Constitutive Agonist-independent CCR5 Oligomerization and Antibody-mediated Clustering Occurring at Physiological Levels of Receptors
2002; Elsevier BV; Volume: 277; Issue: 38 Linguagem: Inglês
10.1074/jbc.m202386200
ISSN1083-351X
AutoresHassan Issafras, Stéphane Angers, Sébastien Bulenger, Cédric Blanpain, Marc Parmentier, Catherine Labbé‐Jullié, Michel Bouvier, Stéfano Marullo,
Tópico(s)Chemokine receptors and signaling
ResumoAlthough homo-oligomerization has been reported for several G protein-coupled receptors, this phenomenon was not studied at low concentrations of receptors. Furthermore, it is not clear whether homo-oligomerization corresponds to an intrinsic property of nascent receptors or if it is a consequence of receptor activation. Here CCR5 receptor oligomerization was studied by bioluminescence resonance energy transfer (BRET) in cells expressing physiological levels of receptors. A strong energy transfer could be observed, in the absence of ligands, in whole cells and in both endoplasmic reticulum and plasma membrane subfractions, supporting the hypothesis of a constitutive oligomerization that occurs early after biosynthesis. No change in BRET was observed upon agonist binding, indicating that the extent of oligomerization is unrelated to the activation state of the receptor. In contrast, a robust increase of BRET, induced by a monoclonal antibody known to promote receptor clustering, suggests that microaggregation of preformed receptor homo-oligomers can occur. Taken together, our data indicate that constitutive receptor homo-oligomerization has a biologically relevant significance and might be involved in the process of receptor biosynthesis. Although homo-oligomerization has been reported for several G protein-coupled receptors, this phenomenon was not studied at low concentrations of receptors. Furthermore, it is not clear whether homo-oligomerization corresponds to an intrinsic property of nascent receptors or if it is a consequence of receptor activation. Here CCR5 receptor oligomerization was studied by bioluminescence resonance energy transfer (BRET) in cells expressing physiological levels of receptors. A strong energy transfer could be observed, in the absence of ligands, in whole cells and in both endoplasmic reticulum and plasma membrane subfractions, supporting the hypothesis of a constitutive oligomerization that occurs early after biosynthesis. No change in BRET was observed upon agonist binding, indicating that the extent of oligomerization is unrelated to the activation state of the receptor. In contrast, a robust increase of BRET, induced by a monoclonal antibody known to promote receptor clustering, suggests that microaggregation of preformed receptor homo-oligomers can occur. Taken together, our data indicate that constitutive receptor homo-oligomerization has a biologically relevant significance and might be involved in the process of receptor biosynthesis. G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; BRET, bioluminescence resonance energy transfer; FRET, fluorescence resonance energy transfer; GABA, γ-amino butyric acid; hRluc, "humanized" Renilla luciferase; MIP-1β, macrophage inflammatory polypeptide 1β; RANTES, regulated on activation normal T cell expressed and secreted; YFP, yellow mutant of the enhanced green fluorescent protein; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorting. constitute the largest family of membrane receptors. They are involved in the regulation of most biological functions and represent, collectively, one of the most important targets for therapeutic intervention. A rapidly growing number of studies indicate that GPCRs may be organized as oligomers (1Angers S. Salahpour A. Bouvier M. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 409-435Crossref PubMed Scopus (515) Google Scholar). The biological function of this phenomenon was questioned for some time until it was reported that the hetero-oligomerization of two isoforms of the GABABreceptor was indispensable for the formation of functional GABAB binding sites in tissues (2Kaupmann K. Malitschek B. Schuler V. Heid J. Froestl W. Beck P. Mosbacher J. Bischoff S. Kulik A. Shigemoto R. Karschin A. Bettler B. Nature. 