Báo cáo Y học: Probing intermolecular protein–protein interactions in the calcium-sensing receptor homodimer using bioluminescence resonance energy transfer (BRET) potx

Eur. J. Biochem. 269, 5076–5087 (2002) Ó FEBS 2002

doi:10.1046/j.1432-1033.2002.03218.x

Probing intermolecular protein–protein interactions in the
calcium-sensing receptor homodimer using bioluminescence
resonance energy transfer (BRET)
Anders A. Jensen1*, Jakob L. Hansen2*, Søren P. Sheikh2 and Hans Brauner-Osborne1
ă
1

NeuroScience PharmaBiotec Research Centre, Department of Medicinal Chemistry, The Royal Danish School of Pharmacy,
Copenhagen, Denmark; 2Laboratory 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 noncovalent 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 agonistinduced intermolecular movements in the CaR homodimer
using the new bioluminescence resonance energy transfer
technique, BRET2, which is based on the transference of
energy from Renilla luciferase (Rluc) to the green fluorescent
protein mutant GFP2. We tagged CaR with Rluc and GFP2

Family C of the G-protein coupled receptor (GPCR)

superfamily consists of eight metabotropic glutamate
receptors (mGluR1-8) [1–3], a calcium-sensing receptor
(CaR) [4], two c-aminobutyric acid type B receptors
(GABABR1-2) [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

Correspondence to A. A. Jensen, Department of Medicinal Chemistry,
The Royal Danish School of Pharmacy, 2 Universitetsparken,
DK-2100 Copenhagen, Denmark.
Fax: + 45 3530 6040, Tel.: + 45 3530 6491,
E-mail:
Abbreviations: GPCR, G-protein coupled receptor; mGluR, metabotropic glutamate receptor; CaR, calcium-sensing receptor;
GABABR, c-aminobutyric acid receptor type B; ATD, amino
terminal domain; 7TM, 7 transmembrane moiety; BRET, bioluminescence resonance energy transfer; FRET, fluorescence resonance
energy transfer; Rluc, Renilla luciferase; GFP, green fluorescent
protein; EGFP, enhanced green fluorescent protein; EYFP, enhanced
yellow fluorescent protein; IP, inositol phosphate; WT, wild type;
i1/i2/i3, intracellular loop 1, 2 and 3.
Note: *Co-first authors
(Received 19 June 2002, revised 13 August 2002,
accepted 29 August 2002)

at different intracellular locations. Stable and highly
receptor-specific BRET signals were obtained in tsA cells
transfected with Rluc- and GFP2-tagged CaRs under basal
conditions, indicating that CaR is constitutively dimerized.

However, the signals were not enhanced by the presence of
agonist. These results could indicate that at least parts of the
two 7TMs of the CaR homodimer are in close proximity in
the inactivated state of the receptor and do not move much
relative to one another upon agonist activation. However,
we cannot exclude the possibility that the BRET technology
is unable to register putative conformational changes in the
CaR homodimer induced by agonist binding because of the
bulk sizes of the Rluc and GFP2 molecules.
Keywords: family C GPCR; CaR; BRET; dimerization;
homodimerization.

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
GABABR1 and GABABR2 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 equilibrates between a resting and an active state, which differs by
a 70° 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
the classical two-state model for family A GPCR function

[30,31]. Speculating on the following steps in the signal
transduction, Kunishima et al. have proposed that this
activation twist in the relative ATD–ATD conformation
could cause a contraction of the two 7TMs in the
homodimer thereby creating a new structural motif recognizable to the G-protein [20]. A similar signal mechanism
has been proposed for certain cytokine receptors signalling
through a JAK/STAT pathway [32,33].
Bioluminescence resonance energy transfer (BRET) is
the product of nonradiative transfer of energy from a


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Homodimerization of CaR in living cells (Eur. J. Biochem. 269) 5077

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),
and the interaction between the two proteins gives rise to
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 substrate. Recently, a new BRET2 technology has been
introduced, where the emission of fluorescence caused by
the proximity of Rluc and the GFP mutant GFP2 is

measured using DeepBlueCTM, a modified form of
coelenterazine h, as the substrate (Packard Bioscience).
The BRET2 assay has very recently been applied in a
study of the homo- and heterodimerization of opioid and
adrenergic receptors [41].
In the present study, we have applied the BRET2
technology to investigate the intermolecular arrangement
of the 7TMs in the family C GPCR homodimer, exemplified
by the CaR.

