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Distinct Conformational Dynamics of Three G Protein-Coupled Receptors Measured Using FlAsH-BRET Biosensors

View Article: PubMed Central - PubMed

ABSTRACT

A number of studies have profiled G protein-coupled receptor (GPCR) conformation using fluorescent biaresenical hairpin binders (FlAsH) as acceptors for BRET or FRET. These conformation-sensitive biosensors allow reporting of movements occurring on the intracellular surface of a receptor to investigate mechanisms of receptor activation and function. Here, we generated eight FlAsH-BRET-based biosensors within the sequence of the β2-adrenergic receptor (β2AR) and compared agonist-induced responses to the angiotensin II receptor type I (AT1R) and the prostaglandin F2α receptor (FP). Although all three receptors had FlAsH-binding sequences engineered into the third intracellular loops and carboxyl-terminal domain, both the magnitude and kinetics of the BRET responses to ligand were receptor-specific. Biosensors in ICL3 of both the AT1R and FP responded robustly when stimulated with their respective full agonists as opposed to the β2AR where responses in the third intracellular loop were weak and transient when engaged by isoproterenol. C-tail sensors responses were more robust in the β2AR and AT1R but not in FP. Even though GPCRs share the heptahelical topology and are expressed in the same cellular background, different receptors have unique conformational fingerprints.

No MeSH data available.


BRET kinetics in the C-terminal β2-adrenergic receptor (β2AR) FlAsH constructs as well as in AT1R and FP biosensors. HEK 293 SL cells transiently transfected with the three C-tail β2AR recombinant biosensors or with FP ICL3 p4 or AT1R ICL3 p3 and C-tail p1 and then labeled with the FlAsH reagent. (A) C-tail p1, (B) C-tail p2, (C) C-tail p3, (D) FP ICL3 p4, (E) AT1R ICL3 p3, and (F) AT1R C-taol p1. Open boxes refer to vehicle and solid boxes refer to agonist treatment. Basal BRET was captured prior to the injection of each receptor’s respective full agonist, 10 µM isoproterenol, 1 µM PGF2α, or 1 µM angiotensin II. After ligand stimulation, data were continuously captured to observe the corresponding change in the BRET signal. The BRET ratio was calculated by dividing the fluorescence by the luminescence and plotted as a function of time. The dotted line represents the time at which the injection took place. The inset at the top right corner of each graph zooms in at the time points close to the injection. Measurements were recorded on 40,000 cells except for the AT1R C-tail p1 where 30,000 cells were used. All readings were taken using the Tristar multimode plate reader (Berthold Technologies) except the AT1R C-tail p1 which was assayed on the Victor X Light (Perkin Elmer). The data are representative of three or more independent experiments.
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Figure 6: BRET kinetics in the C-terminal β2-adrenergic receptor (β2AR) FlAsH constructs as well as in AT1R and FP biosensors. HEK 293 SL cells transiently transfected with the three C-tail β2AR recombinant biosensors or with FP ICL3 p4 or AT1R ICL3 p3 and C-tail p1 and then labeled with the FlAsH reagent. (A) C-tail p1, (B) C-tail p2, (C) C-tail p3, (D) FP ICL3 p4, (E) AT1R ICL3 p3, and (F) AT1R C-taol p1. Open boxes refer to vehicle and solid boxes refer to agonist treatment. Basal BRET was captured prior to the injection of each receptor’s respective full agonist, 10 µM isoproterenol, 1 µM PGF2α, or 1 µM angiotensin II. After ligand stimulation, data were continuously captured to observe the corresponding change in the BRET signal. The BRET ratio was calculated by dividing the fluorescence by the luminescence and plotted as a function of time. The dotted line represents the time at which the injection took place. The inset at the top right corner of each graph zooms in at the time points close to the injection. Measurements were recorded on 40,000 cells except for the AT1R C-tail p1 where 30,000 cells were used. All readings were taken using the Tristar multimode plate reader (Berthold Technologies) except the AT1R C-tail p1 which was assayed on the Victor X Light (Perkin Elmer). The data are representative of three or more independent experiments.

Mentions: 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.


