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 dimer
Trang 1Probing intermolecular protein–protein interactions in the
calcium-sensing receptor homodimer using bioluminescence
resonance energy transfer (BRET)
Anders A Jensen1*, Jakob L Hansen2*, Søren P Sheikh2and Hans Bra¨uner-Osborne1
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 newbioluminescence 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
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 GFP2molecules
Keywords: family C GPCR; CaR; BRET; dimerization; homodimerization
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
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 equili-brates 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 newstructural motif recog-nizable 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
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: aaj@dfh.dk
Abbreviations: GPCR, G-protein coupled receptor; mGluR,
metabo-tropic glutamate receptor; CaR, calcium-sensing receptor;
GABA B R, c-aminobutyric acid receptor type B; ATD, amino
terminal domain; 7TM, 7 transmembrane moiety; BRET,
biolumi-nescence resonance energy transfer; FRET, fluorescence resonance
energy transfer; Rluc, Renilla luciferase; GFP, green fluorescent
protein; EGFP, enhanced green fluorescent protein; EYFP, enhanced
yellowfluorescent 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)
Trang 2luminescent 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 betw een the tw o 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 yellowfluorescent
protein (EYFP) as luminescent donor and fluorescent
acceptor, respectively, and coelenterazine h as the
sub-strate 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
E X P E R I M E N T A L P R O C E D U R E S
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)
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
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–ApaI fragment of CaRD1036-Rluc and CaRD886-Rluc into the pCDNA6-V5/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 nucleo-tides 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 subse-quent 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 Ser65fi Thr mutation in the GFP2part 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 GABAB 1a-EGFP 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
Fig 1 The Rluc-, GFP2- and EGFP-tagged receptors (A) The topo-logy of the Rluc-, GFP 2 - or EGFP-tagged GPCRs used in the present study (B) The fusion regions of the Rluc- and GFP2/EGFP-tagged receptors GFP 2 and EGFP are given as GFP.
Trang 3except 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-GFP2were 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)
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 UÆmL)1),
streptomycin (100 lgÆmL)1), 10% dialyzed fetal calf serum
and 1 lCiÆmL)1myo-[2–3H]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) AT1areceptor 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· 105cells per well) were split into wells of a 6
well-culture plate and transfected with 0.4 lg of a GFP2
-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
NaCl/Pi, resuspended in 300 lL NaCl/Piand distributed in
black optiplates (Packard) Fluorescence and luminescence
recordings were performed in a FusionTMreader (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
experi-ments 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 polyD-lysine coated 24-well
tissue culture plate in DMEM with reduced
concentra-tions of CaCl2(0.9 mM) and MgCl2(0.8 mM)
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/Piand 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
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/Pisupplemented with 10% fetal calf serum the cells were incubated for 1 h with secondary Cy3-conjugated affinity-purified goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA; 1 : 200) Then the cells were washed (2· 5 min) with NaCl/Pi and view ed through a Leica
DM IRB fluorescence microscope
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
mGluR1D877-EGFP 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 concen-trations of CaCl2 (0.9 mM) and MgCl2 (0.8 mM), supple-mented with penicillin (100 UÆmL)1), streptomycin (100 lgÆmL)1) and 10% dialyzed calf serum The follow-ing day, sfollow-ingle 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/Piand resuspended in 500 lL NaCl/Piin a cuvette DeepBlueCTMwas added to a final concentration of
5 lM, and light emission acquisition (340–600 nM) w as 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 GFP2the 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
Trang 4following day (5 lg of one plasmid, 2.5 lg of each of tw o
plasmids, or otherwise indicated) The day after
transfec-tion the medium was changed The following day, the cells
w ere w ashed 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
DeepBlueCTMwas 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
Rluc-tagged CaRs, the cells were mechanically lyzed
immedi-ately 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
R E S U L T S
Pharmacological characterization of
Rluc-and EGFP-tagged CaRs
In excellent agreement with a previous study of
EGFP-tagged 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
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
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
fluo-rescence and luminescence in cells cotransfected with
various combinations of GFP2- and Rluc-constructs Cells
were transfected with similar amounts of cDNA of
Rluc-and GFP2-constructs as those used in the BRET
experi-ments
The levels of fluorescence in cells transfected with the
GFP2-tagged receptors were comparable in size, whereas
GFP2was expressed at slightly higher levels (Fig 3A) The
luminescence levels in CaRD1036-Rluc and CaRD886-Rluc
transfected cells were similar, whereas cells expressing Rluc
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-GFP2and CaR-Rluc fusion proteins, respectively, and visualized these using immunofluorescence microscopy (Fig 4) No fluo-rescence 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 immunofluores-cence technique further, we took advantage of the well-established 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
Fig 2 Pharmacological characterization of EGFP- and Rluc-tagged CaRs (A) Concentration-response curves of Ca2+-induced IP accu-mulation in tsA cells transfected with WT CaR, CaRD1036-V5/His, CaRD1036-Rluc, CaRD1036-EGFP, 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 R and At1aD359-GFP 2 Data are given fold response [R/
R basal ].