1998; 396: 683-687Crossref PubMed Scopus (1017) Google Scholar, 3Jones K.A. Borowsky B. Tamm J.A. Craig D.A. Durkin M.M. Dai M. Yao W.J. Johnson M. Gunwaldsen C. Huang L.Y. Tang C. Shen Q. Salon J.A. Morse K. Laz T. Smith K.E. Nagarathnam D. Noble S.A. Branchek T.A. Gerald C. Nature. 1998; 396: 674-679Crossref PubMed Scopus (925) Google Scholar, 4White J.H. Wise A. Main M.J. Green A. Fraser N.J. Disney G.H. Barnes A.A. Emson P. Foord S.M. Marshall F.H. Nature. 1998; 396: 679-682Crossref PubMed Scopus (1015) Google Scholar). Hetero-oligomerization of other GPCRs has also been proposed as a mean to increase pharmacological diversity and expand signaling modes for this class of receptors (5Jordan B.A. Devi L.A. Nature. 1999; 399: 697-700Crossref PubMed Scopus (978) Google Scholar, 6Rocheville M. Science. 2000; 288: 154-157Crossref PubMed Scopus (751) Google Scholar). However, very little is known about the role that receptor homo-oligomerization could play. The fact that homo-oligomerization has been studied mainly with receptors overexpressed in heterologous systems has even raised concerns about the biological relevance of this phenomenon. Whether oligomeric complexes form at the plasma membrane or in other subcellular compartments and whether or not oligomers result from a dynamic, regulated, and reversible assembly of monomers following receptor activation are two other important and still open questions. The latter question, in particular, was the object of several studies using different experimental approaches that gave different interpretations. For example, it was concluded that agonists could increase homo-oligomerization of β2-adrenergic (7Hebert T.E. Moffett S. Morello J.P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem. 1996; 271: 16384-16392Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar) and TRH (8Kroeger K.M. Hanyaloglu A.C. Seeber R.M. Miles L.E. Eidne K.A. J. Biol. Chem. 2001; 276: 12736-12743Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) receptors, decrease the homo-dimerization of the δ-opioid receptor (9Cvejic S. Devi L.A. J. Biol. Chem. 1997; 272: 26959-26964Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar), or have no effect on the oligomeric state of M3-muscarinic receptors (10Zeng F.Y. Wess J. J. Biol. Chem. 1999; 274: 19487-19497Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). The issue of receptor oligomerization and its potential regulation by agonists is of particular interest for the chemokine receptor CCR5, one of the two major co-receptors for the human immunodeficiency virusin vivo, since it has been suggested that CCR5 dimerization might prevent human immunodeficiency virus infection (11Vila-Coro A.J. Mellado M. Martin De Ana A. Lucas P. del Real G. Martinez A.C. Rodriguez-Frade J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3388-3393Crossref PubMed Scopus (130) Google Scholar). In one study, CCR5 homo-oligomerization was detected after chemical cross-linking and immunoprecipitation of solubilized receptors but only if cells were previously treated with an agonist or a divalent antibody. The authors concluded that the CCR5 receptor exists exclusively as monomeric entities under basal conditions and that it oligomerizes only upon receptor activation or antibody-mediated reticulation (11Vila-Coro A.J. Mellado M. Martin De Ana A. Lucas P. del Real G. Martinez A.C. Rodriguez-Frade J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3388-3393Crossref PubMed Scopus (130) Google Scholar). These conclusions are, however, difficult to reconcile with a previous study in which constitutive CCR5 dimers were resolved under low stringent conditions of protein denaturation (12Benkirane M. Jin D.Y. Chun R.F. Koup R.A. Jeang K.T. J. Biol. Chem. 1997; 272: 30603-30606Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). The fact that two similar studies, both based on immunoprecipitation experiments from cells expressing uncontrolled levels of receptors, reached such divergent conclusions prompted us to develop another experimental approach to study CCR5 homo-oligomerization in living cells at physiological concentrations of receptor. In the present study, CCR5 receptor oligomerization was investigated in intact HEK-293 cells using bioluminescence resonance energy transfer (BRET). Unambiguous constitutive homo-oligomerization, evidenced by a robust energy