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Interleukin-5 Receptor Subunit Oligomerization and Rearrangement Revealed by Fluorescence Resonance Energy Transfer Imaging

2008; Elsevier BV; Volume: 283; Issue: 19 Linguagem: Inglês

10.1074/jbc.m710230200

ISSN

1083-351X

Autores

Meirav Zaks‐Zilberman, Adrian Harrington, Tetsuya Ishino, Irwin Chaiken,

Tópico(s)

Immunotherapy and Immune Responses

Resumo

Interleukin (IL)-5 exerts hematopoietic functions through binding to the IL-5 receptor subunits, α and βc. Specific assembly steps of full-length subunits as they occur in cell membranes, ultimately leading to receptor activation, are not well understood. We tracked the oligomerization of IL-5 receptor subunits using fluorescence resonance energy transfer (FRET) imaging. Full-length IL-5Rα and βc were expressed in Phoenix cells as chimeric proteins fused to enhanced cyan or yellow fluorescent protein (CFP or YFP, respectively). A time- and dose-dependent increase in FRET signal between IL-5Rα-CFP and βc-YFP was observed in response to IL-5, indicative of heteromeric receptor α-βc subunit interaction. This response was inhibited by AF17121, a peptide antagonist of IL-5Rα. Substantial FRET signals with βc-CFP and βc-YFP co-expressed in the absence of IL-5Rα demonstrated that βc subunits exist as preformed homo-oligomers. IL-5 had no effect on this βc-alone FRET signal. Interestingly, the addition of IL-5 to cells co-expressing βc-CFP, βc-YFP, and nontagged IL-5Rα led to further increase in FRET efficiency. Observation of preformed βc oligomers fits with the view that this form can lead to rapid cellular responses upon IL-5 stimulation. The IL-5-induced effects on βc assembly in the presence of nontagged IL-5Rα provide direct evidence that IL-5 can cause higher order rearrangements of βc homo-oligomers. These results suggest that IL-5 and perhaps other βc cytokines (IL-3 and granulocyte/macrophage colony-stimulating factor) trigger cellular responses by the sequential binding of cytokine ligand to the specificity receptor (subunit α), followed by binding of the ligand-subunit α complex to, and consequent rearrangement of, a ground state form of βc oligomers. Interleukin (IL)-5 exerts hematopoietic functions through binding to the IL-5 receptor subunits, α and βc. Specific assembly steps of full-length subunits as they occur in cell membranes, ultimately leading to receptor activation, are not well understood. We tracked the oligomerization of IL-5 receptor subunits using fluorescence resonance energy transfer (FRET) imaging. Full-length IL-5Rα and βc were expressed in Phoenix cells as chimeric proteins fused to enhanced cyan or yellow fluorescent protein (CFP or YFP, respectively). A time- and dose-dependent increase in FRET signal between IL-5Rα-CFP and βc-YFP was observed in response to IL-5, indicative of heteromeric receptor α-βc subunit interaction. This response was inhibited by AF17121, a peptide antagonist of IL-5Rα. Substantial FRET signals with βc-CFP and βc-YFP co-expressed in the absence of IL-5Rα demonstrated that βc subunits exist as preformed homo-oligomers. IL-5 had no effect on this βc-alone FRET signal. Interestingly, the addition of IL-5 to cells co-expressing βc-CFP, βc-YFP, and nontagged IL-5Rα led to further increase in FRET efficiency. Observation of preformed βc oligomers fits with the view that this form can lead to rapid cellular responses upon IL-5 stimulation. The IL-5-induced effects on βc assembly in the presence of nontagged IL-5Rα provide direct evidence that IL-5 can cause higher order rearrangements of βc homo-oligomers. These results suggest that IL-5 and perhaps other βc cytokines (IL-3 and granulocyte/macrophage colony-stimulating factor) trigger cellular responses by the sequential binding of cytokine ligand to the specificity receptor (subunit α), followed by binding of the ligand-subunit α complex to, and consequent rearrangement of, a ground state form of βc oligomers. It is generally thought that most cytokines trigger cellular signal transduction by inducing the association of receptor subunits that leads to increased proximity of associated intracellular kinases and initiation of phosphorylation cascades. Recent studies of human growth hormone and erythropoietin have shown that the assembly of their receptor subunits leads to conformational rearrangement and that this process plays a role in receptor activation (1Brown R.J. Adams J.J. Pelekanos R.A. Wan Y. McKinstry W.J. Palethorpe K. Seeber R.M. Monks T.A. Eidne K.A. Parker M.W. Waters M.J. Nat. Struct. Mol. Biol. 2005; 12: 814-821Crossref PubMed Scopus (295) Google Scholar, 2Seubert N. Royer Y. Staerk J. Kubatzky K.F. Moucadel V. Krishnakumar S. Smith S.O. Constantinescu S.N. Mol. Cell. 2003; 12: 1239-1250Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 3Kubatzky K.F. Ruan W. Gurezka R. Cohen J. Ketteler R. Watowich S.S. Neumann D. Langosch D. Klingmuller U. Curr. Biol. 2001; 11: 110-115Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). However, evidence for such cytokine-induced receptor rearrangement for other cytokine-receptor complexes remains limited. In the case of interleukin (IL) 2The abbreviations used are: IL, interleukin; GM-CSF, granulocyte/macrophage colony-stimulating factor; βc, common receptor β subunit; FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; ROI, region of interest. 