Trang 5HA-tagged GABAB1 receptors, when these were
cotrans-fected with WT GABAB2(data not shown)
The c-myc-CaR/HA-CaR, c-myc-CaRD1036-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
CaRD886-receptors and that of WT CaR appeared to be similar
The cellular expression patterns of the GFP-tagged
receptors were investigated in greater detail using
confo-cal microscopy The expression patterns of
CaRD1036-EGFP and CaRD886-CaRD1036-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
ex-periments 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-GFP2was 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 fewamino 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
Fig 4 Immunofluorescence analysis of 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-GFP2and 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.
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 2 -combinations The ratio is given as [Fluorescence/Luminescence].
Trang 6WT 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
signal-to-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/GFP2transfected 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(estimatedS (max EGFP)
55 000 cm)1ÆM )1(Clontech) and estimatedS (max GFP2)
21 000 cm)1ÆM )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 GFP2 -tagged receptors alone no luminescence signals were detec-ted (data not shown)
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).
Trang 7Significant BRET signals were obtained for all
CaR-GFP2and CaR-Rluc combinations (Fig 7B) BRET ratios
between 0.11 and 0.17 were obtained for every combination
including CaRD1036-Rluc or CaRD1036-GFP2, w hereas
the CaRD886-Rluc/CaRD886-GFP2combination 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) Further-more, the BRET signal obtained with CaRD886-Rluc and CaRD886-GFP2was reduced considerably by coexpression
Fig 6 Spectral properties of DeepBlueCTMillumination (A) Light-emission acquisition spectrum of Cos7 cells transfected with Rluc/EGFP, Rluc/ GFP2(pBRET+) and pRluc-N2 Cells were incubated with 5 l M 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/GFP 2 (pBRET+) and Rluc-N2 The BRET ratio is given as emission500)530 nM/emission370)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.
Trang 8of 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-GFP2with
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-GFP2transfected 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 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/GFP2 combinations
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 (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 2 ) or Rluc/EGFP in absence and presence of 20 m M CaCl 2 (B) BRET in tsA cells transfected with Rluc- and GFP 2 -tagged CaRs (C) Receptor specificity of the BRET In [1], BRET obtained in tsA cells transfected with CaRD886-Rluc or CaRD886-GFP 2 and pRluc-N2, pGFP 2 -N3 or AT1aD359-GFP2were recorded In [2], two 10 cm culture dishes of tsA cells were transfected with 2.5 lg CaRD886-Rluc and 2.5 lg CaRD886-GFP 2 , 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 GFP2or Rluc Cells were transfected with 0.5 lg CaRD886-Rluc, 0.5 lg CaRD886-GFP2and 4 lg of various plasmids (pSI, CaR-pSI, CaRD1036-V5/His, CaRD886-V5/His, m1-pCD, H1-pCDNA3 and GABA B 2-pCDNA3) and assayed as described in Experimental Procedures.
Trang 9(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)
D I S C U S S I O N
Evaluation of the BRET2assay
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/GFP2system 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 BRET2assay The fact
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/coelen-terazine h [34,40], may hold some practical advantages for future studies of GFP fusion proteins
As the obtained BRET signal patterns using GFP2and EGFP as fluorescent acceptor proteins were similar, GFP 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-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/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 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/His-tagged 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
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 (emission 515 nm )
back-ground 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 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.
Trang 10Agonist-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 GFP-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
homo-dimer, we have also studied CaRs with Rluc and GFP
molecules tagged to the intracellular loop 1 (i1) (Jensen,
Hansen, Sheikh and Bra¨uner-Osborne, unpublished data)
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
pharmaco-logical observations support this speculation: the trivalent
cation Gd3+has been show n to activate CaR directly at its
7TM [18], the somatic Ala843fi 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
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 GABABreceptor 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 dimerizademonstra-tion 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 technical perspective, we have demonstrated that interac-tions between Rluc- and GFP2/EGFP-tagged proteins can
be recorded using DeepBlueCTM as the substrate The BRET2assay 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
A C K N O W L E D G E M E N T S
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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