Nature Chemical Biology 1, 25-28 (2005)
doi: 10.1038/nchembio705
Molecular basis of inverse agonism in a G
protein−coupled receptor
Jean-Pierre Vilardaga1, Ralf Steinmeyer2, Greg S Harms2 and Martin J Lohse1
G protein−coupled receptors (GPCRs) recognize a wide variety of extracellular ligands to
control diverse physiological processes. Compounds that bind to such receptors can either
stimulate, fully or partially (full or partial agonists), or reduce (inverse agonists) the
receptors' basal activity and receptor-mediated signaling. Various studies have shown that
the activation of receptors through binding of agonists proceeds by conformational
changes as the receptor switches from a resting to an active state leading to G protein
signaling1, 2, 3, 4, 5. Yet the molecular basis for differences between agonists and inverse
agonists is unclear. These different classes of compounds are assumed to switch the
receptors' conformation in distinct ways. It is not known, however, whether such
switching occurs along a linear 'on-off' scale or whether agonists and inverse agonists
induce different switch mechanisms. Using a fluorescence-based approach to study the
2A-adrenergic receptor ( 2AAR), we show that inverse agonists are differentiated from
agonists in that they trigger a very distinct mode of a receptor's switch. This switch
couples inverse agonist binding to the suppression of activity in the receptor.
Discerning the basic mechanisms by which agonists and inverse agonists exert their
distinct effects on receptor function is fundamental to understanding signal transduction
mediated by GPCRs. To examine whether binding of inverse agonists to GPCRs causes
conformational changes resulting in inverse agonism6, 7, 8 (that is, inverse agonist effects),
we used a recently established fluorescence resonance energy transfer (FRET) approach
to monitor the activation of GPCRs directly in living cells5. This approach relies on the
capacity of a GPCR sensor based on cyan and yellow fluorescent proteins, GPCRCFP/YFP,
to report in real time, through a fast decrease in FRET, the intramolecular conformational
rearrangements associated with receptor activation (see Supplementary Fig. 1 online). We
applied this strategy to the 2AAR, a receptor that couples to the G proteins Gi and Go and
binds to several well-characterized inverse agonists9.
We applied saturating concentrations of norepinephrine (agonist) or yohimbine (inverse
agonist) sequentially to a single cultured human embryonic kidney (HEK293) cell
expressing 2AARCFP/YFP and recorded FRET signals over time (Fig. 1a). Norepinephrine
induced a fast decline of the FRET signal, whereas yohimbine increased the FRET signal.
This opposite change in the signal suggests that in response to yohimbine the receptor
undergoes a conformational change distinct from that seen in response to norepinephrine.
Similar signals obtained with three other distinct inverse agonists, rauwolscine,
RX821002 and MK-912 (partial response) (Figs. 1b and 2a), confirmed that the increase
in the FRET signal represented a general response to inverse agonists.
Figure 1: Conformational changes of the
2AAR
in response to full and inverse agonists.
(a) Emission intensities of YFP (yellow fluorescent protein) and CFP (cyan fluorescent
protein) were recorded simultaneously from a single HEK293 cell expressing
CFP/YFP
, and with FRET calculated as the ratio of emission intensities FYFP/FCFP
2AAR
(red). Shown are the changes induced by rapid superfusion with norepinephrine (NE; 100
M) or yohimbine (Yoh; 300 M). Traces are representative of at least ten separate
experiments. (b) Percentage change in the FRET ratio (left) and anisotropy values (right)
of 2AARCFP/YFP in intact HEK293 cells measured in the absence (-) or presence of NE
(100 M) or rauwolscine (Rau; 300 M). Data indicate the mean s.e.m. of four
separate experiments.
Full figure and legend (104K) Figures, schemes & tables index
Figure 2: Action of inverse agonists on
2AAR
CFP/YFP
.