transfer in the absence of ligands, could be demonstrated in HEK-293 cells expressing the same density of CCR5 receptors as found in human monocytes and lymphocytes. Furthermore, consistent with the notion of constitutive oligomerization, BRET was detected within the endoplasmic reticulum, indicating that oligomerization occurs early after receptor biosynthesis. Antibodies but not agonists could increase the basal energy transfer, probably as a result of preformed oligomer clustering. If not otherwise specified, all chemicals and reagents were from Sigma. The CCR5 and CXCR4 coding sequences without a stop codon were amplified using sense and antisense primers harboring unique HindIII and BamHI sites. The fragments were then subcloned in frame into theHindIII/BamHI sites of the pGFP-N1-Topaz, (PerkinElmer Life Sciences) and a humanized form ofRenilla luciferase (hRluc)-N3, (BioSignal; PerkinElmer Life Sciences) vectors encoding the YFP yellow variant of green fluorescent protein, and of the humanizedRenilla luciferase. In the resulting constructs, YFP and hRluc were directly fused to the 3′-end of the receptor cDNAs. Peripheral mononuclear cells were isolated from freshly collected heparinized blood of healthy adult donors by Ficoll density gradient centrifugation (Eurobio Biotechnology, Les Ulis, France) as previously described (13Ferrante A. Thong Y.H. J. Immunol. Methods. 1980; 36: 109-117Crossref PubMed Scopus (447) Google Scholar). CD4+ and CD14+ populations were identified by fluorescein isothiocyanate-conjugated monoclonal antibody binding (BD Pharmingen, Heidelberg, Germany). Lymphocytes, separated from monocytes by adherence on plastic, were cultured in RPMI 1640 supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm glutamine, at 37 °C in an atmosphere of 5% CO2. They were stimulated by 3-day incubation with anti-CD3 antibodies (UCHT-1; Seralab) bound to dynabeads (Dynal) precoated with sheep anti-mouse IgG. Binding to beads was achieved by incubating 0.5 μg of anti-CD3 antibodies with 107 beads in PBS, 0.1% bovine serum albumin for 1 h at room temperature. Human embryonic kidney (HEK-293) and HeLa cells, maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and streptomycin (all from Invitrogen), were seeded at a density of 3 × 105 cells in 35-mm plates. Transient transfections were performed the following day using Fugene (Roche Molecular Biochemicals) according to the manufacturer's protocol. Cells were harvested and used 24 h after transfection. Stable cell lines expressing wild-type CCR5 or CCR5 fused to either YFP or hRluc were also selected using 600 μg/ml G418 in the culture medium. The expression level of stable clones was assessed by quantitative FACS analysis as described below. Cyclic AMP assays were conducted on stable HEK-293 clones expressing wild-type CCR5 or CCR5 fused to either YFP or hRluc. The accumulation of cAMP in intact cells was determined using the Alpha-Screen system (PerkinElmer Life Sciences). This immunoassay is based on the competition between the cAMP contained within the cells to be tested and biotinylated cAMP. Biotinylated cAMP is complexed by streptavidin molecules, which are covalently bound to "donor" beads, whereas an anti-cAMP monoclonal antibody is bound to "acceptor" beads. The interaction between biotinylated cAMP and the antibody bridges donor and acceptor beads. Upon appropriate irradiation (680 nm) of the donor beads, singlet oxygen is released and diffuses to activate the acceptor beads only if donor and acceptor beads are in close proximity. Activated acceptor beads then emit at 520–620 nm. The emission signal, recorded using a polyvalent fluorescence-luminescence detector (Fusion; PerkinElmer Life Sciences) decreased linearly with the increase of competing cellular cAMP concentration. Calibration curves were constructed using known concentrations of added cAMP. Adenylyl cyclase inhibition, promoted by the activation of wild-type CCR5, CCR5-YFP, and CCR5-hRluc, was measured in stable clones incubated with increasing concentrations of MIP-1β (Preprotech Inc., Rocky Hill, NJ), after enzyme prestimulation with 10 μmforskolin. Receptor internalization was assessed by flow cytometry (FACS). HEK-293 cells, expressing either wild-type CCR5, CCR5-YFP, or CCR5-hRluc were incubated at 37 °C