EXPERIMENTAL PROCEDURES
Materials
Culture media, serum, antibiotics and buffers for cell
culture were obtained from Life Technologies (Paisley,
UK). All other chemicals were obtained from Sigma
(St. Louis, MO). The rCaR-pRK5 [42] and pmGluR1a
[43] plasmids were generous gifts from Professor Solomon
H. Snyder (The Johns Hopkins University School of
Medicine, Baltimore, MD) and Professor Shigetada
Nakanishi (Kyoto University, Japan), respectively. The
pSI and pEGFP-N2 vectors were obtained from Promega
(Madison, WI) and Clontech (Palo Alto, CA), respectively.
DeepBlueCTM, pGFP2-N3, pRluc-N1, pRluc-N2 and the
pBRET+ vector (a Rluc/GFP2 fusion protein) were
purchased from Biosignal Packard (Montreal, Canada).
The tsA cells (a transformed human embryonic kidney
(HEK) 293 cell line) [44] and the c-myc- and HA-tagged
GABAB 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).

ApaI–XbaI fragment of EGFP-N2 and Rluc-N2 into CaRpSI 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. CaRD886EGFP and CaRD886-Rluc were constructed by subcloning
of EcoRI–ApaI digested PCR products into CaRD1036EGFP and CaRD1036-Rluc digested with EcoRI and ApaI,
respectively. CaRD1036-V5/His and CaRD886-V5/His were
created by subcloning of XhoI–ApaI fragment of
CaRD1036-Rluc and CaRD886-Rluc into the pCDNA6V5/His-A vector (Invitrogen, San Diego, CA). The
mGluR1D877-EGFP and mGluR1D877-Rluc plasmids
were created by subcloning of BspEI–XbaI digested PCR
products of EGFP-N2 and Rluc-N2 into mGluR1a-pSI
digested with BspEI (an endogenous site covering nucleotides 2627–2632 in mGluR1a) and XbaI. Receptor-GFP2
fusion plasmids were created in a similar fashion as described
above. AT1aD359-GFP2 was created by PCR using the
angiotensin II receptor subtype 1a as template and subsequent subcloning into pGFP2-N3 using HindIII and BamHI
as restriction enzymes. The pRluc/EGFP plasmid was
created from pRluc/GFP2 (pBRET+) by the introduction
of a Ser65 fi Thr mutation in the GFP2 part of the plasmid.
For the construction of the c-myc-CaR and HA-CaR
constructs, a MluI site was introduced after the signal
peptide in CaR (covering nucleotides 55–60) using the
QuickChange mutagenesis kit. Following digestion with

restriction enzymes MluI and NotI, CaR was subcloned
into c-myc-GABAB1a-EGFP and HA-GABAB1b-EGFP,
respectively. The MluI–NotI digestion cut out GABAB1aEGFP and GABAB1b-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

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 GFP2, which are the F64L/S65T and
F64L mutants of GFP, respectively [45]. CaRD1036-EGFP
and CaRD1036-Rluc were created by subcloning of the

Fig. 1. The Rluc-, GFP2- and EGFP-tagged receptors. (A) The topology of the Rluc-, GFP2- or EGFP-tagged GPCRs used in the present
study. (B) The fusion regions of the Rluc- and GFP2/EGFP-tagged
receptors. GFP2 and EGFP are given as ÔGFPÕ.


Ó FEBS 2002

5078 A. A. Jensen et al. (Eur. J. Biochem. 269)

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-GFP2
and HA-CaRD886-GFP2 were created by subcloning of the
EcoRI–NotI segment of the respective GFP2-tagged CaRs
into HA-CaR.