(a) FRET signals mediated by the inverse agonists yohimbine (Yoh; 100 M),
rauwolscine (Rau; 100 M) and RX821002 (RX; 100 M) and by the agonist
norepinephrine (NE; 100 M). (b) Effect of the antagonist phentolamine (Phe; 100 M)
or the inverse agonist Yoh (100 M) on the FRET signal caused by NE. (c) Relationship
between the apparent rate constant kobs and NE ( ) or Yoh ( ) concentrations. kobs values
obtained from fitting the kinetic data of experiments like those of Figure 1a with a
monoexponential equation. Data indicate the mean s.e.m. of ten experiments for NE
and at least three for Yoh. Results for NE values comparison originate from our previous
study5. (d) Plot of kobs values for diverse types of ligands versus the percentage changes
they induce in the ratio FYFP/FCFP. Data obtained at saturating concentrations of full
agonists: NE (n = 10), UK-14,304 (UK; n = 4); partial agonists: moxonidine (Mox, n =
8), dopamine (DA, n = 8), oxymetazoline (Oxy, n = 4), clonidine (Clo, n = 5); inverse
agonists: MK-912 (MK; n = 3), Rau (n = 4), Yoh (n = 7) and RX (n = 4). Note that data
for Rau, Yoh and RX were similar, and only data for Yoh are represented.
Full figure and legend (35K) Figures, schemes & tables index
We further tested whether yohimbine could reverse the receptor's conformational change
mediated by norepinephrine. After continuous exposure of a cell to norepinephrine, the
addition of yohimbine reversed the FRET signal change generated by norepinephrine and
resulted in a signal similar to that caused by yohimbine in the absence of norepinephrine
(Fig. 2b). This contrasts with the effect of an antagonist such as phentolamine, which also
prevents the signal mediated by norepinephrine (Fig. 2b) but does not cause a
conformational rearrangement per se5. Therefore, yohimbine and phentolamine both
suppressed the signal produced by norepinephrine, in accordance with their competitive
binding nature, but the inverse agonist differentiated itself from the antagonist by causing
a conformational rearrangement of the receptor rather than just blocking the binding site.
To clarify whether the FRET changes arose from changes in the orientation of the
polarization of the fluorophores or changes in distance, we simultaneously measured in
CFP/YFP
fluorophore anisotropies and FRET ratios in response to agonist
2AAR
(norepinephrine) or inverse agonist (rauwolscine) with a microscope setup as described in
a published study10 (Fig. 1b). The anisotropy values for CFP and YFP agreed well with
those previously established for pure CFP11 and YFP12 in aqueous solution, and they
remained unchanged in response to norepinephrine or rauwolscine (Fig. 1b, right),
whereas the FRET signal changed in opposite directions (Fig. 1b, left). Thus, the
fluorophores showed similar dipole-dipole orientations when the receptor responded to
agonist or inverse agonist, suggesting that changes in FRET reflected changes in the
distances between the fluorophore moieties. Given the position of the fluorophores in the
receptor, we assume that the decrease in FRET in response to agonist reflects a distance
separation between the third intracellular loop and the C terminus, whereas the increase
in FRET in response to the inverse agonist is consistent with the view that these two
receptors' domains come closer together.
The temporal resolution of FRET with increasing ligand concentrations showed that
yohimbine-induced conformational switching of the receptor follows a hyperbolic
saturation of the rate constant (Fig. 2c). This behavior is consistent with a simple process
whereby at low ligand concentration the rate constant (kobs) increased linearly with ligand
concentration, indicating that ligand binding was the rate-limiting step. At high ligand
concentrations the rate constants approached a constant value, suggesting that a step other
than the association of ligand and receptor became rate limiting. This step is consistent
with a ligand-mediated conformational change of the receptor. We observed a similar
hyperbolic dependence on norepinephrine concentrations in earlier work5, indicating a
similar model but for a different conformational change (Fig. 2c). However, the
conformational switch responded 35 times slower to yohimbine than to norepinephrine
(Fig. 2c). Thus, not only does the switch induced by the inverse agonist produce a signal
opposite to the agonist-induced signal, but the vastly different kinetics suggest that the
inverse agonist-induced switch involves a specific conformational change relating to a
distinct kinetic pathway.
In the next experiment we tested whether the slow kinetics of receptor conformational
changes were characteristic of inverse agonists. The kinetic comparison between
structurally distinct low- and high-affinity full agonists (norepinephrine and UK-14,304),
partial agonists (dopamine, moxonidine, clonidine and oxymetazoline) and inverse
agonists (rauwolscine, yohimbine, RX821002 and MK-912) (see Supplementary Fig. 2
online for chemical structures) suggests that ligands with different efficacies induce a
range of conformational changes in the receptor with distinct kinetics (Fig. 2d and
Supplementary Fig. 3 online). These different kinetics contrast with structural differences
between ligands and with their affinities but are in good agreement with ligand efficacies.
In contrast to other GPCRs that show constitutive activity, we could not detect basal
activity of either the wild-type 2AAR or the 2AARCFP/YFP in HEK293 cells at the level of
cAMP or G protein inwardly-rectifying K+ channel (GIRK) current (data not shown).