with 100 nm RANTES (R & D Systems Europe, Abingdon, UK) or a 10 μg/ml concentration of the anti-CCR5 MC-1 monoclonal antibody for the indicated time. After washing with ice-cold PBS, cells were incubated for 2 min at 37 °C in an acidic buffer, containing 100 mm NaCl and 50 mm glycine, pH 3, to remove bound RANTES or the MC-1 antibody. Cells were incubated at 4 °C for 1 h with the monoclonal phycoerythrin-conjugated 2D7 antibody, directed against CCR5 (PharMingen, San Diego, CA). Cells were then washed with ice-cold PBS and fixed with 1% formaldehyde. Samples were analyzed with a Becton-Dickinson flow cytometer. Base-line fluorescence, determined from untransfected HEK-293 cells, was subtracted from each sample, and the cell surface expression was defined as the mean fluorescence signal multiplied by the number of positive cells. 24 h after transfection with the indicated cDNAs encoding BRET donors and acceptors, HEK-293 or HeLa cells were detached with PBS/EDTA and washed in PBS. Aliquots of 1 × 105 cells were distributed in the wells of 96-well microplates (White Optiplate from PerkinElmer Life Sciences) in the presence or absence of CCR5 ligands (100 nm) or antibodies (10 μg/ml). The monoclonal MC-1, MC-5, and MC-6 antibodies, directed against various epitopes of the CCR5 receptor, were generated in the laboratory of Dr. Matthias Mack (University of Munich). Single-chain antibody and Fab fragments from the MC-1 antibody were prepared as described (14Blanpain C. Vanderwinden J.M. Cihak J. Wittamer V., Le Poul E. Issafras H. Stangassinger M. Vassart G. Marullo S. Schlöndorff D. Parmentier M. Mack M. Mol. Biol. Cell. 2002; 13: 723-737Crossref PubMed Scopus (124) Google Scholar). The luciferase substrate, coelenterazine h(Molecular Probes Europe, Leiden, The Netherlands), was added at a final concentration of 5 μm, and emitted luminescence and fluorescence were measured simultaneously using the FUSION fluorescence-luminescence detector (PerkinElmer Life Sciences). Cells expressing BRET donors alone (receptors fused to hRluc) were used to determine background. Filter sets were 485 ± 10 nm for luciferase emission and 530 ± 12.5 nm for YFP emission. Mean values from five consecutive measurements were used to determine BRET ratios that were calculated as described (15Angers S. Salahpour A. Joly E. Hilairet S. Chelsky D. Dennis M. Bouvier M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3684-3689PubMed Google Scholar). Cell surface expression of CCR5 and CXCR4 was quantified by flow cytometry using the phycoerythrin-conjugated 2D7 and 12G5 monoclonal antibodies (PharMingen), respectively. The number of PE molecules bound on the surface of lymphocytes, monocytes, and transfected HEK-293 cells was determined in parallel, using the Quanti-BRITE PE Fluorescence Quantitation kit (Becton Dickinson Immunocytometry Systems, San Jose, CA) and the software provided by the manufacturer. For each experiment, fluorescence calibration curves were established with beads coated with known amounts of epitopes. Receptor density was expressed as the number of epitopes recognized by the antibodies per mg of total proteins, measured using the BCA protein assay (Pierce). To determine the proportion of receptors at the cell surface, comparative FACS analyses were conducted on intact cells and cells permeabilized with 0.05% saponin for 1 h at 4 °C. HEK-293 cells grown in 10-cm dishes were transiently transfected with CCR5-hRluc and CCR5-YFP plasmids. 48 h after transfection, the cells were washed three times with ice-cold PBS, scraped off the dish, and lysed with 1 ml of ice-cold hypotonic lysis buffer (20 mm HEPES, pH 7.4, 2 mm EDTA, 2 mm EGTA, 6 mmmagnesium chloride, 1 mm phenylmethylsulfonyl fluoride, 10 μm leupeptin, 10 μm aprotinin, 1 mm benzamidine). The cell lysate was homogenized with 50 strokes of a Dounce homogenizer. Cellular debris and unlysed cells were removed by centrifuging at 1000 × g for 5 min at 4 °C. The supernatant was collected, and sucrose was added to obtain a final concentration of 0.2 m sucrose. The discontinuous sucrose step gradient was made using the above hypotonic lysis buffer with the addition of sucrose at the following final molar concentrations: 0.5, 0.9, 1.2, 1.35, 1.5, and 2.0. Each step in the gradient had a total volume of 