Constitutive Agonist-independent CCR5 Oligomerization and Antibody-mediated Clustering Occurring at Physiological Levels of Receptors

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. 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 and a form Life the yellow of green fluorescent and of the In the and were to the of the receptor cells were from of by density as previously A. J. PubMed Scopus Google Scholar). and were by monoclonal antibody binding from monocytes by on were in with and at in an of They were by with to with to was by of with in PBS, for at and in with and from were at a density of cells in were the following using to the were and used after cell expressing CCR5 or CCR5 to or were also using in the The of was by as were on HEK-293 expressing CCR5 or CCR5 to or The of in intact cells was using the (PerkinElmer Life is based on the the within the cells to be and is by which are to an monoclonal antibody is to The and the antibody and of the is and to the only if and are in then at The using a Life with the increase of were using known concentrations of by the activation of CCR5, and was in with concentrations of after with was by HEK-293 expressing CCR5, or were at with or a of the monoclonal antibody for the with PBS, cells were for at in an and to or the antibody. were at for with the monoclonal CCR5 were then with and with were with a from HEK-293 was from and the cell was as the mean fluorescence by the number of after with the BRET and HEK-293 or cells were with and in of cells were in the of from Life in the or absence of CCR5 or The monoclonal and of the CCR5 were in the of of antibody and fragments from the antibody were as C. J.M. J. M. G. S. D. M. M. Biol. 2002; PubMed Scopus Google Scholar). The The was at a of and and fluorescence were using the (PerkinElmer Life expressing BRET to were used to were for and for from were used to BRET that were as S. Salahpour A. S. D. M. Bouvier M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: Google Scholar). of CCR5 and CXCR4 was by using the and monoclonal The number of on the of and HEK-293 cells was in using the and the by the For fluorescence were with with known of density was expressed as the number of by the of using the protein the of receptors at the cell were on intact cells and cells with for at HEK-293 cells in were with and after the cells were with PBS, the and with of The cell was with of a and cells were by at for at The was and was to a of The was using the with the of at the following and in the a of The were for at in a A of of were from the of the BRET was on of The of the to the plasma membrane from the endoplasmic reticulum was by was with a monoclonal antibody that the of the membrane at a of and a antibody reticulum at a of cells expressing or the mutant of both under the of a regulated were in the of were in with and and of and was induced by from the after the with the and BRET and the were in after a of could be as by on with a monoclonal antibody from the of For studies, cells were on in with a and used for the cells were and for fluorescence as previously A. S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). were under an to a were using the studies, based on immunoprecipitation after cell that CCR5 receptors might form and (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, 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, M. Rodriguez-Frade J.M. A.J. S. Martin Ana A. Martinez A.C. J. 2001; PubMed Scopus Google Scholar), as reported for a growing number of GPCRs in M. Rev. 2001; PubMed Scopus Google Scholar). However, studies were on important such as the constitutive or induced of CCR5 In an to CCR5 oligomerization in living of a based on BRET, which was to of Johnson Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar) and to the study of GPCRs oligomerization M. Rev. 2001; PubMed Scopus Google for a on the K. G. R. B. 1998; PubMed Scopus Google Scholar), it was that the energy transfer BRET is on the of the of a BRET two receptors a that can be by the formation of receptor dimers or oligomers S. Salahpour A. S. D. M. Bouvier M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: Google Scholar). For this were CCR5 and or of intact and HEK-293 expressing a subcellular similar to that of CCR5 about of both or receptors was found at the plasma membrane after not receptors functional as by the similar of to CCR5, and of and agonist binding induced a similar extent and of receptor of receptor The and were in HEK-293 and the energy transfer was after the of the of in a energy transfer was observed and upon in the absence of CCR5 No such was observed in cells expressing or was by a chemokine to The CXCR4 receptor was to the as a for BRET, as it was reported that the latter receptor might form functional with CCR5 M. Rodriguez-Frade J.M. A.J. S. Martin Ana A. Martinez A.C. J. 2001; PubMed Scopus Google Scholar). The of BRET and not result from the of CXCR4 to as a BRET since a strong was observed upon of and The observed with CXCR4 not a of receptors in constitutive since it could result from in BRET The of the was also demonstrated by BRET experiments the BRET was studied in the of concentrations of receptors. of CCR5 but not of CXCR4 the transfer of energy and BRET which the of constitutive CCR5 oligomerization, are in with a previous in which CCR5 oligomers could be from cell of cells and by (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 CCR5 oligomers only intact cells were with agonists or chemical 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, M. Rodriguez-Frade J.M. A.J. S. Martin Ana