This impeded our ability to link structural rearrangements caused by inverse agonists to
signal suppression. Therefore, to facilitate the measurement of suppression of basal
receptor activities by inverse agonists, we took advantage of a well-characterized
threonine-to-lysine point mutation, T373K, in the third intracellular loop adjacent to helix
6 of the 2AAR, which generates a constitutively active mutant (CAM) receptor,
CFP/YFP
was thus converted into a CAM receptor,
2AARCAM (ref. 13). The 2AAR
2AARCFP/YFPCAM. We demonstrated the constitutive activity of 2AARCFP/YFPCAM
by three distinct approaches. First, radioligand binding experiments showed that the
2AARCFP/YFPCAM. had an increased affinity for the agonist norepinephrine and a
slightly lower affinity for inverse agonists such as yohimbine (Supplementary Fig. 4
online) or rauwolscine (data not shown). These data are in agreement with similar studies
done with the wild-type 2AAR and 2AARCAM (ref. 9). Second, cAMP synthesis was
significantly suppressed in forskolin-stimulated cells expressing 2AARCFP/YFPCAM
(Fig. 3a). Third, coexpression of the parathyroid hormone (PTH) receptor (a Gs-coupled
receptor) and the 2AARCFP/YFPCAM reduced the ability of PTH to mediate cAMP
accumulation even in the absence of the 2AAR agonist UK-14,304 (Supplementary Fig.
4 online). These data emphasize that 2AARCFP/YFPCAM is constitutively active.
Figure 3: Characterization of the constitutively active receptor.
(a) cAMP measurement of cells expressing 2AARCFP/YFP (wild type (WT), 1 pmol mg-1)
or 2AARCFP/YFPCAM (CAM, 0.9 pmol mg-1) and incubated with 10 M forskolin
(FSK). FSK-stimulated cAMP in mock-transfected cells was set as 100%, and bars
represent the mean s.e.m. of four independent experiments. (b) Energy transfer
efficiency (E.T.) values for 2AARCFP/YFP (WT, n = 9) and 2AARCFP/YFPCAM (CAM, n
= 6). Significant differences from WT at P < 0.01 (**) by t-test indicated. (c) Comparing
switches of 2AARCFP/YFP (dotted trace) and 2AARCFP/YFPCAM (solid trace) in
response to norepinephrine (NE; 100 M) or yohimbine (Yoh; 100 M). The traces are
representative of three separate experiments. (d) FRET signals of 2AARCFP/YFPCAM
for diverse concentration of Yoh. (e) Concentration-response relation for the change in
FRET (from data similar to those in Figure 3d, ) and FSK-stimulated cAMP ( ) in
cells expressing 2AARCFP/YFPCAM. Data indicate the mean s.e.m. of three separate
experiments.
Full figure and legend (36K) Figures, schemes & tables index
Photodestruction (that is, photobleaching) of YFP leads to recovery of the CFP emission
that corresponds to the energy transfer (Supplementary Fig. 1 online), and this made it
possible to determine and to compare the energy transfer efficiencies (and structural
consequences) of 2AARCFP/YFP and 2AARCFP/YFPCAM. The mutant receptor showed a
lower efficiency of energy transfer ( 38%) than did 2AARCFP/YFP ( 45%) (Fig. 3b). This
decrease in energy transfer efficiency agrees with a decrease in the level of forskolinstimulated cAMP in 2AARCFP/YFPCAM −expressing cells (Fig. 3a). The single
substitution T373K in the receptor triggers a structural rearrangement that leads to an
alteration in the distance between the two fluorophores, apparently resulting in a lower
FRET efficiency. These data provide compelling evidence that 2AARCFP/YFP and
2AARCFP/YFPCAM have distinct conformations and that constitutive activation of
2AARCFP/YFPCAM is tightly coupled to the conformational difference observed
between the receptor and its constitutively active variant.
Antagonists such as phentolamine or idazoxan induced only insignificant changes in the
FRET signal of 2AARCFP/YFP or 2AARCFP/YFPCAM (data not shown). In contrast, in
response to saturating concentrations of norepinephrine or yohimbine, the constitutively
active receptor showed a pattern of FRET changes similar to those observed for
CFP/YFP
but of different amplitude: in 2AARCFP/YFPCAM, changes in FRET were
2AAR
smaller for norepinephrine but higher for yohimbine (Fig. 3c). These differences are
understandable if we assume that in the constitutively active receptor much of the
conformational activation occurs before agonist binding. Then, the receptor switches
from an intermediate active to a fully active state, giving rise to a smaller conformational
change.