5 ml. The samples were centrifuged for 16 h at 27,000 rpm in a Beckman SW28 rotor. A total of 31 fractions of 1 ml each were collected from the top of the tube. BRET was measured on 100 μl of each fraction. The efficiency of the sucrose gradient to resolve the plasma membrane from the endoplasmic reticulum was verified by immunoblotting. Each fraction was probed with a mouse monoclonal antibody that specifically recognizes the α subunit of the Na/K-ATPase pump (Sigma A-276) (plasma membrane marker) at a dilution of 1:250 and a polyclonal anti-calnexin antibody (Stressgen Biotechnologies SPA-860) (endoplasmic reticulum marker) at a dilution of 1:8000. HeLa cells expressing either wild-type dynamin or the K44A dominant negative mutant of dynamin, both under the control of a regulated tetracyclin promoter (Tet-off system), were generated in the laboratory of Dr. Sandra Schmid (Scripps Clinic, San Diego, CA). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml each penicillin and streptomycin, 400 μg/ml G418, 200 ng/ml puromycin, and 1 μg/ml doxycyclin. Overexpression of wild-type and dominant negative K44A dynamin was induced by removing doxycyclin from the medium 12 h after the transfection with the cDNAs encoding CCR5-hRluc and CCR5-YFP. BRET analysis and the endocytosis assay were conducted in parallel 48 h after transfection. Under these conditions, a ∼20-fold overexpression of dynamin could be obtained, as determined by Western blots carried on whole-cell lysates with a monoclonal anti-dynamin antibody from the laboratory of Dr. Sandra Schmidt. For immunofluorescence studies, HeLa cells were seeded on coverslips in six-well plates, transfected with a plasmid encoding CCR5-YFP, and used for immunofluorescence 2 days post-transfection. After the indicated treatments, cells were fixed and processed for fluorescence microscopy as described previously (16Scott M.G. Benmerah A. Muntaner O. Marullo S. J. Biol. Chem. 2002; 277: 3552-3559Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Samples were examined under an epifluorescence microscope (Leica DM-IRB) attached to a cooled CCD camera (Micromax 1300YHS; Princeton Instruments). Images were processed using the Metamorph™ software. Previous studies, based on immunoprecipitation assays after cell solubilization, indicated that CCR5 receptors might form homo- and hetero-oligomers (12Benkirane M. Jin D.Y. Chun R.F. Koup R.A. Jeang K.T. J. Biol. Chem. 1997; 272: 30603-30606Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 11Vila-Coro A.J. Mellado M. Martin De Ana A. Lucas P. del Real G. Martinez A.C. Rodriguez-Frade J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3388-3393Crossref PubMed Scopus (130) Google Scholar, 17Mellado M. Rodriguez-Frade J.M. Vila-Coro A.J. Fernandez S. Martin de Ana A. Jones D.R. Toran J.L. Martinez A.C. EMBO J. 2001; 20: 2497-2507Crossref PubMed Scopus (380) Google Scholar), as reported for a growing number of GPCRs (reviewed in Ref. 18Bouvier M. Nat. Rev. Neurosci. 2001; 2: 274-286Crossref PubMed Scopus (581) Google Scholar). However, these studies were discrepant on important issues such as the constitutive or induced nature of CCR5 oligomerization. In an effort to better characterize CCR5 oligomerization in living cells, we took advantage of a biophysical assay based on BRET, which was initially developed to monitor homodimerization of cyanobacteria clock proteins (19Xu Y. Piston D.W. Johnson C.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 151-156Crossref PubMed Scopus (506) Google Scholar) and more recently applied to the study of GPCRs oligomerization (see Ref. 18Bouvier M. Nat. Rev. Neurosci. 2001; 2: 274-286Crossref PubMed Scopus (581) Google Scholar for a review). Based on the Förster equation (20Mahajan N.P. Linder K. Berry G. Gordon G.W. Heim R. Herman B. Nat. Biotechnol. 