Homo- and Hetero-oligomerization of β-Arrestins in Living Cells

Arrestins are important proteins, which regulate the function of serpentine heptahelical receptors and contribute to multiple signaling pathways downstream of receptors. The ubiquitous β-arrestins are believed to function exclusively as monomers, although self-association is assumed to control the activity of visual arrestin in the retina, where this isoform is particularly abundant. Here the oligomerization status of β-arrestins was investigated using different approaches, including co-immunoprecipitation of epitope-tagged β-arrestins and resonance energy transfer (BRET and FRET) in living cells. At steady state and at physiological concentrations, β-arrestins constitutively form both homo- and hetero-oligomers. Co-expression of β-arrestin2 and β-arrestin1 prevented β-arrestin1 accumulation into the nucleus, suggesting that hetero-oligomerization may have functional consequences. Our data clearly indicate that β-arrestins can exist as homo- and hetero-oligomers in living cells and raise the hypothesis that the oligomeric state may regulate their subcellular distribution and functions. Arrestins are important proteins, which regulate the function of serpentine heptahelical receptors and contribute to multiple signaling pathways downstream of receptors. The ubiquitous β-arrestins are believed to function exclusively as monomers, although self-association is assumed to control the activity of visual arrestin in the retina, where this isoform is particularly abundant. Here the oligomerization status of β-arrestins was investigated using different approaches, including co-immunoprecipitation of epitope-tagged β-arrestins and resonance energy transfer (BRET and FRET) in living cells. At steady state and at physiological concentrations, β-arrestins constitutively form both homo- and hetero-oligomers. Co-expression of β-arrestin2 and β-arrestin1 prevented β-arrestin1 accumulation into the nucleus, suggesting that hetero-oligomerization may have functional consequences. Our data clearly indicate that β-arrestins can exist as homo- and hetero-oligomers in living cells and raise the hypothesis that the oligomeric state may regulate their subcellular distribution and functions. Arrestins play a central role in the regulation and signaling of serpentine heptahelical G protein-coupled receptors (GPCRs). 5The abbreviations used are: GPCRG protein-coupled receptorβarrβ-arrestinBRETbioluminescence resonance energy transferFRETfluorescescence resonance energy transferNESnuclear export signalYFPyellow fluorescent proteinTSCSPCtime- and space-correlated single photon countingFLIMfluorescence lifetime imaging microscopyGFPgreen fluorescent proteinCFPcyan fluorescent proteinJNKc-Jun NH2-terminal kinase. Arrestin 1 and 4 are restricted to retinal rods and cones where they regulate rhodopsin (1Wilden U. Hall S.W. Kuhn H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1174-1178Crossref PubMed Scopus (577) Google Scholar). In contrast, arrestin 2 and 3, also referred to as β-arrestin 1 (βarr1) and 2 (βarr2), respectively, are ubiquitous and translocate to a large variety of ligand-activated GPCRs. Originally identified as negative regulators of GPCR function, promoting desensitization (2Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (918) Google Scholar), βarrs were subsequently shown to be adaptor proteins connecting GPCRs to the endocytic machinery (3Goodman O.B.J. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1178) Google Scholar, 4Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (528) Google Scholar). βarrs also serve as signaling scaffolds linking receptors to a growing number of effector pathways (5Lefkowitz R.J. Shenoy S.K. Science. 2005; 308: 512-517Crossref PubMed Scopus (1436) Google Scholar). For example, βarrs act as scaffolds for the activation of ERK and JNK3 (5Lefkowitz R.J. Shenoy S.K. Science. 