To verify that structural rearrangements upon inverse agonist binding contribute to
inverse agonism, we compared the effects of different concentrations of yohimbine on
forskolin-stimulated cAMP in cells expressing 2AARCFP/YFPCAM with changes in
FRET. As the extent of FRET increased, the constitutive inhibition of cAMP synthesis
was suppressed (Fig. 3d,e). The notable parallel effect between the FRET signal
magnitude and changes in cAMP signaling strengthened the conclusion that inverse
agonism depends on the receptor's structural rearrangement mediated by inverse agonists.
In summary, inverse agonists signaled a conformational rearrangement in the receptor
that accompanied inverse agonism. The differences in the kinetics and character of the
conformational changes induced by full agonists and inverse agonists, and also by partial
agonists (see ref. 5 and Supplementary Fig. 3 online), imply that these different classes of
drugs use distinct molecular switches in the receptor. This demonstrates that the receptors
do not function merely as simple 'on-off' switches but rather have several distinct
conformational states and can be switched into these states with distinct kinetics.
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Methods
Molecular biology and cell culture.
Constructions of the 2A-adrenergic receptor sensor 2AARCFP/YFP and its CAM
14
2AARCFP/YFPCAM were carried out by established PCR strategies . Receptor cDNAs
were cloned into pCDNA3 (Invitrogen) for transient and stable expression in mammalian
cells. HEK293 cells served as the expression systems, and the procedure for the selection
of stable cell line has been described15.
Pharmacology.
Ligand binding, receptor number determination and measurement of cAMP were
measured as described15, 16. Saturation and competition binding studies were analyzed
with Prism 4.0 (GraphPad).
Microscopic FRET measurements.
FRET experiments were done as described5. In brief, cells grown on coverslips were
maintained in HEPES buffer (137 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 20
mM HEPES, 0.1% (wt/vol) BSA, pH 7.4) at room temperature and placed on a Zeiss
inverted microscope (Axiovert135) equipped with an oil immersion 100 objective and a
dual-emission photometric system (Till Photonics). Samples were excited with light from
a polychrome IV (Till Photonics). To minimize photobleaching, the illumination time
was set to 5−10 ms applied with a frequency between 1 and 75 Hz depending on agonist
concentration. FRET was monitored as the emission ratio of YFP to CFP, FYFP/FCFP,
where FYFP and FCFP are the emission intensities at 535 15 nm and 480 20 nm (beam
splitter dichroic long-pass (DCLP) 505 nm) upon excitation at 436 10 nm (beam splitter
DCLP 460 nm). The emission ratio was corrected by the respective spillover of CFP into
the 535-nm channel (spillover of YFP into the 480-nm channel was negligible) to give a
corrected ratio FYFP/FCFP. FRET between CFP and YFP in cells stably expressing the
receptor constructs was also determined by donor recovery after acceptor bleaching.
FRET efficiency was calculated according to the equation:
where CFPbefore and CFPafter are CFP emissions before and after photobleaching the YFP
by 3−5 min illumination at 500 nm.
Recording of ligand-induced changes in FRET.
To determine ligand-induced changes in FRET, cells were continuously superfused with
the HEPES buffer and ligand was applied using a computer-assisted solenoid
valve−controlled rapid superfusion device (ALA-VM8 from ALA Scientific Instruments;
solution exchange 5−10 ms). Signals detected by avalanche photodiodes were digitalized
using an AD converter (Digidata1322A; Axon Instruments) and stored on a PC using
Clampex 9.0 software (Axon Instruments). The change in FRET ratio was fitted to the
equation r(t) = A (1 - e-t/ ), where is the time constant (s) and A is the magnitude of the
signal. When necessary for calculating , ligand-independent changes in FRET due to
photobleaching were subtracted.
Simultaneous measurements of FRET and anisotropy.
Fluorescence anisotropy and FRET were measured with a microscopy setup as
described10. The fluorophores were excited with a linear polarized light at 458 nm for the
donor and 514 nm for the acceptor. The fluorescence emission was split to its parallel and
perpendicular components by a Wollaston prism and into the two emission wavelength
ranges for CFP (465−500 nm) and YFP (550−610 nm) by a wedge with dichroic
reflective coated surfaces. Intensities were recorded at the plasma membrane, and the full
intensity for each fluorophore was calculated as I + 2 g I . The anisotropy was
calculated as A = (I - g I )/(I + 2 g I ), where g is a correction factor for the relative
detection efficiencies between the parallel and the perpendicular channel (g = / ) and
I and I are the intensities of parallel and perpendicular fluorescence, respectively.
