Probing intermolecular protein–protein interactions in the
calcium-sensing receptor homodimer using bioluminescence
resonance energy transfer (BRET)
Anders A. Jensen
1
*, Jakob L. Hansen
2
*, Søren P. Sheikh
2
and Hans Bra¨ uner-Osborne
1
1
NeuroScience PharmaBiotec Research Centre, Department of Medicinal Chemistry, The Royal Danish School of Pharmacy,
Copenhagen, Denmark;
2
Laboratory of Molecular Cardiology, Copenhagen University Hospital, University of Copenhagen,
Copenhagen, Denmark
The calcium-sensing receptor (CaR) belongs to family C of
the G-protein coupled receptor superfamily. The receptor is
believed to exist as a homodimer due to covalent and non-
covalent interactions between the two amino terminal
domains (ATDs). It is well established that agonist binding
to family C receptors takes place at the ATD and that this
causes the ATD dimer to twist. However, very little is known
about the translation of the ATD dimer twist into G-protein
coupling to the 7 transmembrane moieties (7TMs) of these
receptor dimers.
In this study we have attempted to delineate the agonist-
induced intermolecular movements in the CaR homodimer
using the new bioluminescence resonance energy transfer
technique, BRET

(GABA
B
R1-2) [5], several families of putative pheromone
and taste receptors [6,7], and four recently cloned orphan
receptors [8–11]. With the exception of the orphan
receptors, all family C GPCRs are characterized by
unusually large extracellular amino terminal domains
(ATDs) of up to 600 amino acid residues to which
agonist binding takes place [12–20]. The subsequent
translation of the activation signal from the ATD into
G-protein coupling to the 7 transmembrane moiety (7TM)
is poorly understood.
All family C GPCRs, to which an endogenous ligand has
been identified, are believed to exist as dimers. Whereas
GABA
B
R1 and GABA
B
R2 undergo heterodimerization
[21–23], the mGluRs and CaR form homodimers [24,25].
The crystal structures of the mGluR1 ATD homodimer
have confirmed the findings from immunoblot studies of
CaR and mGluRs that the ATD dimer interface is
constituted by intermolecular noncovalent interactions
and a disulfide bridge [20,26–29]. Furthermore, the crystal
structures have revealed that the ATD homodimer equili-
brates between a resting and an active state, which differs by
a70° twist in the relative orientation of the two ATDs [20].
Agonist binding to one of the ATDs appears to stabilize the
active dimer conformation, a principle closely resembling

Eur. J. Biochem. 269, 5076–5087 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03218.x
luminescent donor to a fluorescent acceptor protein. In the
sea pansy Renilla reniformis the energy from the catalytic
degradation of coelenterazine h by Renilla luciferase
(Rluc) is transferred to green fluorescent protein (GFP),
andtheinteractionbetweenthetwoproteinsgivesriseto
emission of fluorescence. BRET is a derivation technique
of fluorescence resonance energy transfer (FRET), and the
two techniques have been applied repeatedly in studies of
the oligomerization of GPCRs and other protein–protein
interactions [34–40]. In these studies, BRET has been
measured using Rluc and enhanced yellow fluorescent
protein (EYFP) as luminescent donor and fluorescent
acceptor, respectively, and coelenterazine h as the sub-
strate.Recently,anewBRET
2
technology has been
introduced, where the emission of fluorescence caused by
the proximity of Rluc and the GFP mutant GFP
2
is
measured using DeepBlueC
TM
, a modified form of
coelenterazine h, as the substrate (Packard Bioscience).
The BRET
2
assay has very recently been applied in a
study of the homo- and heterodimerization of opioid and
adrenergic receptors [41].

