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*, Sứren P Sheikh2 and Hans Brauner-Osborne1 ă 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 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, 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, metabotropic glutamate receptor; CaR, calcium-sensing receptor; GABABR, c-aminobutyric acid receptor type B; ATD, amino terminal domain; 7TM, 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, and 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 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 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 Ó FEBS 2002 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 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 ƠGFPÕ Ĩ 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; : 500) or anti-HA (clone 12CA5, Roche Molecular Biolabs; : 100) monoclonal Igs for h Following · 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 h with secondary Cy3-conjugated affinity-purified goat antimouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA; : 200) Then the cells were washed (2 · 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 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 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 The cells were washed · with NaCl/Pi, incubated with 500 lL NaCl/Pi supplemented with 10% fetal calf serum for 20 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 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 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 nm, increment 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 lg plasmid the Ó FEBS 2002 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 · 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 lM, and measurements were performed in a FusionTM reader (Packard Bioscience) (read time 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 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 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 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 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 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 Ó FEBS 2002 Homodimerization of CaR in living cells (Eur J Biochem 269) 5081 Fig 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 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 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 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 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 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 lg pSI (vector alone) or CaR-pSI (WT CaR), respectively, and assayed as described in Experimental Procedures (compare Figs 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 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 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 (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 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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... 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... 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