All amplified receptor DNAs were sequenced on an ABI
Prism 310 using Big Dye Terminator Cycle Sequencing kit
(Perkin-Elmer, Warrington, UK).

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/Pi and a 5-min
incubation with 500 lL NaCl/Pi supplemented with 10%
fetal calf serum the cells were incubated for 1 h with
secondary Cy3-conjugated affinity-purified goat antimouse IgG (Jackson ImmunoResearch Laboratories,
West Grove, PA; 1 : 200). Then the cells were washed
(2 · 5 min) with NaCl/Pi and viewed through a Leica
DM IRB fluorescence microscope.

Inositol phosphate (IP) assay
The tsA cells (3 · 105) were split into a 6-cm tissue culture
plate and transfected the following day. The day after
transfection, the cells were split into 16 wells of a poly
D-lysine coated 48-well tissue culture plate in inositol-free
DMEM (Dulbecco’s modified Eagle’s medium) with
reduced concentrations of CaCl2 (0.9 mM) and MgCl2
(0.8 mM), supplemented with penicillin (100 mL)1),
streptomycin (100 lgỈmL)1), 10% dialyzed fetal calf serum
and 1 lCiỈmL)1 myo-[2–3H]inositol (Amersham, Buckinghamshire, UK). Sixteen to twenty-four hours after application of the radioligand, the cells were assayed as previously
described [46,47]. The pharmacological characterization of
wild type (WT) AT1a receptor and AT1aD359-GFP2 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 GFP2-constructs, tsA
cells (1.5 · 105 cells per well) were split into wells of a 6 wellculture plate and transfected with 0.4 lg of a GFP2construct 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
NaCl/Pi, resuspended in 300 lL NaCl/Pi and distributed in
black optiplates (Packard). Fluorescence and luminescence
recordings were performed in a FusionTM reader (Packard).
Fluorescence excitation was performed at 425/20 nm and
emission was measured at 530/10 nm. Luminescence was
assayed by addition of coelenterazine h and measured
without any filter.
Immunofluorescence studies
The tsA cells (3 · 105) were split into a 6-cm tissue culture
plate and transfected with a total of 1.7 lg plasmid
(pCDNA3 or GABAB receptors for the control experiments or various combinations of c-myc- and HA-tagged
CaRs) the following day. The day after transfection, the
cells were split into wells of a poly D-lysine coated 24-well
tissue culture plate in DMEM with reduced concentrations of CaCl2 (0.9 mM) and MgCl2 (0.8 mM) supplemented
with
penicillin
(100 mL)1),
streptomycin
)1
(100 lgỈmL ) and 10% dialyzed fetal calf serum. The
following day the medium was aspirated, the cells were
washed twice with NaCl/Pi and fixed by incubation with
500 lL methanol for 5 min. The cells were washed
5 · 2 min with NaCl/Pi, incubated with 500 lL NaCl/Pi
supplemented with 10% fetal calf serum for 20 min and

Single cell fluorescence measurements
The tsA cells (3 · 105) were split into a 6-cm tissue culture
plate and transfected with a total of 1.7 lg plasmid
(CaRD1036-EGFP, CaRD886-EGFP, mGluR1D877EGFP or AT1aD359-EYFP) the following day. The day
after transfection, the cells were split into poly D-lysine
coated 3.5 cm wells containing a glass slide (MatTek
Corp., Ashland, MA) in DMEM with reduced concentrations of CaCl2 (0.9 mM) and MgCl2 (0.8 mM), supplemented with penicillin (100 mL)1), streptomycin
(100 lgỈmL)1) and 10% dialyzed calf serum. The following 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 · 106) were split into a 10-cm tissue
culture plate and transfected with 15 lg plasmid (pRluc-N2,
pRluc/GFP2 (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/Pi and resuspended in 500 lL NaCl/Pi in a
cuvette. DeepBlueCTM was added to a final concentration of
5 lM, and light emission acquisition (340–600 nM) was
performed with a delay 30 s to assure dark adaption using
a SPEX Fluoromax-2 spectrofluorometer (Jobin Yvon
Inc., Edison, NJ) with the lamp turned off connected to a
PC equipped with the Datamax 2.2 software package
(emission slit 25 nm, increment 2 nm, integration time
0.5 s).