Accession codes.
BIND identifiers (http://bind.ca/): 261940, 261941, 261942, 261943, 261944, 261945,
261946, 261947, 261948, 261949.
Note: Supplementary information is available on the Nature Chemical Biology website.
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Acknowledgments
We thank M. Bernhard for technical support and K.-N. Klotz and M. Bünemann for
comments. The Deutsche Forschungsgemeinschaft and the Fonds der Chemischen
Industrie supported this work.
Competing interests
The authors declared no competing interests.
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1. Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher
Strasse 9, D-97078 Würzburg, Germany.
2. Rudolf-Virchow Center, University of Würzburg, Versbacher Strasse 9, D-97078
Würzburg, Germany.
3. Email: vilardaga@toxi.uni-wuerzburg.de
Correspondence to: Jean-Pierre Vilardaga1 Email: vilardaga@toxi.uni-wuerzburg.de
(a) Emission intensities of YFP (yellow fluorescent protein) and CFP (cyan fluorescent
protein) were recorded simultaneously from a single HEK293 cell expressing
CFP/YFP
, and with FRET calculated as the ratio of emission intensities FYFP/FCFP
2AAR
(red). Shown are the changes induced by rapid superfusion with norepinephrine (NE; 100
M) or yohimbine (Yoh; 300 M). Traces are representative of at least ten separate
experiments. (b) Percentage change in the FRET ratio (left) and anisotropy values (right)
of 2AARCFP/YFP in intact HEK293 cells measured in the absence (-) or presence of NE
(100 M) or rauwolscine (Rau; 300 M). Data indicate the mean s.e.m. of four
separate experiments.
(a) FRET signals mediated by the inverse agonists yohimbine (Yoh; 100 M),
rauwolscine (Rau; 100 M) and RX821002 (RX; 100 M) and by the agonist
norepinephrine (NE; 100 M). (b) Effect of the antagonist phentolamine (Phe; 100 M)
or the inverse agonist Yoh (100 M) on the FRET signal caused by NE. (c) Relationship
between the apparent rate constant kobs and NE ( ) or Yoh ( ) concentrations. kobs values
obtained from fitting the kinetic data of experiments like those of Figure 1a with a
monoexponential equation. Data indicate the mean s.e.m. of ten experiments for NE
and at least three for Yoh. Results for NE values comparison originate from our previous
study5. (d) Plot of kobs values for diverse types of ligands versus the percentage changes
they induce in the ratio FYFP/FCFP. Data obtained at saturating concentrations of full
agonists: NE (n = 10), UK-14,304 (UK; n = 4); partial agonists: moxonidine (Mox, n =
8), dopamine (DA, n = 8), oxymetazoline (Oxy, n = 4), clonidine (Clo, n = 5); inverse
agonists: MK-912 (MK; n = 3), Rau (n = 4), Yoh (n = 7) and RX (n = 4). Note that data
for Rau, Yoh and RX were similar, and only data for Yoh are represented.
(a) cAMP measurement of cells expressing 2AARCFP/YFP (wild type (WT), 1 pmol mg-1)
or 2AARCFP/YFPCAM (CAM, 0.9 pmol mg-1) and incubated with 10 M forskolin
(FSK). FSK-stimulated cAMP in mock-transfected cells was set as 100%, and bars
represent the mean s.e.m. of four independent experiments. (b) Energy transfer
efficiency (E.T.) values for 2AARCFP/YFP (WT, n = 9) and 2AARCFP/YFPCAM (CAM, n
= 6). Significant differences from WT at P < 0.01 (**) by t-test indicated. (c) Comparing
switches of 2AARCFP/YFP (dotted trace) and 2AARCFP/YFPCAM (solid trace) in
response to norepinephrine (NE; 100 M) or yohimbine (Yoh; 100 M). The traces are
representative of three separate experiments. (d) FRET signals of 2AARCFP/YFPCAM
for diverse concentration of Yoh. (e) Concentration-response relation for the change in
FRET (from data similar to those in Figure 3d, ) and FSK-stimulated cAMP ( ) in
cells expressing
experiments.
2AARCFP/YFPCAM.
Data indicate the mean
s.e.m. of three separate