receptors were generous gifts from Penelope
S. V. Jones (University of California, San Diego, CA) and
Bernhard Bettler, (University of Basel, Switzerland),
respectively. All transfections in this study were performed
with Polyfect as a DNA carrier according to the protocol
of the manufacturer (Qiagen, Hilden, Germany). Point
mutations were made using the Quick-Change mutagenesis
kit according to the manufacturer’s instructions (Stratagene,
La Jolla, CA).
Construction of tagged receptors
CaR and mGluR1a were subcloned from their original
vectors as described previously [17]. Two different GFP
mutants were used in this study: Enhanced green fluorescent
protein (EGFP) and GFP
2
, which are the F64L/S65T and
F64L mutants of GFP, respectively [45]. CaRD1036-EGFP
and CaRD1036-Rluc were created by subcloning of the
ApaI–XbaI fragment of EGFP-N2 and Rluc-N2 into CaR-
pSI digested with ApaI (an endogenous site covering
nucleotides 3103–3108 in CaR) and XbaI, respectively
(Fig. 1). Using the endogenous ApaI site for the constructs
results in the truncation of the last 43 amino acid residues in
the 212 residues-long carboxy terminal of rCaR. CaRD886-
EGFP and CaRD886-Rluc were constructed by subcloning
of EcoRI–ApaI digested PCR products into CaRD1036-
EGFP and CaRD1036-Rluc digested with EcoRI and ApaI,
respectively. CaRD1036-V5/His and CaRD886-V5/His were
created by subcloning of XhoI–ApaIfragmentof
CaRD1036-Rluc and CaRD886-Rluc into the pCDNA6-

B
1b-EGFP,
respectively. The MluI–NotI digestion cut out GABA
B
1a-
EGFP and GABA
B
1b-EGFP parts of the original plasmids.
Hence, c-myc-CaR and HA-CaR consisted of the signal
peptide for mGluR5, HA or c-myc and the entire CaR
Fig. 1. The Rluc-, GFP
2
- and EGFP-tagged receptors. (A) The topo-
logy of the Rluc-, GFP
2
-orEGFP-taggedGPCRsusedinthepresent
study. (B) The fusion regions of the Rluc- and GFP
2
/EGFP-tagged
receptors. GFP
2
and EGFP are given as ÔGFPÕ.
Ó FEBS 2002 Homodimerization of CaR in living cells (Eur. J. Biochem. 269) 5077
except for its signal peptide. The c-myc-CaRD1036-Rluc,
c-myc-CaRD886-Rluc receptors were created by subcloning
of the EcoRI–NotI segments of the respective Rluc-tagged
CaRs into c-myc-CaR. Analogously, HA-CaRD1036-GFP
2
and HA-CaRD886-GFP
2

), 10% dialyzed fetal calf serum
and 1 lCiÆmL
)1
myo-[2–
3
H]inositol (Amersham, Bucking-
hamshire, UK). Sixteen to twenty-four hours after applica-
tion of the radioligand, the cells were assayed as previously
described [46,47]. The pharmacological characterization of
wild type (WT) AT
1a
receptor and AT1aD359-GFP
2
was
performed analogously, except that HEK 293 cells were
used instead of tsA cells.
Fluorescence and luminescence measurements
For the measurements of fluorescence and luminescence in
cells cotransfected with Rluc- and GFP
2
-constructs, tsA
cells (1.5 · 10
5
cells per well) were split into wells of a 6 well-
culture plate and transfected with 0.4 lgofaGFP
2
-
construct and 0.4 lg of a Rluc-construct the following day.
The day after the transfection the medium was changed.
The following day, the cells were washed three times in