For excitation and emission spectra measurements of
EGFP and GFP2 the Cos7 cells were handled as described
above, except that they were transfected with pEGFP-N1 or
pGFP2-N1. Excitation spectra were recorded from 340 to
520 nm acquiring emission at 530 nm (emission/excitation
slit of 1 nm, increment 2 nm, integration time 0.1 s).
Emission spectra were recorded from 450 to 600 nm by
exciting at 425 nm using the same conditions as above,
where background was subtracted using nontransfected
cells, and the spectra were normalized.
BRET assay
The tsA cells (1 · 106) were split into a 10-cm tissue
culture plate and transfected with 5 lg plasmid the


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Homodimerization of CaR in living cells (Eur. J. Biochem. 269) 5079

following day (5 lg of one plasmid, 2.5 lg of each of two
plasmids, or otherwise indicated). The day after transfection the medium was changed. The following day, the cells
were washed in NaCl/Pi and detached. Approximately
1 · 106 cells per well were distributed in a 96-well
optiplate in the presence or absence of 20 mM CaCl2.
DeepBlueCTM was added to a final concentration of 5 lM,
and measurements were performed in a FusionTM reader
(Packard Bioscience) (read time 1 s, gain 50, dual bands
410/80 nm and 515/30 nm). BRET ratios was calculated
as (emission515 nm ) background515 nm)/(emission410 nm )
background410 nm). The background signal was assessed

in each experiment by measuring the signal of a sample of
nontransfected cells. In the BRET measurements using
lyzed tsA cells transfected with various GFP2- and Rluctagged CaRs, the cells were mechanically lyzed immediately before the measurements by sucking the cell
suspension up and down 12 times with a tuberculin
syringe with a 27 gauge needle.
All experiments were performed at least three times, and
the data shown reflects the results of all experiments.

RESULTS
Pharmacological characterization of Rlucand EGFP-tagged CaRs
In excellent agreement with a previous study of EGFPtagged CaRs [48], CaRD1036-EGFP, CaRD1036-Rluc,
CaRD886-EGFP and CaRD886-Rluc were all functional
in an IP assay, demonstrating that all of these receptors
were expressed at the cell surface (Fig. 2A). However, the
fold responses of particularly CaRD886-Rluc and
CaRD886-EGFP were significantly decreased compared
to that of WT CaR, and Ca2+ displayed significant lower
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 displayed 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
GFP2-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 fluorescence and luminescence in cells cotransfected with
various combinations of GFP2- and Rluc-constructs. Cells
were transfected with similar amounts of cDNA of Rlucand GFP2-constructs as those used in the BRET experiments.
The levels of fluorescence in cells transfected with the
GFP2-tagged receptors were comparable in size, whereas
GFP2 was expressed at slightly higher levels (Fig. 3A). The
luminescence levels in CaRD1036-Rluc and CaRD886-Rluc
transfected cells were similar, whereas cells expressing Rluc

Fig. 2. Pharmacological characterization of EGFP- and Rluc-tagged
CaRs. (A) Concentration-response curves of Ca2+-induced IP accumulation in tsA cells transfected with WT CaR, CaRD1036-V5/His,
CaRD1036-Rluc, CaRD1036-EGFP, CaRD886-V5/His, CaRD886Rluc and CaRD886-EGFP. Data are given as disintegration per
minute (DPM) per well. (B) Concentration-response curves of angiotensin II-induced IP accumulation in HEK 293 cells transfected with
WT At1aR and At1aD359-GFP2. Data are given fold response [R/
Rbasal].

itself displayed a significantly higher luminescent signal
(Fig. 3B). These data indicates that the overall cellular
expression levels of the Rluc- and GFP2-tagged CaRs and
AT1aRs are similar.
To evaluate the cell surface expression, we tagged HA
and c-myc epitopes to the N-terminal of the CaR-GFP2 and
CaR-Rluc fusion proteins, respectively, and visualized these
using immunofluorescence microscopy (Fig. 4). No fluorescence was observed for mock transfected cells, when
either anti-HA or antic-myc antibodies were used (data not
shown). To validate the reliability of the immunofluorescence technique further, we took advantage of the wellestablished heterodimerization of the GABAB receptors
[5,21–23]. In agreement with a previous study [50], cell
surface staining was only observed for c-myc and