1998; 16: 547-552Crossref PubMed Scopus (267) Google Scholar), it was estimated that the distance allowing energy transfer between BRET pairs is on the order of 25–100 Å. Therefore, the detection of a specific BRET between two receptors demonstrates a physical proximity that can be explained best by the formation of receptor dimers or oligomers (15Angers S. Salahpour A. Joly E. Hilairet S. Chelsky D. Dennis M. Bouvier M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3684-3689PubMed Google Scholar). For this purpose, fusion proteins were constructed between CCR5 and either hRluc or YFP. FACS analysis of intact and permeabilized HEK-293 cells, transiently expressing each chimeric receptor, revealed a subcellular distribution similar to that of control wild-type CCR5 receptor: about 70% of both native or chimeric receptors was found at the plasma membrane 24 h after transfection (data not shown). Fusion receptors maintained their functional properties, as indicated by the similar potencies of MIP-1β to inhibit forskolin-promoted cAMP production via wild-type CCR5, CCR5-hRluc, and CCR5-YFP (IC50 of 1.61 ± 0.08, 1.5 ± 0.07, and 0.83 ± 0.03 nm, respectively; Fig.1 A). Also, agonist binding induced a similar extent and rate of receptor internalization of each receptor construct (Fig. 1 B). The cDNAs encoding CCR5-hRluc and CCR5-YFP were transiently co-expressed in HEK-293 cells, and the energy transfer was measured after the addition of coelenterazine h, the cell-permeable substrate of hRluc. As shown in Fig.2 A, a significant (p < 0.01) energy transfer was observed between CCR5-hRluc and CCR5-YFP upon coelenterazine h addition in the absence of any CCR5 ligand. No such signal was observed in cells expressing CCR5-hRLuc alone or when CCR5-YFP was replaced by a closely related chemokine receptor, fused to YFP (CXCR4-YFP). The CXCR4 receptor was preferred to the CCR2 as a negative control for BRET, as it was reported that the latter receptor might form functional hetero-oligomers with CCR5 (17Mellado M. Rodriguez-Frade J.M. Vila-Coro A.J. Fernandez S. Martin de Ana A. Jones D.R. Toran J.L. Martinez A.C. EMBO J. 2001; 20: 2497-2507Crossref PubMed Scopus (380) Google Scholar). The lack of BRET between CCR5-hRluc and CXCR4-YFP did not result from the inability of CXCR4 to serve as a BRET acceptor, since a particularly strong signal was observed upon co-expression of CXCR4-YFP and CXCR4-hRluc. The stronger signal observed with CXCR4 does not necessarily reflect a higher proportion of receptors engaged in constitutive oligomers, since it could result from differences in orientation and/or distance between BRET partners. The selectivity of the interaction was also demonstrated by BRET competition experiments where the BRET signal was studied in the presence of increasing concentrations of native receptors. Overexpression of CCR5 but not of CXCR4 inhibited the transfer of energy between CCR5-hRluc and CCR5-YFP (Fig. 2 B). BRET data, which support the existence of constitutive CCR5 oligomerization, are in agreement with a previous report in which CCR5 oligomers could be immunoprecipitated from cell lysates of untreated cells and visualized by Western blot (12Benkirane M. Jin D.Y. Chun R.F. Koup R.A. Jeang K.T. J. Biol. Chem. 1997; 272: 30603-30606Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). However, in other reports, CCR5 oligomers became apparent only when intact cells were preincubated with agonists or bivalent antibodies before chemical cross-linking, solubilization and immunoprecipitation (11Vila-Coro A.J. Mellado M. Martin De Ana A. Lucas P. del Real G. Martinez A.C. Rodriguez-Frade J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3388-3393Crossref PubMed Scopus (130) Google Scholar, 17Mellado M. Rodriguez-Frade J.M. Vila-Coro A.J. Fernandez S. Martin de Ana A. Jones D.R. Toran J.L. Martinez A.C. EMBO J. 2001; 20: 2497-2507Crossref PubMed Scopus (380) Google Scholar). By comparing the experimental protocols used in these studies, we noticed that the authors, who could not detect the presence of constitutive dimers, used more stringent denaturing conditions. We therefore conducted a series of immunoprecipitation studies on solubilized CCR5, in which we varied the composition of the sample buffer, and confirmed that the relative proportion of oligomeric receptors markedly decreased as the denaturing conditions became more stringent. CCR5 oligomers completely disappeared after resuspension of immunoprecipitated material in a classical Laemmli buffer, whereas, under low stringent conditions, oligomers were visible even in the absence of agonist treatment (see supplementary material). These data suggest a different interpretation of the actual role that agonists and antibodies play in the visualization