Distinct Conformational Dynamics of Three G Protein-Coupled Receptors Measured Using FlAsH-BRET Biosensors
BRET kinetics in the C-terminal β2-adrenergic receptor (β2AR) FlAsH constructs as well as in AT1R and FP biosensors. HEK 293 SL cells transiently transfected with the three C-tail β2AR recombinant biosensors or with FP ICL3 p4 or AT1R ICL3 p3 and C-tail p1 and then labeled with the FlAsH reagent. (A) C-tail p1, (B) C-tail p2, (C) C-tail p3, (D) FP ICL3 p4, (E) AT1R ICL3 p3, and (F) AT1R C-taol p1. Open boxes refer to vehicle and solid boxes refer to agonist treatment. Basal BRET was captured prior to the injection of each receptor’s respective full agonist, 10 µM isoproterenol, 1 µM PGF2α, or 1 µM angiotensin II. After ligand stimulation, data were continuously captured to observe the corresponding change in the BRET signal. The BRET ratio was calculated by dividing the fluorescence by the luminescence and plotted as a function of time. The dotted line represents the time at which the injection took place. The inset at the top right corner of each graph zooms in at the time points close to the injection. Measurements were recorded on 40,000 cells except for the AT1R C-tail p1 where 30,000 cells were used. All readings were taken using the Tristar multimode plate reader (Berthold Technologies) except the AT1R C-tail p1 which was assayed on the Victor X Light (Perkin Elmer). The data are representative of three or more independent experiments.
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Figure 6: BRET kinetics in the C-terminal β2-adrenergic receptor (β2AR) FlAsH constructs as well as in AT1R and FP biosensors. HEK 293 SL cells transiently transfected with the three C-tail β2AR recombinant biosensors or with FP ICL3 p4 or AT1R ICL3 p3 and C-tail p1 and then labeled with the FlAsH reagent. (A) C-tail p1, (B) C-tail p2, (C) C-tail p3, (D) FP ICL3 p4, (E) AT1R ICL3 p3, and (F) AT1R C-taol p1. Open boxes refer to vehicle and solid boxes refer to agonist treatment. Basal BRET was captured prior to the injection of each receptor’s respective full agonist, 10 µM isoproterenol, 1 µM PGF2α, or 1 µM angiotensin II. After ligand stimulation, data were continuously captured to observe the corresponding change in the BRET signal. The BRET ratio was calculated by dividing the fluorescence by the luminescence and plotted as a function of time. The dotted line represents the time at which the injection took place. The inset at the top right corner of each graph zooms in at the time points close to the injection. Measurements were recorded on 40,000 cells except for the AT1R C-tail p1 where 30,000 cells were used. All readings were taken using the Tristar multimode plate reader (Berthold Technologies) except the AT1R C-tail p1 which was assayed on the Victor X Light (Perkin Elmer). The data are representative of three or more independent experiments.
Mentions: 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.

View Article: PubMed Central - PubMed

ABSTRACT

A number of studies have profiled G protein-coupled receptor (GPCR) conformation using fluorescent biaresenical hairpin binders (FlAsH) as acceptors for BRET or FRET. These conformation-sensitive biosensors allow reporting of movements occurring on the intracellular surface of a receptor to investigate mechanisms of receptor activation and function. Here, we generated eight FlAsH-BRET-based biosensors within the sequence of the β2-adrenergic receptor (β2AR) and compared agonist-induced responses to the angiotensin II receptor type I (AT1R) and the prostaglandin F2α receptor (FP). Although all three receptors had FlAsH-binding sequences engineered into the third intracellular loops and carboxyl-terminal domain, both the magnitude and kinetics of the BRET responses to ligand were receptor-specific. Biosensors in ICL3 of both the AT1R and FP responded robustly when stimulated with their respective full agonists as opposed to the β2AR where responses in the third intracellular loop were weak and transient when engaged by isoproterenol. C-tail sensors responses were more robust in the β2AR and AT1R but not in FP. Even though GPCRs share the heptahelical topology and are expressed in the same cellular background, different receptors have unique conformational fingerprints.

No MeSH data available.