5080 A. A. Jensen et al. (Eur. J. Biochem. 269)

Ó FEBS 2002

Fig. 3. Measurements of fluorescence and luminescence in cells cotransfected with GFP2- 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 lM
coelenterazine h as substrate. (C) The ratio between the fluorescence and luminescence signals in the various Rluc-/GFP2-combinations. The ratio is
given as [Fluorescence/Luminescence].

Fig. 4. Immunofluorescence analysis of c-myc- and HA-tagged CaRs. Visualization of cell surface expression of tsA cells transfected with c-mycCaR/HA-CaR, c-myc-CaRD1036-Rluc/HA-CaRD1036-GFP2 and c-myc-CaRD886-Rluc/HA-CaRD886-GFP2, 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.

HA-tagged GABAB1 receptors, when these were cotransfected with WT GABAB2 (data not shown).
The c-myc-CaR/HA-CaR, c-myc-CaRD1036-Rluc/
HA-CaRD1036-GFP2
and
c-myc-CaRD886-Rluc/
HA-CaRD886-GFP2 transfected tsA cells all displayed
substantial degrees of cell surface staining both when
labeled with anti-(c-myc) and anti-HA Ig (Fig. 4). The
fraction of cells expressing the CaRD1036- and CaRD886receptors and that of WT CaR appeared to be similar.
The cellular expression patterns of the GFP-tagged
receptors were investigated in greater detail using confocal microscopy. The expression patterns of CaRD1036EGFP and CaRD886-EGFP were recorded in several
cells. Cells representing the predominant expression
pattern of the respective receptors are depicted in
Fig. 5. In agreement with the immunofluorescence experiments and previous studies of similar EGFP-tagged
CaRs, CaRD1036-EGFP and CaRD886-EGFP were

localized in the cell membrane as well as intracellularly
(Fig. 5) [48,51].

Cellular expression of GFP- and Rluc-tagged mGluR1
and AT1aR
The receptors mGluR1D877-EGFP, mGluR1D877-GFP2
and mGluR1D877-Rluc were originally constructed as
control receptors for the BRET experiments. However,
confocal microscopy revealed that mGluR1D877-EGFP
was trapped in vesicles inside the tsA cell (Fig. 5). Hence,
the Rluc/GFP-tagged mGluR1D877 constructs were
determined to be unsuitable for the BRET experiments,
and AT1aD359-GFP2 was used instead.
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 GFP2, it is reasonable to assume
that the expression pattern of AT1aD359-GFP2 is similar
to that of AT1aD359-EYFP.
In the IP assay angiotensin II displayed a potency at
AT1aD359-GFP2 not significantly different from that at


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Homodimerization of CaR in living cells (Eur. J. Biochem. 269) 5081

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).

WT AT1aR, albeit the fold response of the GFP2-tagged
receptor was attenuated compared to that of the WT
receptor (Fig. 2B). Furthermore, WT AT1aR and
AT1aD359-GFP2 displayed similar binding characteristics
in a [I125]angiotensin II whole cell binding assay (data not
shown). These observations are in excellent agreement with
the findings of another group [52] and suggest that
AT1aD359-GFP2 is functional and expressed at the cell
surface to a degree comparable to that of WT AT1aR.
BRET in living cells
To evaluate the ability of our assay to detect BRET
caused by protein–protein interactions, light emission
spectra were recorded from Cos7 cells transfected with
pRluc-N2 or the two fusion proteins pRluc/GFP2
(pBRET+) and pRluc/EGFP (Fig. 6). The signalto-noise ratio using DeepBlueCTM 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/GFP2
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/GFP2 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/GFP2 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 GFP2 (estimated S (max EGFP)
55 000 cm)1ỈM)1 (Clontech) and estimated S (max GFP2)
21 000 cm)1ỈM)1 (Packard, unpublished data)). Secondly,
the spectral overlap for EGFP occurs at higher wavelength,
where the electric field drops off more slowly and energy
transfer can occur at further distances [53, 54].
BRET experiments with Rluc- and GFP2-tagged
receptors
We did not detect any BRET signal in cells transfected
exclusively with a Rluc-tagged or a GFP2-tagged CaR. A
BRET ratio of  0.05 was observed from cells transfected
with CaRD1036-Rluc or CaRD886-Rluc (Fig. 7B). This
signal corresponds to no energy transfer, and this fraction of
the BRET ratio is caused by background emission from
Rluc into the GFP filter. In cells transfected with the GFP2tagged receptors alone no luminescence signals were detected (data not shown).