of CCR5 oligomers following covalent cross-linking (11Vila-Coro A.J. Mellado M. Martin De Ana A. Lucas P. del Real G. Martinez A.C. Rodriguez-Frade J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3388-3393Crossref PubMed Scopus (130) Google Scholar, 17Mellado M. Rodriguez-Frade J.M. Vila-Coro A.J. Fernandez S. Martin de Ana A. Jones D.R. Toran J.L. Martinez A.C. EMBO J. 2001; 20: 2497-2507Crossref PubMed Scopus (380) Google Scholar); the chemical bridging of adjacent CCR5 molecules, which maintains the complexes in denaturing conditions, may only occur when receptors adopt a specific conformation promoted by the agonist or when receptors are preclustered by antibodies. BRET experiments, indicating that both CCR5 and CXCR4 receptors form constitutive homo-oligomers but not CCR5-CXCR4 hetero-oligomers, are also consistent with recent electron microscopy observations indicating that chemokine receptors, including CCR5, CXCR4, and CCR2, are found within closely apposed but distinct clusters of 50–100 nm at the plasma membrane of macrophages, T cells, and fibroblasts (21Singer I.I. Scott S. Kawka D.W. Chin J. Daugherty B.L. DeMartino J.A. DiSalvo J. Gould S.L. Lineberger J.E. Malkowitz L. Miller M.D. Mitnaul L. Siciliano S.J. Staruch M.J. Williams H.R. Zweerink H.J. Springer M.S. J. Virol. 2001; 75: 3779-3790Crossref PubMed Scopus (140) Google Scholar). One of the major concerns when studying receptor oligomerization is the level of expression necessary to monitor oligomer formation, since a high concentration of receptors might cause random interactions between overexpressed receptor molecules. A recent analysis of the theory for FRET indicated a way to distinguish between FRET due to specific "clustering" of donors and acceptors and FRET due to high concentrations of donors and acceptors that are randomly distributed in the membrane. If donor and acceptor fluorophores are clustered, FRET is independent of absolute donor and acceptor surface densities but is dependent on the donor/acceptor ratio. In contrast, if donor and acceptor fluorophores are randomly distributed, FRET varies with acceptor density, and it is insensitive to the donor/acceptor ratio (22Kenworthy A.K. Edidin M. J. Cell Biol. 1998; 142: 69-84Crossref PubMed Scopus (404) Google Scholar, 23Pentcheva T. Edidin M. J. Immunol. 2001; 166: 6625-6632Crossref PubMed Scopus (68) Google Scholar). Since FRET and BRET are based on the same physical principle, the same rules determined for membrane protein clustering in FRET experiments should apply for BRET. Therefore, BRET studies were conducted in HEK-293 cells transfected with either increasing amounts of a fixed ratio of donor/acceptor cDNAs or with decreasing donor/acceptor ratios (Fig. 3). In the first set of experiments, the energy transfer remained stable for surface receptor levels ranging from 40 to 100 fmol of receptor/mg of membrane proteins (Fig. 3 A). In contrast, the BRET signal increased linearly over the same range of total surface receptors, when a fixed amount of donor DNA was coexpressed with increasing concentrations of the acceptor DNA (Fig. 3 B). These data indicate that the constitutive energy transfer between CCR5-hRluc and CCR5-YFP reflects a nonrandom association between the two receptor species. In addition, to determine whether specific BRET signals could be detected at physiologically relevant expression levels, precise assessments of cell surface receptors were carried out by quantitative FACS analysis. The receptor densities in transfected HEK-293 cells were then compared with those existing in human hematopoietic cells prepared from healthy donors. Specific BRET was detected in cells expressing as little as 40 fmol of receptor per mg of membrane proteins, corresponding to 8–10 × 103 surface receptors/cell (Fig. 3 A). These receptor concentrations fall well within the range measured in human activated lymphocytes and monocytes (Fig.3 C). Another important issue in studies on GPCR oligomerization has been to determine whether ligands can modulate the amount of oligomers. We found that incubation with RANTES or MIP-1β for up to 10 min had no effect on the energy transfer between CCR5-hRluc and CCR5-YFP (Fig.4 A), indicating that CCR5 agonists c
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