each of the eight β₂AR FlAsH biosensors and the β₂AR-WT-RlucII construct supplemented with 0.5 µg of the previously described H188 EPAC FRET sensor (21) using Lipofectamine 2000 (Invitrogen) follow-ing the manufacturer’s instructions. The day following transfection, cells were detached with 0.25% Trypsin–EDTA (Wisent) and 4.0 × 10⁴ cells were re-plated onto a poly-l-ornithine (Sigma-Aldrich) treated black flat bottom 96-well plate (Costar, 3916). The day of the experiment, cells were washed once in 150 µL of Krebs buffer and the cells then sat in 90 µL of Krebs at 37°C prior to the start of the assay. A Synergy 2 plate reader (Biotek) was used to assay coupling of the β₂AR FlAsH biosensors to Gαs by investigating accumulation of cAMP. The temperature of the instrument was set at 37°C and kinetic measurements were taken. The 420/50 excitation filter was used to excite the donor molecule, mTurquoise2, and light was captured by the emission filters 485/20 (mTurquoise2) and 528/20 (Venus). Basal FRET was measured continuously every 5 s for a total of 20 s. Cells were then treated with either the vehicle (ascorbic acid) or the full agonist, 10 µM isoproterenol (in ascorbic acid) using the injector module. Stimulated FRET readings were then captured every 5 s for a total time of 2 min. FRET ratios were computed by dividing the Venus emission channel by the mTurquoise2 emission channel. ΔFRET ratios were calculated by subtracting the averaged isoproterenol stimulated FRET ratio by the averaged basal FRET ratio, as shown; ΔFRET = (avgFRET_stimulated − avgFRET_basal).

Twenty-four hours post-transfection, cells were detached with 0.25% Trypsin–EDTA (Wisent) and 400 µL of cell suspension was re-plated onto a clear 12-well plate (Costar, 3513). On the day of the experiment, the cells were starved in DMEM without serum supplementation for 5 h. Afterward, cells were stimulated with either vehicle or 10 µM isoproterenol for 5 min at 37°C. The plate was then placed on ice, where the cells were washed once with an ice-cold PBS solution. The cells were lysed in 200 µL of 4× Laemmli buffer (2% SDS, 10% glycerol, 60 mM Tris pH 6.8, 0.02% bromophenol blue, 5% β-mercaptoethanol). In order to shear the genomic DNA, lysates were sonicated three times, each repetition for 5 s at 3 W using a Sonicatior 3000 (Misonix). Lysates were then heated at 65°C for 15 min.

MAP kinase activation was measured by western blot. Correspondingly, 30 µL of cell lysate was loaded and proteins were, respectively, separated by SDS-PAGE and then transferred onto a PVDF membrane via a wet transfer technique. To prevent non-specific binding of the primary antibody, the membrane was blocked in a 5% non-fat milk solution in Tris-buffered saline and 0.0005% Tween20 solution. An anti-phospho-ERK1/2 rabbit primary antibody was used (Cell Signalling Technologies, 1:1,000) followed by an anti-rabbit polyclonal IgG peroxidase secondary antibody (Santa Cruz Biotechnology, 1:20,000). Immuno-detection was accomplished via chemiluminescence using Western Lightning plus-ECL (Perkin Elmer) or ECL-Select western blotting detection reagent (GE Healthcare) given that the secondary antibody was conjugated to the horseradish peroxidase enzyme.

Twenty-four hours post-transfection, cells were detached with 0.25% Trypsin–EDTA (Wisent) and 4.0 × 10⁴ cells were re-plated onto a poly-l-ornithine (Sigma-Aldrich) treated white 96-well plate (Thermo Scientific, 236105). The next morning, a 25 mM solution of 1,2-ethanedithiol (EDT) was prepared by diluting it in dimethyl sulfoxide. Then, one volume of FlAsH reagent (2 mM) was added to two volumes of EDT to make a 667 µM FlAsH (Invitrogen) solution, which was incubated for 10 min at room temperature. Following the incubation, 100 µL of Hank’s balan-ced salt solution (HBSS) without phenyl red, with sodium bicarbonate, calcium, and magnesium was added to the 667 µM FlAsH solution and further incubated for 5 min at room temperature (Wisent). Then, HBSS was added to make a solution with final concentration of 750 nM FlAsH-EDT₂. In parallel, cells were washed in 150 µL of HBSS prior to the FlAsH labeling. Subsequently, 60 µL of the 750 nM FlAsH-EDT₂ solution was added to the cells and incubated for 1 h at 37°C, protected from any source of direct light. Following the incubation, cells were washed once with 100 µL of l M 2,3-dimercapto-1-propanol (BAL, Invitrogen) diluted in HBSS buffer and then incubated for 10 min at 37°C. The cells were washed once again with BAL without incubation. Afterward, cells were washed once with 150 µL of the assay buffer: Krebs (146 mM NaCl, 4.2 mM KCl, 0.5 mM MgCl₂, 1 mM CaCl₂, 10 mM HEPES pH 7.4, 0.1% glucose). The cells then sat in 80 µL of Krebs for 2 h at room temperature, in an environment protected from light, prior to the BRET assay. The FlAsH labeling procedure has been previously described elsewhere (11).

All statistical analysis was performed using GraphPad Prism 7.0 software. Data are reported as mean ± SE. The Prism software performed a Brown–Forsythe test to determine if parame-tric or non-parametric statistics should be performed. The degree of Gαs coupling was evaluated using a one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test comparing the various FlAsH positions to the wild type (Figures 3A,B). When determining the basal BRET exhibited by each of the recombinant β₂AR biosensors, a one-way ANOVA was performed. A Dunnett’s post hoc test was successively completed with the purpose of comparing the basal BRET of the eight recombinant constructs to the wild-type receptor (Figure 4A). When evaluating the agonist-induced BRET response, a two-way ANOVA was carried out followed by a Bonferroni corrected Student’s t-test aimed at comparing the response of the vehicle to the response of the agonist for each individual sensor position (Figure 4B).