M
)andMgCl
2
(0.8 m
M
) supplemen-
ted with penicillin (100 UÆmL
)1
), streptomycin
(100 lgÆmL
)1
) and 10% dialyzed fetal calf serum. The
following day the medium was aspirated, the cells were
washed twice with NaCl/P
i
and fixed by incubation with
500 lL methanol for 5 min. The cells were washed
5 · 2 min with NaCl/P
i
, incubated with 500 lLNaCl/P
i
supplemented with 10% fetal calf serum for 20 min and
labeled with anti-myc (clone 9E10, Roche Molecular
Biolabs; 1 : 500) or anti-HA (clone 12CA5, Roche
Molecular Biolabs; 1 : 100) monoclonal Igs for 1 h.
Following 2 · 5 min washes with NaCl/P
i
and a 5-min
incubation with 500 lLNaCl/P
i

mented with penicillin (100 UÆmL
)1
), streptomycin
(100 lgÆmL
)1
) and 10% dialyzed calf serum. The follow-
ing day, single cell fluorescence was viewed with an
Axiovert 100M confocal microscope (Zeiss, Jena, Germany)
using the objective Plan-Achromat 63 · 14 W Oil (DiC)
and an excitation wavelength of 488 nm. The cellular
expression of each of the fusion proteins was determined
in at least four individual cells.
Emission and excitation spectral measurements
For emission spectral measurement of fusion Rluc/GFP
proteins Cos7 cells (1 · 10
6
) were split into a 10-cm tissue
culture plate and transfected with 15 lg plasmid (pRluc-N2,
pRluc/GFP
2
(pBRET +) or pRluc/EGFP) the following
day. The day after the transfection the medium was
changed. The following day, the cells were washed three
times in NaCl/P
i
and resuspended in 500 lLNaCl/P
i
in a
cuvette. DeepBlueC
TM

culture plate and transfected with 5 lg plasmid the
5078 A. A. Jensen et al.(Eur. J. Biochem. 269) Ó FEBS 2002
following day (5 lg of one plasmid, 2.5 lgofeachoftwo
plasmids, or otherwise indicated). The day after transfec-
tion the medium was changed. The following day, the cells
werewashedinNaCl/P
i
and detached. Approximately
1 · 10
6
cells per well were distributed in a 96-well
optiplate in the presence or absence of 20 m
M
CaCl
2
.
DeepBlueC
TM
was added to a final concentration of 5 l
M
,
and measurements were performed in a Fusion
TM
reader
(Packard Bioscience) (read time 1 s, gain 50, dual bands
410/80 nm and 515/30 nm). BRET ratios was calculated
as (emission
515 nm
) background
515 nm

potencies at these two receptors (Fig. 2A). The less
efficient G-protein coupling of the Rluc/EGFP-tagged
CaRs compared to WT CaR appeared to arise from an
interference of the Rluc/EGFP molecule in the coupling
process, as CaRD1036-V5/His and CaRD886-V5/His dis-
played WT-like agonist pharmacologies (Fig. 2A). The
observation that fusion of a 26 amino acid residue peptide
to residues 1036 and 886 of CaR did not alter the
pharmacological properties of the receptor is in excellent
agreement with a previous study of CaRs truncated in the
carboxy termini [49].
Cellular expression of the GFP- and Rluc-tagged CaRs
To estimate the overall expression levels of Rluc- and
GFP
2
-tagged CaRs and the control constructs in the cells
and to compare the overall cellular donor/acceptor ratios
within the different experiments, we measured the fluo-
rescence and luminescence in cells cotransfected with
various combinations of GFP
2
- and Rluc-constructs. Cells
were transfected with similar amounts of cDNA of Rluc-
and GFP
2
-constructs as those used in the BRET experi-
ments.
The levels of fluorescence in cells transfected with the
GFP
2

CaRs. (A) Concentration-response curves of Ca
2+
-induced IP accu-
mulation in tsA cells transfected with WT CaR, CaRD1036-V5/His,
CaRD1036-Rluc, CaRD1036-EGFP, CaRD886-V5/His, CaRD886-
Rluc and CaRD886-EGFP. Data are given as disintegration per
minute (DPM) per well. (B) Concentration-response curves of angio-
tensin II-induced IP accumulation in HEK 293 cells transfected with
WT At
1a
RandAt1aD359-GFP
2
. Data are given fold response [R/
R
basal
].
Ó FEBS 2002 Homodimerization of CaR in living cells (Eur. J. Biochem. 269) 5079
HA-tagged GABA
B1
receptors, when these were cotrans-
fected with WT GABA
B2
(data not shown).
The c-myc-CaR/HA-CaR, c-myc-CaRD1036-Rluc/
HA-CaRD1036-GFP
2
and c-myc-CaRD886-Rluc/
HA-CaRD886-GFP
2
transfected tsA cells all displayed