A. Martinez A.C. J. 2001; PubMed Scopus Google Scholar). the experimental used in studies, that the could not the of constitutive used stringent a of immunoprecipitation studies on solubilized CCR5, in which the of the and that the of oligomeric receptors as the conditions CCR5 oligomers after of in a under low stringent oligomers were even in the absence of agonist These data a different of the role that agonists and in the of CCR5 oligomers following 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, M. Rodriguez-Frade J.M. A.J. S. Martin Ana A. Martinez A.C. J. 2001; PubMed Scopus Google the chemical of CCR5 which the complexes in may only receptors a by the agonist or receptors are by BRET indicating that both CCR5 and CXCR4 receptors form constitutive homo-oligomers but not are also consistent with indicating that chemokine receptors, CCR5, and are found within but of at the plasma membrane of T and S. J. J.A. J. M.J. J. 2001; PubMed Scopus Google Scholar). of the major concerns receptor oligomerization is the of to oligomer since a of receptors might overexpressed receptor A of the for a to to of and and to concentrations of and that are in the If and are is of and but is on the In contrast, if and are with and it is to the M. J. Biol. 1998; PubMed Scopus Google Scholar, T. M. J. 2001; PubMed Scopus Google Scholar). and BRET are based on the same the same for membrane protein in experiments for BRET studies were in HEK-293 cells with of a of or with In the of the energy transfer for receptor levels from to of of membrane In contrast, the BRET the same of receptors, a of was with concentrations of the These data indicate that the constitutive energy transfer and a the two receptor In to whether BRET could be detected at relevant of cell receptors were by The receptor in HEK-293 cells were then with in human cells from BRET was detected in cells expressing as little as of receptor of membrane to These receptor concentrations within the in human and monocytes important issue in studies on oligomerization has been to whether can the of found that with or for to no effect on the energy transfer and indicating that CCR5 agonists the constitutive oligomerization of The of CCR5 agonists to BRET be to since BRET observed for to the previous energy studies on oligomerization or a similar to the was observed for the receptor Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) and for the human δ-opioid receptor M. 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Chem. 1997; 272: 30603-30606Abstract Full Text Full Text PDF PubMed Scopus (321) Google R.A. 1997; PubMed Scopus Google Scholar, T. A. A. A. J. 2001; PubMed Scopus Google Scholar). Although our that receptors may be as not the that oligomers may to form such as of a is by studies microaggregation of the receptor A. J.A. G. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). can are known to or of membrane and it was reported that an antibody could the of CCR5 oligomeric in experiments (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). investigated the effect of monoclonal on constitutive the of the were in of with chemokine binding, receptor and C. J.M. J. M. G. S. D. M. M. Biol. 2002; PubMed Scopus Google Scholar). The which to the The which to the a strong and receptor and the which to a induced receptor in the not the the or enhanced the BRET in a a after of with the antibody at to was reported for the CCR5 C. J.M. J. M. G. S. D. M. M. Biol. 2002; PubMed Scopus Google Scholar), only the antibody could the BRET and a of a of BRET However, receptor was not involved in the of BRET by the it was still observed after the of the mutant of A and a mutant known to both and T. J. Biol. PubMed Scopus Google Scholar, Annu. Rev. Biol. 2000; PubMed Scopus Google Scholar), which receptor by the antibody The fact that only and not or induced a BRET all the suggests a of only the antibody to receptor even in the of the C. J.M. J. M. G. S. D. M. M. 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However, our that the constitutive BRET was to which and was previously to from clustering, it an of and on BRET by the antibody. cells expressing the or the mutant of under the of the were with and at a BRET was then as in A following a with of the antibody or the in the or absence of and in cells or not the mutant of BRET in the of overexpressed were similar to in the of The data are expressed as increase in BRET by the and the mean of was in cells or not the mutant of of the CCR5 receptor was by following a with a of the antibody or the The is a the of after with cells expressing cells and cells expressing and were with the after the cells were and or not and with for at and then and or not and with the antibody for at cells were for as under The In the of CCR5 induced by the antibody were at the plasma membrane and receptor in both cells and cells expressing and and and in cells and that in cells expressing a strong of was in the which probably corresponds to the of from the The present study at physiological CCR5 receptors as constitutive oligomers that form early after in the endoplasmic These as demonstrated in the of the GABAB receptor homo-oligomerization might a role in endoplasmic reticulum The fact that CCR5 agonists not the constitutive BRET that activation is not by a change in the receptor oligomerization data also the of previous studies, which concluded that oligomer formation by cross-linking receptor that most the of for for of the and for The the of CCR5 and CXCR4 were by The and monoclonal were by with

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