2005; 308: 512-517Crossref PubMed Scopus (1436) Google Scholar). In addition, βarr2 redistributes the ubiquitin ligase Mdm2 and the kinase JNK3 from the nucleus to the cytoplasm, a property related to the presence of a leucine-rich nuclear export signal (NES) in βarr2 (6Scott M.G. Le Rouzic E. Perianin A. Pierotti V. Enslen H. Benichou S. Marullo S. Benmerah A. J. Biol. Chem. 2002; 277: 37693-37701Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 7Wang P. Wu Y. Ge X. Ma L. Pei G. J. Biol. Chem. 2003; 278: 11648-11653Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). This signal is absent from βarr1, determining some differences in both subcellular distribution and functional roles between the two isoforms (6Scott M.G. Le Rouzic E. Perianin A. Pierotti V. Enslen H. Benichou S. Marullo S. Benmerah A. J. Biol. Chem. 2002; 277: 37693-37701Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 7Wang P. Wu Y. Ge X. Ma L. Pei G. J. Biol. Chem. 2003; 278: 11648-11653Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 8Wang P. Gao H. Ni Y. Wang B. Wu Y. Ji L. Qin L. Ma L. Pei G. J. Biol. Chem. 2003; 278: 6363-6370Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). G protein-coupled receptor β-arrestin bioluminescence resonance energy transfer fluorescescence resonance energy transfer nuclear export signal yellow fluorescent protein time- and space-correlated single photon counting fluorescence lifetime imaging microscopy green fluorescent protein cyan fluorescent protein c-Jun NH2-terminal kinase. Crystal structures of visual arrestin (9Granzin J. Wilden U. Choe H.W. Labahn J. Krafft B. Buldt G. Nature. 1998; 391: 918-921Crossref PubMed Scopus (211) Google Scholar, 10Hirsch J.A. Schubert C. Gurevich V.V. Sigler P.B. Cell. 1999; 97: 257-269Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar) revealed that this molecule contains two globular domains and an extended COOH-terminal tail locking the molecule into an inactive state. Upon binding to receptors, the arrestin C-tail is released, leading to an open active conformation (11Gurevich V.V. Gurevich E.V. Trends Pharmacol. Sci. 2004; 25: 105-111Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). In crystals, visual arrestin is a tetramer composed of two asymmetric dimers (9Granzin J. Wilden U. Choe H.W. Labahn J. Krafft B. Buldt G. Nature. 1998; 391: 918-921Crossref PubMed Scopus (211) Google Scholar, 10Hirsch J.A. Schubert C. Gurevich V.V. Sigler P.B. Cell. 1999; 97: 257-269Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). In vitro experiments showed that, in solution, tetramers are in equilibrium with monomers at physiological concentrations (12Shilton B.H. McDowell J.H. Smith W.C. Hargrave P.A. Eur. J. Biochem. 2002; 269: 3801-3809Crossref PubMed Scopus (34) Google Scholar, 13Schubert C. Hirsch J.A. Gurevich V.V. Engelman D.M. Sigler P.B. Fleming K.G. J. Biol. Chem. 1999; 274: 21186-21190Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), and it was proposed that self-association might regulate arrestin activity by limiting availability of active monomeric species (13Schubert C. Hirsch J.A. Gurevich V.V. Engelman D.M. Sigler P.B. Fleming K.G. J. Biol. Chem. 1999; 274: 21186-21190Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The crystal structure of βarr1 is very similar to that of visual arrestin, but unlike visual arrestin, full-length βarr1 was found to be monomeric (14Milano S.K. Pace H.C. Kim Y.M. Brenner C. Benovic J.L. Biochemistry. 2002; 41: 3321-3328Crossref PubMed Scopus (172) Google Scholar). In addition, because of their lower intracellular concentration, falling far below that leading to equilibrium between arrestin monomers and tetramers in solution, it was postulated that βarr1 and βarr2 only exist as monomers in cells (14Milano S.K. Pace H.C. Kim Y.M. Brenner C. Benovic J.L. Biochemistry. 2002; 41: 3321-3328Crossref PubMed Scopus (172) Google Scholar, 15Han M. Gurevich V.V. Vishnivetskiy S.A. Sigler P.B. Schubert C. Structure (Camb.). 2001; 9: 869-880Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). However, βarr1 truncated of its COOH-terminal tail was found to form dimers in crystals (15Han M. Gurevich V.V. Vishnivetskiy S.A. Sigler P.B. Schubert C. Structure (Camb.). 2001; 9: 869-880Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). Therefore, the possibility that βarrs oligomerize in vivo cannot be excluded based on the existing evidence. This hypothesis was investigated here using a combination of biochemical and biophysical approaches in vitro and in living cells. Materials—If not otherwise specified, all chemicals and reagents were from Sigma-Aldrich (Saint-Quentin Fallavier, France). Polyclonal anti-βarr2 antibodies were a generous gift from Prof. J. L. Benovic (Thomas Jefferson University, Philadelphia, PA). Leptomycin B was a kind gift from Minoru Yoshida (University of Tokyo, Japan). The anti-Myc polyclonal antibody was from Santa Cruz Biotechnology, monoclonal and polyclonal anti-FLAG antibodies were from Sigma. Alexa-594-conjugated goat anti-mouse immunoglobin was from Molecular Probes (Molecular Probes