We constructed a number of FlAsH-BRET biosensors in the β₂AR with a FlAsH-binding site engineered into various intra-cellular sites and Renilla luciferase placed on the carboxy terminus (Figure 1). If the biosensor components are positioned at appropriate sites within the receptor then this would allow profiling of conformational changes in the receptor upon ligand stimulation. In order for our intramolecular BRET constructs to be meaningful tools for the study of receptor conformational dynamics, recombinant receptors must maintain their native function. If they do not function in a manner similar to the wildtype receptor, then conformational analysis will be meaningless. Immunofluorescence was first used to verify the surface localization of the recombinant receptors generated. An anti-HA antibody was used to label the recombinant receptors, followed by an Alexa fluor-488 conjugated secondary antibody. As illustrated in Figure 2, almost all the FlAsH-tagged β₂AR constructs trafficked to the cell surface. Receptors tagged within the second intracellular loop were less robustly expressed compared to the wild type. However, the fluorescence intensity for all other positions was similar to the wild type providing us with at least six positions to carry forward.

Next, to further validate the functionality of each construct, we measured isoproterenol-mediated cAMP accumulation as well as ERK1/2 MAPK activation. We studied the relative accumulation of cAMP as an indication of Gαs activation using the H188 FRET-based EPAC sensor. FRET was used as BRET-based EPAC biosensors could not be used with the BRET-based conformational biosensors (as both would be activated). As shown in Figures 3A,B, the majority of constructs displayed similar levels of cAMP accumulation (measured as a decrease in FRET) as compared to the untagged wild-type receptor. As the β₂AR shows agonist-independent basal activity, we examined both basal (Figure 3A) and agonist-stimulated FRET (Figure 3B). We observed that certain sensor positions exhibited high basal activity as shown as the reduced FRET ratio at baseline. This may have been as a result of higher expression levels of these biosensors. For example, the C-tail P3 position may seem as though there is less cAMP production than the wild type in response to agonist; however, the lack of a robust decrease in FRET as a response to agonist is probably due to having attained the threshold of detection at basal levels.

We then examined a more distal readout of receptor functionality; the β₂AR-mediated MAPK (Raf/Ras/MEK/ERK) signaling pathway that has been previously characterized by various groups (22, 23). As demonstrated by Figure 3C, all the recombinant β₂AR constructs exhibited MAPK activation at similar intensities as the wild-type receptor. As a result, the third intracellular loop sensors as well as the C-tail sensors passed the validation stage although some caution again must be taken when interpreting results using the sensors engineered into the second intracellular loop. It must be noted here that all our transfections were transient and no attempt was made to normalize levels of expression per se.

Next, we measured basal BRET between the FlAsH-labeled receptors and the C-tail luciferase. Basal BRET or the BRET ratio after it has been corrected for spectral overlap of the donor and acceptor channels was determined by subtracting BRET where cells were not labeled with FlAsH. All receptor biosensors showed basal BRET to varying degrees (Figure 4A). The larger the basal BRET, the closer the donor–acceptor pair was at the outset. As a result, there is a greater dynamic range to capture relative changes in receptor conformation. The β₂AR biosensor with the greatest basal BRET was the third position within the C-tail. There was a position-dependent increase in the basal BRET, as one moves farther down the tail of the receptor, as acceptor and donor moieties get closer together. As for the third loop, the second position showed the largest basal BRET which is in accordance to its position in the middle of the loop (Figure 4A).

ΔBRET in response to ligand was measured by subtracting the averaged post-injection BRET from the averaged pre-injection BRET readings. BRET ratios could potentially increase or decrease depending on the ligand used and the subsequent conforma-tion adopted by the receptor. It was hoped that our biosensors would differentially respond to ligands and provide a conformational fingerprint to better understand the dynamic nature of the receptor which could be exploited for validating new drugs in early phases of development. Of all the biosensors tested, only the C-tail positions P2 and P3 showed a robust conformational change upon isoproterenol stimulation (Figure 4B). The lack of response in ICL3 was somewhat of a surprise but the functional data (Figures 2 and 3) suggested that ICL2 sensors may not be correctly folded.

In order to make a comprehensive assessment of the isopro-terenol induced responses of the β₂AR biosensors, we also examined the underlying kinetics. As mentioned, neither the second or third loop positions captured a sustained conformational change in response to isoproterenol (Figures 4B and 5). Oddly, a small spike was a consistent feature of the ligand-induced response in these sensors with the exception of ICL3 P1 (Figures 5D–F). The presence of this spike was not an artifact originating from the sampling instrument as no such spikes were seen when vehicle was similarly injected and it was also absent from kinetic traces of the wild-type receptor expressing RlucII with no FlAsH-binding sequences (Figure 5A). The C-tail P1 sensor displayed similar features as the second and third loop positions (Figures 5 and 6A). However, the responses in the other C-tail sensors were much more robust and sustained (Figures 6B,C). We have previously analyzed responses to ligand in both FP (11) and in the AT1R (17). Responses in ICL3 in AT1R and FP were both robust and sustained (Figures 6D,E) compared to the β₂AR. Further, robust sustained responses have also been detected in both ICL2 and the C-terminus of AT1R [(17); Figure 6F]. Interestingly, no responses were detected in similar constructs built into either ICL2 or the C-tail of FP (data not shown). Taken together, our data paint a picture which highlights the conformational heterogeneity of different GPCRs in response to ligand stimulation.