Confocal microscopy of cells transfected with
AT1aD359-EYFP demonstrated that this receptor was
expressed at the cell surface as well as intracellularly
(Fig. 5). Considering the few amino acid residues differing
in EYFP compared to GFP
2
, it is reasonable to assume
that the expression pattern of AT1aD359-GFP
2
is similar
to that of AT1aD359-EYFP.
In the IP assay angiotensin II displayed a potency at
AT1aD359-GFP
2
not significantly different from that at
Fig. 4. Immunofluorescence analysis of c-myc- and HA-tagged CaRs. Visualization of cell surface expression of tsA cells transfected with c-myc-
CaR/HA-CaR, c-myc-CaRD1036-Rluc/HA-CaRD1036-GFP
2
and c-myc-CaRD886-Rluc/HA-CaRD886-GFP
2
, respectively. The transfected tsA
cells were prepared as described in Experimental Procedures. All cell culture dishes with transfected cells were 80–90% confluent on the day of
viewing. The upper row of images was labeled with anti-(c-myc) Ig and the bottom row with anti-HA Ig.
Fig. 3. Measurements of fluorescence and luminescence in cells cotransfected with GFP
2
- and Rluc-constructs. The tsA cells were prepared and
assayed as described in Experimental Procedures. (A) Fluorescence measurements: excitation was performed at 425/20 nm, and emission was
measured at 530/10 nm. Data are given as CFU. (B) Luminescence measurements performed at 530 nm using a final concentration of 5 l
M
coelenterazine h as substrate. (C) The ratio between the fluorescence and luminescence signals in the various Rluc-/GFP

spectra were recorded from Cos7 cells transfected with
pRluc-N2 or the two fusion proteins pRluc/GFP
2
(pBRET+) and pRluc/EGFP (Fig. 6). The signal-
to-noise ratio using DeepBlueC
TM
as Rluc substrate
turned out to be considerably higher than that reported
for coelenterazine h forms used in other studies [34,40]. In
the window of 500–530 nm the emission of Rluc/GFP
2
transfected cells was  7.4 times higher than that of Rluc
transfected cells (Fig. 6A).
Interestingly, the BRET ratio obtained in Rluc/EGFP
transfected cells was only  20% lower than that in the
Rluc/GFP
2
transfected cells (Fig. 6A,B). At a glance this
was intriguing, as the normalized spectral overlap between
the donor emission and the acceptor excitation was
significantly higher for the Rluc/GFP
2
pair than for the
Rluc/EGFP pair (Fig. 6C). However, this may be explained
by two factors: Firstly, EGFP has a 2.6 times higher
excitation coefficient than GFP
2
(estimated S (max EGFP)
55 000 cm
)1

Fig. 5. Confocal microscopy of EGFP-tagged receptors. Confocal microscopy of tsA cells transfected with CaRD1036-EGFP, CaRD886-EGFP,
mGluR1D877-EGFP and AT1aD359-EYFP. All images were recorded as described in Experimental Procedures using an excitation wavelength of
488 nm. No fluorescence was detected in mock-transfected cells, and the fluorescence in cells transfected with EGFP and EYFP were uniformly
distributed over the entire cell (data not shown).
Ó FEBS 2002 Homodimerization of CaR in living cells (Eur. J. Biochem. 269) 5081
Significant BRET signals were obtained for all CaR-
GFP
2
and CaR-Rluc combinations (Fig. 7B). BRET ratios
between 0.11 and 0.17 were obtained for every combination
including CaRD1036-Rluc or CaRD1036-GFP
2
,whereas
the CaRD886-Rluc/CaRD886-GFP
2
combination gave rise
to a BRET signal of substantial higher intensities (BRET
ratios between 0.31 and 0.47).
No changes in the BRET signal were observed for any of
the combinations by addition of Ca
2+
(Fig. 7B). In these
experiments Ca
2+
was unable to reach the intracellular pool
of receptors. Hence, in order to investigate whether
exposure of all receptors in the cell to Ca
2+
would result
in an increased BRET signal, experiments were also