5082 A. A. Jensen et al. (Eur. J. Biochem. 269)

Ó FEBS 2002

Fig. 6. Spectral properties of DeepBlueCTM illumination. (A) Light-emission acquisition spectrum of Cos7 cells transfected with Rluc/EGFP, Rluc/
GFP2 (pBRET+) and pRluc-N2. Cells were incubated with 5 lM DeepBlueCTM, 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/GFP2 (pBRET+) and Rluc-N2. The
BRET ratio is given as emission500)530 nM/emission370)450nM. (C) Excitation and emission spectra measurements of EGFP and GFP2. 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.

Significant BRET signals were obtained for all CaRGFP2 and CaR-Rluc combinations (Fig. 7B). BRET ratios
between 0.11 and 0.17 were obtained for every combination
including CaRD1036-Rluc or CaRD1036-GFP2, whereas
the CaRD886-Rluc/CaRD886-GFP2 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 Ca2+ (Fig. 7B). In these
experiments Ca2+ was unable to reach the intracellular pool
of receptors. Hence, in order to investigate whether
exposure of all receptors in the cell to Ca2+ would result
in an increased BRET signal, experiments were also
performed on mechanically lyzed tsA cells transfected with
various combinations of GFP2- and Rluc-tagged
CaRD1036 and CaRD886. However, the BRET ratios

in these experiments were comparable to the similar
experiments using whole cells, and no Ca2+-induced BRET
could be detected (data not shown). It was also verified that
the BRET2 assay itself was not sensitive to Ca2+
concentration changes (Fig. 7A).
Several experiments were performed in order to confirm
that the BRET signals obtained in CaR-Rluc/CaR-GFP2
transfected cells were receptor-specific. Co-expression of
CaRD886-GFP2 and pRluc-N2 did not give rise to any
BRET signal, and coexpression of CaRD886-Rluc and
pGFP2-N3 elicited only a weak signal (Fig. 7C). No
significant BRET was recorded in cells expressing
CaRD886-Rluc and the angiotensin II receptor 1a tagged
with GFP2 (AT1aD359-GFP2) either (Fig. 7C). Furthermore, the BRET signal obtained with CaRD886-Rluc and

CaRD886-GFP2 was reduced considerably by coexpression


Ó FEBS 2002

Homodimerization of CaR in living cells (Eur. J. Biochem. 269) 5083

Fig. 7. BRET in tsA cells transfected with Rluc- and GFP2-tagged receptors. The experiments were performed as described in Experimental
Procedures, and the BRET ratio is given as (emission515 nm ) background515 nm)/(emission410 nm ) background410 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/GFP2) or Rluc/EGFP in absence and presence of 20 mM CaCl2. (B) BRET in tsA cells transfected with Rluc- and GFP2-tagged CaRs. (C)
Receptor specificity of the BRET. In [1], BRET obtained in tsA cells transfected with CaRD886-Rluc or CaRD886-GFP2 and pRluc-N2, pGFP2N3 or AT1aD359-GFP2 were recorded. In [2], two 10 cm culture dishes of tsA cells were transfected with 2.5 lg CaRD886-Rluc and 2.5 lg
CaRD886-GFP2, respectively, and cells from the two dishes were mixed immediately prior to the BRET recording. The mixture of the two
population of cells is indicated with brackets around each cell line. The two experiments depicted in Fig. 7C were performed independently of each
other. (D) Competitive inhibition of BRET by coexpression of receptors not tagged with GFP2 or Rluc. Cells were transfected with 0.5 lg
CaRD886-Rluc, 0.5 lg CaRD886-GFP2 and 4 lg of various plasmids (pSI, CaR-pSI, CaRD1036-V5/His, CaRD886-V5/His, m1-pCD, H1pCDNA3 and GABAB2-pCDNA3) and assayed as described in Experimental Procedures.