Europe, Leiden, The Netherlands). Expression Vectors—The construction of pβarr2-FLAG, pβarr2-YFP, pβarr2-GFP, pβarr2-L395A-GFP, and pβarr2-R396A-GFP have been described previously (6Scott M.G. Le Rouzic E. Perianin A. Pierotti V. Enslen H. Benichou S. Marullo S. Benmerah A. J. Biol. Chem. 2002; 277: 37693-37701Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The plasmid pβarr2-CFP was generated by subcloning the βarr2 coding region into ECFPN1 (Clontech Europe, Erembodegem, Belgium). βarr2 and βarr1 cDNAs were amplified by PCR and subcloned in pCMV-Tag3A (Stratagene) to create pMyc-βarr2 and pMyc-βarr1. To obtain Rluc-βarr1 and βarr2-Rluc, appropriate forms of βarr were subcloned in frame, in the phRluc-C1 or -N3 vector, respectively, encoding the humanized Renilla luciferase (BioSignal, Montreal, Canada). YFP-βarr1 and YFP-βarr2 were obtained by sucloning corresponding cDNAs in the pEYFP-C1 vector (Clontech). All constructs were verified by nucleotide sequencing. Cell Culture and Transfection—COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm glutamine (all from Invitrogen, Cergy Pontoise, France), at 37 °C in an atmosphere of 5% CO2. Cells were seeded at a density of 3 × 105 cells in the 35-mm diameter wells of 6-well plates. Transient transfections were performed the following day using FuGENE (Roche, Meylan, France), according to the manufacturer's protocol. Cells were harvested 24 h after transfection and either used for BRET experiments or grown on coverslips for FRET or immunofluorescence experiments. About 50-70% cells were fluorescent at the time of the experiments. Co-immunoprecipitation Experiments—48 h post-transfection cells were lysed in 1 ml of cold glycerol lysis buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 2 mm EDTA 1% Trition X-100, 10% glycerol, 100 μm Na3VO4, 1 mm NaF, supplemented with protease inhibitors) and clarified by centrifugation at 13, 000 × g for 20 min at 4 °C. Immunoprecipitations were performed using 20 μl of a 50% slurry of monoclonal M2 anti-FLAG-affinity agarose, with constant agitation overnight at 4 °C. Following incubation, immune complexes were washed four times with lysis buffer. Immunoprecipitated proteins were subjected to SDS-PAGE and Western blot analysis was performed using a polyclonal anti-Myc antibody. The chemiluminescence reaction was performed using the ECL reagent (Amersham Biosciences). BRET Assay—COS cells were transfected with 10 ng/well of the DNA construct coding for BRET donor and increasing (10-250 ng/well) amounts of the construct coding for BRET acceptor (or control YFP). 24 h after transfection, cells were detached with phosphate-buffered saline/EDTA and washed in phosphate-buffered saline. Aliquots of 1 × 105 cells were distributed in 96-well microplates (White Optiplate, PerkinElmer Life Sciences/Packard Biosciences). 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 Mithras™ fluorescence-luminescence detector (Berthold, Germany). Cells expressing BRET donors alone were used to determine background. Filter sets were 485 ± 10 nm for luciferase emission and 530 ± 12.5 nm for YFP emission. BRET ratios were calculated as described (16Angers 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). Fluorescence Lifetime Imaging Microscopy—Fluorescence decays were measured in cells expressing the FRET donor alone (βarr2 fused to CFP) or both FRET donor and acceptor (βarr2 fused to YFP). The microscopy system, based on time- and space-correlated single photon counting (TSCSPC), has been described elsewhere (17Emiliani V. Sanvitto D. Tramier M. Piolot T.Z.P. Kemnitz K. Durieux C. Coppey-Moisan M. Appl. Phys. Lett. 2003; 83: 2471-2473Crossref Scopus (63) Google Scholar). Briefly, a mode-locked titanium sapphire laser (Millennia 5W/Tsunami 3960-M3BB-UPG kit, Spectra-Physics, France) delivering picosecond pulses was tuned at 880 nm to obtain a 440 nm excitation wavelength after frequency doubling. The repetition was 4 after The laser was and into an France) for The fluorescence emitted by the was with a at the of the detector Germany). Fluorescence imaging was by counting for 10 min and single emitted according the time between their and the laser time their × and their The was to emission nm was to the donor fluorescence and to the acceptor Fluorescence decays were from different of in cells expressing the fluorescence donor alone or both donor and were with a of at by using two as were obtained by fluorescence decays with a cells were seeded on coverslips in 6-well transfected with appropriate and used for immunofluorescence 1 day Cells were and for fluorescence microscopy as described previously M.G. Benmerah A. Marullo S. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). were a were using and for using βarr2 and βarr1 were to βarr1 and βarr2 not βarrs were the identified with the of the βarr1 also with a βarr2 hetero-oligomerization between the different βarr The were by biochemical which were to very of L. Ferguson S.S. Zhang J. Lefkowitz R.J. Caron M.G. Barak L.S. Pharmacol. PubMed Scopus Google Scholar), were transfected with encoding epitope-tagged βarr2 for co-immunoprecipitation experiments βarr2 with the hypothesis that βarr2 exist in cells. approaches can in living cells at physiological Trends 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In BRET between Renilla luciferase the and a yellow of the green fluorescence protein the BRET has been used to GPCR oligomerization M. 2001; PubMed Scopus Google Scholar, 2004; PubMed Scopus Google Scholar) and βarr2 to receptors (16Angers 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, S. Bouvier M. 2005; PubMed Scopus Google Scholar). BRET experiments Salahpour A. S. A. Bouvier M. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar) were in cells to βarr2 constant of encoding a in which is to the of was with increasing amounts of cDNAs coding for βarr2 fused either downstream or of YFP alone was used to determine BRET Salahpour A. S. A. Bouvier M. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar) BRET were measured with in either the presence of βarr2 BRET were in experiments with with that BRET an oligomerization of βarr2 and not its in the of BRET have been of donor and acceptor The of the BRET to not the of βarr2 to form the of at which BRET was obtained is similar for both experiments were to the of YFP-βarr2 and in of in which BRET were obtained The of with anti-βarr2 antibodies was by and was found to be times the in cells. In experiments of the BRET the of which was based on luciferase and fluorescence was below the of βarr2 in the Western blot of In addition, have been to to βarr2 cells F. Penn R.B. Gagnon A.W. Benovic J.L. Keen J.H. J. Cell Sci. 2000; PubMed Google Scholar), that βarr2 may form in living cells expressing physiological concentrations of this The BRET cannot the subcellular in which the between the donor and the acceptor can be obtained by using FRET the approaches, picosecond fluorescence lifetime imaging microscopy was to be particularly to in vivo because it excitation and of fluorescent proteins to be M. Piolot S. Kemnitz K. J. Durieux C. V. Coppey-Moisan M. J. 2002; 83: Full Text Full Text PDF PubMed Scopus Google Scholar, M. Piolot V. J. Kemnitz K. Durieux C. Coppey-Moisan M. 2003; PubMed Scopus Google Scholar). is based on the that the of the donor fluorescence is FRET between the donor and an Fluorescence lifetime of the FRET the cyan of the was measured at steady state in cells either expressing alone or and βarr2 fused downstream to the FRET acceptor YFP fluorescence is fluorescence were calculated from The in fluorescence decays for 10 min the of single cells. The of by FRET in cells and with cells expressing the presence of βarr2 FRET was measured in cells expressing and YFP not a a of was measured between cells expressing a protein which FRET is and cells expressing alone not The can be by on a shown in βarr2 distributed the in cells. In with the nuclear of βarr2 at steady state (6Scott M.G. Le Rouzic E. Perianin A. Pierotti V. Enslen H. Benichou S. Marullo S. Benmerah A. J. Biol. Chem. 2002; 277: 37693-37701Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), βarr2 was in the Co-immunoprecipitation and BRET experiments were also with βarr1 constructs with and BRET were obtained using and YFP-βarr1 as donor and BRET and were with measured for BRET on βarr2 that the two βarr isoforms have a similar for βarrs the that βarr1 and βarr2 have the to form in investigated hetero-oligomerization between the two different βarr isoforms in living cells. of this hypothesis from experiments with epitope-tagged βarrs that, can be with BRET were also in cells expressing a donor and an acceptor of βarr species BRET in and to obtained in the experiments were measured the βarr species used as BRET donor or indicate that βarrs have the to into either or hetero-oligomers in living cells. of βarr and determine βarr oligomerization may be with of a that βarr2 between the and the nucleus in cells and that of the nuclear export machinery