(AT1aD359-GFP
2
) either (Fig. 7C). Further-
more, the BRET signal obtained with CaRD886-Rluc and
CaRD886-GFP
2
was reduced considerably by coexpression
Fig. 6. Spectral properties of DeepBlueC
TM
illumination. (A) Light-emission acquisition spectrum of Cos7 cells transfected with Rluc/EGFP, Rluc/
GFP
2
(pBRET+) and pRluc-N2. Cells were incubated with 5 l
M
DeepBlueC
TM
, and light-emission acquisition was measured with a delay of 30 s.
The normalized luminescence is given. (B) BRET ratios in Cos7 cells transfected with Rluc/EGFP, Rluc/GFP
2
(pBRET+) and Rluc-N2. The
BRET ratio is given as emission
500)530 nM
/emission
370)450nM
. (C) Excitation and emission spectra measurements of EGFP and GFP
2
. Cos7 cells
were transfected with pEGFP-N1 or pGFP2-N1. Excitation spectra were recorded from 340 to 520 nm acquiring emission at 530 nm. Emission
spectra were recorded from 450 to 600 nm by exciting at 425 nm. The recording of the light-emission spectrum of Rluc is described above.
5082 A. A. Jensen et al.(Eur. J. Biochem. 269) Ó FEBS 2002

and
CaRD1036-Rluc/CaRD1036-GFP
2
combinations. This
indicated that the overall expression of the fluorescent
acceptor molecule was at least as favourable for the
formation of BRET in the control experiments as in the
regular BRET experiments (Fig. 7B,C). This further sup-
ports that the BRET signal is caused by specific homo-
dimerization of CaR rather than nonspecific interactions
due to overexpression of the proteins.
BRET experiments with Rluc- and EGFP-tagged receptors
Similar BRET patterns were observed for the various Rluc/
EGFP combinations as for the Rluc/GFP
2
combinations
Fig. 7. BRET in tsA cells transfected with Rluc- and GFP
2
-tagged receptors. The experiments were performed as described in Experimental
Procedures, and the BRET ratio is given as (emission
515 nm
) background
515 nm
)/(emission
410 nm
) background
410 nm
). All the experiments were
performed at least three times. Data shown are from a single experiment. (A) BRET in tsA cells transfected with the fusion proteins pBRET+
(Rluc/GFP

(compare Figs 7 and 8). In agreement with the experiments
with the GFP
2
-tagged receptors, no agonist-induced BRET
was detected for any of the Rluc/EGFP–tagged receptor
combinations (data not shown).
DISCUSSION
Evaluation of the BRET
2
assay
The present study is the second publication, where
dimerization between Rluc- and GFP
2
-tagged proteins
has been demonstrated using the modified form of
coelenterazine h DeepBlueC
TM
as the substrate [41]. The
emission of DeepBlueC
TM
catalyzed by Rluc takes place
at a lower wavelength than that of coelenterazine h (390–
400 nm and 475–480 nm, respectively), which gives rise to
a significant increase in spectral resolution (Packard
Bioscience). Because of the higher degree of separation
between the wavelengths of Rluc and Rluc/GFP
2
in the
presence of DeepBlueC
TM