of WT CaR, CaRD1036-V5/His and CaRD886-V5/His
(Fig. 7D). In contrast, the signal was not diminished by
coexpression of CaRD886-Rluc and CaRD886-GFP2 with
family A GPCRs such as the muscarinic acetylcholine
receptor m1 and the histamine H1 receptor or with the
family C GPCR GABAB2 (Fig. 7D). Finally, no significant
BRET signal could be detected, when CaRD886-Rluc
transfected cells and CaRD886-GFP2 transfected cells were
mixed, indicating that the donor and acceptor molecules
had to be present in the same cell in order to elicit BRET
(Fig. 7C).
Another important factor to consider was the ratio
between the fluorescence signal and the luminescence signal

for the various GFP2/Rluc-combinations. As can be seen
from Fig. 3C, this ratio was higher for the CaRD886-Rluc/

GFP2 and CaRD886-Rluc/AT1D359-GFP2 combinations
than for the CaRD886-Rluc/CaRD886-GFP2 and
CaRD1036-Rluc/CaRD1036-GFP2 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 supports that the BRET signal is caused by specific homodimerization 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/GFP2 combinations


Ó FEBS 2002

5084 A. A. Jensen et al. (Eur. J. Biochem. 269)

that EGFP, the most widely used GFP variant, can be
used as fluorescent acceptor in this BRET2 assay in
contrast to the original BRET assay using Rluc/coelenterazine h [34,40], may hold some practical advantages for
future studies of GFP fusion proteins.
As the obtained BRET signal patterns using GFP2 and
EGFP as fluorescent acceptor proteins were similar, GFP
will be used as a common reference point in the following
sections.
Receptor specificity of BRET

Fig. 8. BRET in tsA cells transfected with Rluc- and EGFP-tagged
receptors. The experiments were performed as described in Experimental
Procedures, and the BRET ratio is given as (emission515 nm ) background515 nm)/(emission410 nm ) background410 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 lg CaRD886-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.

(compare Figs 7 and 8). In agreement with the experiments
with the GFP2-tagged receptors, no agonist-induced BRET
was detected for any of the Rluc/EGFP–tagged receptor
combinations (data not shown).

DISCUSSION
Evaluation of the BRET2 assay
The present study is the second publication, where
dimerization between Rluc- and GFP2-tagged proteins
has been demonstrated using the modified form of
coelenterazine h DeepBlueCTM as the substrate [41]. The
emission of DeepBlueCTM 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/GFP2 in the
presence of DeepBlueCTM than between Rluc and Rluc/
EYFP using coelenterazine h as substrate, the Rluc/
DeepBlueCTM/GFP2 system provides better signal-to-noise

ratios than the Rluc/coelenterazine h/EYFP system
(Fig. 6) [34,40]. Interestingly, the intensity of the BRET
signal caused by proximity of Rluc and EGFP was
comparable to that elicited by Rluc and GFP2 in this
system (Figs 6–8). Thus, GFP2 and EGFP are both
suitable acceptor molecules in the BRET2 assay. The fact