to the nuclear accumulation of βarr2 (6Scott M.G. Le Rouzic E. Perianin A. Pierotti V. Enslen H. Benichou S. Marullo S. Benmerah A. J. Biol. Chem. 2002; 277: 37693-37701Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 7Wang P. Wu Y. Ge X. Ma L. Pei G. J. Biol. Chem. 2003; 278: 11648-11653Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). of the βarr2 by a single nuclear accumulation of the Upon with a of which is in the nucleus was into this where it with lower The of on βarr2 subcellular the of hetero-oligomerization between two species and the nuclear of In to βarr1 has in its tail and is both nuclear and at steady state In cells βarr1 and βarr1 was excluded from the nucleus, of its hetero-oligomerization with βarr2 lower oligomerization of βarrs has not been on visual arrestin in vitro have proposed a in which oligomerization the activation of arrestin and To this hypothesis be for the of and βarr oligomerization on βarr a construct in which a binding was fused downstream of the βarr2 tail and of an living imaging verified that, of the molecule the of βarr2 proteins was and Upon to the receptors and in and with the as and data not data indicate that the oligomeric of βarr2 not their to receptor have biochemical and biophysical for the of βarr homo- and hetero-oligomers. The of βarr2 to and form hetero-oligomers with βarr1 was by co-immunoprecipitation experiments. energy approaches the presence of homo- and hetero-oligomers in cells at physiological concentrations of or forms of βarr2 the subcellular of βarr2 and βarr1, The approaches used cannot between a and of monomers, as tetramers composed of 2 for visual arrestin (9Granzin J. Wilden U. Choe H.W. Labahn J. Krafft B. Buldt G. Nature. 1998; 391: 918-921Crossref PubMed Scopus (211) Google Scholar). The of monomeric they exist in oligomeric βarrs to be as BRET experiments can the of two to but not has been proposed that because of the concentration of visual arrestin in the retina, oligomerization the availability of active monomeric species (13Schubert C. Hirsch J.A. Gurevich V.V. Engelman D.M. Sigler P.B. Fleming K.G. J. Biol. Chem. 1999; 274: 21186-21190Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), which might be for the in L. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). here indicate that oligomerization may in living cells at physiological concentrations of which are far below of visual in the In addition, have shown that βarr2 its to translocate to GPCRs. data are not to the of monomers the active form of an in which might an important functional form of the identified arrestin in receptor binding on the of the two globular domains S.A. Benovic J.L. Gurevich V.V. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). binding to the which are to the NH2-terminal globular which are in the binding to of the receptor the activation and determine the are distributed in both is that βarrs are of with multiple proteins of kinase the globular domains binding to the GPCRs Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). Therefore, the oligomeric of βarrs might their with multiple at the with the hypothesis that βarr oligomerization might contribute to kinase activation is a that of of its is to the activation J. L. Zhang D. D. B. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). the with GPCRs arrestin monomers or as complexes in M.G. Benmerah A. Marullo S. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar), it is to that not are in because of the concentration of of βarr2 have been with its of between the and the In βarr2 JNK3 of the nucleus (6Scott M.G. Le Rouzic E. Perianin A. Pierotti V. Enslen H. Benichou S. Marullo S. Benmerah A. J. Biol. Chem. 2002; 277: 37693-37701Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) and the activity of by the ligase Mdm2 from the nucleus to the P. Gao H. Ni Y. Wang B. Wu Y. Ji L. Qin L. Ma L. Pei G. J. Biol. Chem. 2003; 278: 6363-6370Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). here that βarr2 also βarr1 in the nuclear βarr1 availability and may on the of the two is on the physiological nuclear of in the of the it was that βarr1 activation a in the nucleus H. Y. Wu Y. B. Wang Y. B. Pei G. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). In have shown that the self-association of from in the presence of in living cells. In addition, the subcellular distribution of βarrs can be oligomerization suggesting that the structure of βarrs may control at of their functions. J. for The was by the with