will be used as a common reference point in the following
sections.
Receptor specificity of BRET
Numerous observations support that the BRET signals
obtained in tsA cells transfected with the Rluc- and GFP-
tagged CaRD1036 and CaRD886 were the result of specific
protein–protein interactions between the receptors, rather
than nonspecific diffusive lateral motion or clustering of
overexpressed receptors. First, the lifetime of an excited
Rluc molecule is in the range of 5 nsec (Packard Bioscience),
which limits the contribution of diffusive lateral motion to
negligible levels. Secondly, CaR-Rluc or CaR-GFP recep-
tors expressed alone or together with GFP and Rluc,
respectively, did not give rise to any significant signal
(Fig. 7B,C). Thirdly, CaR-Rluc and CaR-GFP had to be
present in the same cell in order to elicit BRET (Fig. 7C).
Fourthly, the fact that coexpression of CaRD886-Rluc with
AT1aD359-GFP
2
did not give rise to any BRET further
underlines the specificity of the CaR homodimerization
process (Fig. 7C). However, this does not exclude the
possibility that CaR could heterodimerize with other
GPCRs, and recently heterodimerization between CaR
and mGluRs has been reported [55]. Fifthly, the BRET
signal in cells transfected with CaRD886-Rluc/CaRD886-
GFP was significantly reduced by cotransfection with WT
CaR, CaRD1036-V5/His or CaRD886-V5/His (Fig. 7D).
We were unable to suppress the BRET signal to the extent
previously shown in a study of the thyrotropin-releasing

515 nm
)/(emission
410 nm
) background
410 nm
). All data shown
are measured under basal conditions (in the absence of agonist). All the
experiments were performed at least three times. Data shown is from a
single experiment. In the experiments depicted in the two last bars, the
tsA cells were transfected with 0.5 lgCaRD886-Rluc, 0.5 lg
CaRD886-EGFP and 4 lg pSI (vector alone) or CaR-pSI (WT CaR),
respectively, and assayed as described in Experimental Procedures.
5084 A. A. Jensen et al.(Eur. J. Biochem. 269) Ó FEBS 2002
Agonist-induced rearrangement of the 7TMs
in the CaR homodimer?
One of the goals of the present study was to investigate,
whether the activating twist in the ATD dimer of the family
C GPCR homodimer could be detected as agonist-induced
alterations in the BRET signal intensity, reflecting the
7TM)7TM contraction suggested by Kunishima et al. [20].
Because CaR is constitutively dimerized, a certain degree of
constitutive agonist-independent BRET was to be expected.
For us to be able to record agonist-induced BRET, the Rluc
and the GFP molecules would have to be sufficiently
separated in the resting state of the CaR homodimer
compared to in the activated state.
We have not been able to detect agonist-induced BRET
in cells transfected with any of the combinations of GFP-
and Rluc-tagged CaRs (Figs 7 and 8). The recent
demonstration of agonist-induced BRET for the insulin

proposed by Kunishima et al. [20]. A couple of pharmaco-
logical observations support this speculation: the trivalent
cation Gd
3+
hasbeenshowntoactivateCaRdirectlyatits
7TM [18], the somatic Ala843 fi GlumutationinTM7of
CaR causes constitutive activity in the receptor [59], and the
splice variants of mGluR1 and mGluR5 with long carboxy
termini are constitutively active [60,61]. All these pheno-
mena originate exclusively from the 7TM of the family C
GPCR and are unlikely to be accompanied by a conform-
ational change in the ATD dimer. Furthermore, a recent
study of the GABA
B
receptor heterodimer has suggested a
model for signal transduction through the family C GPCR,
where the activation signal is translated by a direct
interaction between the ATDs and the 7TMs of the receptor
dimer [62].
In conclusion, this study represents the first demonstra-
tion of family C GPCR dimerization in living cells. We have
demonstrated that CaR is constitutively dimerized. How-
ever, we have not been able to demonstrate agonist-induced
alterations in BRET signal intensities reflecting 7TM dimer
rearrangement as a result of the activating twist in the ATDs
of the CaR homodimer. Further investigations into the
signal transference from the ATDs to the G-protein
coupling areas of the receptor homodimer are clearly
needed in order to gain a better understanding of the signal
transduction through the family C GPCRs. From a

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