Numerous observations support that the BRET signals
obtained in tsA cells transfected with the Rluc- and GFPtagged 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 receptors 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-GFP2 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/CaRD886GFP 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
hormone receptor [35]. The most likely explanation for the

insuppressible fraction of the BRET signal is that the
cellular distribution patterns of WT CaR, CaRD1036-V5/
His and CaRD886-V5/His are somewhat different from
those of CaRD886-GFP and CaRD886-Rluc. Hence, BRET
could arise from interactions between intracellular
CaRD886-GFP and CaRD886-Rluc proteins in cellular
compartments not expressing WT CaR or the V5/Histagged CaRs.
Constitutive homodimerization of CaR
This study provides the first evidence of dimerization of
CaR or any other family C GPCR in living cells. The
finding that CaR exists as a homodimer under basal
conditions is hardly a surprise. The crystal structure of the
mGluR1 ATD homodimer has strongly suggested
that mGluR1 is constitutively dimerized, and several
groups have demonstrated CaR homodimerization using
coimmunoprecipitation techniques [20,25,26,56]. However,
incomplete solubilization of the receptors prior to the
coimmunoprecipitation step in these experiments could
cause aggregation, which in turn could be misinterpreted as
receptor dimer formation. Hence, this study supplements
the findings from the coimmunoprecipitation studies of
CaR dimerization.


Ó FEBS 2002

Homodimerization of CaR in living cells (Eur. J. Biochem. 269) 5085

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 GFPand Rluc-tagged CaRs (Figs 7 and 8). The recent
demonstration of agonist-induced BRET for the insulin
receptor, which is also constitutively dimerized, proves the
validity of this technique in studies of conformation
changes in dimeric receptor complexes [57]. However, the
intermolecular distances in the CaR homodimer are most
likely quite different from those in the insulin receptor
dimer.
One explanation for the lack of agonist-induced BRET
for CaR is that the chromophore/fluorophore of the Rluc
and GFP molecules are positioned so close in the resting
conformation of the homodimer that maximal BRET
intensity already has been achieved. In an attempt to
probe other intermolecular distances in the CaR homodimer, we have also studied CaRs with Rluc and GFP
molecules tagged to the intracellular loop 1 (i1) (Jensen,
Hansen, Sheikh and Brauner-Osborne, unpublished data).
ă
However, as these fusion proteins were retained in vesicles

inside of the cells, we were not able to use them in the
BRET studies. It would have been interesting to tag Rluc
and GFP molecules to the i2 and i3 of CaR as well.
However, as truncations in these regions of CaR have
been demonstrated to reduce the cell surface expression of
the receptor dramatically [58], we have not made these
constructs.
An alternate interpretation of the lack of agonist-induced
BRET observed in this study is that the translation of
agonist binding to the ATDs of the family C GPCR
homodimer into G-protein coupling of the 7TM)7TM
moiety is mediated by another mechanism than that
proposed by Kunishima et al. [20]. A couple of pharmacological observations support this speculation: the trivalent
cation Gd3+ has been shown to activate CaR directly at its
7TM [18], the somatic Ala843 fi Glu mutation in TM7 of
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 phenomena originate exclusively from the 7TM of the family C
GPCR and are unlikely to be accompanied by a conformational change in the ATD dimer. Furthermore, a recent
study of the GABAB 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 demonstration of family C GPCR dimerization in living cells. We have

demonstrated that CaR is constitutively dimerized. However, 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
technical perspective, we have demonstrated that interactions between Rluc- and GFP2/EGFP-tagged proteins can
be recorded using DeepBlueCTM as the substrate.
The BRET2 assay appears to have a higher signal-to-noise
ratio than previously reported BRET assays and may
represent a small step forward in the study of protein–protein
interactions.

ACKNOWLEDGEMENTS
Søren G. F. Rasmussen and Professor Ulrik Gether are thanked for the
use of the SPEX Fluoromax-2 spectrofluorometer, and Birger Brodin
for technical assistance with the single cell fluorescence measurements.
Mette B. Hermit is thanked for developing the protocol used for the
immunofluorescence experiments.
This work was supported by grants from the Danish Medical
Research Council and the Novo Nordisk Foundation (AAJ, JLH, SPS
and HBO), by the Lundbeck Foundation (AAJ) and by the Danish
Heart Foundation no. 01-1-2-22–22895 and no. 00-2-2–24 A-22838, the
Villadsen Family Foundation, the Birthe and John Meyer Foundation,
and the Foundation of 17.12.1981 (JLH and SPS).

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