2The abbreviations used are: IL, interleukin; GM-CSF, granulocyte/macrophage colony-stimulating factor; βc, common receptor β subunit; FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; ROI, region of interest.-5 and the other βc cytokines IL-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF), the states of receptor subunit assembly in membranes that lead to activation have been proposed, but experimental evidence for these has remained largely indirect. Human IL-5 is a TH2 cell-derived cytokine that regulates hematopoiesis and inflammation. It is implicated in the pathogenesis of allergic disorders, asthma, and other forms of hypereosinophilic syndromes, through its influence on eosinophil maturation, proliferation, activation, expansion, and tissue distribution (4Sanderson C.J. Urwin D. Curr. Opin. Investig. Drugs. 2000; 1: 435-441PubMed Google Scholar, 5Stirling R.G. van Rensen E.L.J. Barnes P.J. Chung K.F. Am. J. Respir. Crit. Care Med. 2001; 164: 1403-1409Crossref PubMed Scopus (97) Google Scholar, 6Kopf M. Brombacher F. Hodgkin P.D. Ramsay A.J. Milbourne E.A. Dai W.J. Ovington K.S. Behm C.A. Kohler G. Young I.G. Matthaei K.I. Immunity. 1996; 4: 15-24Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar, 7Foster P.S. Hogan S.P. Ramsay A.J. Matthaei K.I. Young I.G. J. Exp. Med. 1996; 183: 195-201Crossref PubMed Scopus (1266) Google Scholar). IL-5 exerts its biological functions by binding to a heteromeric cell surface receptor complex that contains two types of subunits, α and βc (8Plaetinck G. Van der Heyden J. Tavernier J. Fache I. Tuypens T. Fischkoff S. Fiers W. Devos R. J. Exp. Med. 1990; 172: 683-691Crossref PubMed Scopus (66) Google Scholar). IL-5Rα confers specificity and is essential for activation of the βc subunit. The IL-5Rα subunit has been shown to bind IL-5 directly, even in the absence of βc, albeit at a lower affinity (9Goodall G.J. Bagley C.J. Vadas M.A. Lopez A.F. Growth Factors. 1993; 8: 87-97Crossref PubMed Scopus (65) Google Scholar). IL-5Rα is structurally related to the receptor α subunits of IL-3 and GM-CSF receptors. The βc receptor subunit is common to the IL-3, IL-5, and GM-CSF receptors and is considered the main signaling component of the activated receptor-ligand complexes (10Lopez A.F. Elliott M.J. Woodcock J. Vadas M.A. Immunol. Today. 1992; 13: 495-500Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 11Tavernier J. Devos R. Cornelis S. Tuypens T. Van der Heyden J. Fiers W. Plaetinck G. Cell. 1991; 66: 1175-1184Abstract Full Text PDF PubMed Scopus (493) Google Scholar). Based on biophysical and high resolution crystallographic data, the βc receptor ectodomain exists as a stable homodimer (12Murphy J.M. Ford S.C. Wiedemann U.M. Carr P.D. Ollis D.L. Young I.G. J. Biol. Chem. 2003; 278: 10572-10577Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 13Murphy J.M. Young I.G. Vitam. Horm. 2006; 74: 1-30Crossref PubMed Scopus (62) Google Scholar). On its own, βc does not measurably bind any of the cytokine ligands (11Tavernier J. Devos R. Cornelis S. Tuypens T. Van der Heyden J. Fiers W. Plaetinck G. Cell. 1991; 66: 1175-1184Abstract Full Text PDF PubMed Scopus (493) Google Scholar, 14Kitamura T. Sato N. Arai K. Miyajima A. Cell. 1991; 66: 1165-1174Abstract Full Text PDF PubMed Scopus (504) Google Scholar, 15Hayashida K. Kitamura T. Gorman D.M. Arai K. Yokota T. Miyajima A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9655-9659Crossref PubMed Scopus (516) Google Scholar), but it cannot be ruled out that βc may have direct contact with IL-5 (or the other cytokines) in ternary cytokine-Rα-βc complexes. Previous binding studies support the view that the IL-5 ligand first binds to IL-5Rα with an apparent Kd value of 0.3-0.6 nm (11Tavernier J. Devos R. Cornelis S. Tuypens T. Van der Heyden J. Fiers W. Plaetinck G. Cell. 1991; 66: 1175-1184Abstract Full Text PDF PubMed Scopus (493) Google Scholar, 16Murata Y. Takaki S. Migita M. Kikuchi Y. Tominaga A. Takatsu K. J. Exp. Med. 1992; 175: 341-351Crossref PubMed Scopus (162) Google Scholar, 17Tavernier J. Tuypens T. Plaetinck G. Verhee A. Fiers W. Devos R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7041-7045Crossref PubMed Scopus (119) Google Scholar), followed by binding of βc into the IL-5·IL-5Rα complex. The affinity of IL-5 when α- and βc-chains are coexpressed in the ternary complex (IL-5·IL-5Rα·βc) is 2-5-fold greater than in the IL-5·IL-5Rα complex (11Tavernier J. Devos R. Cornelis S. Tuypens T. Van der Heyden J. Fiers W. Plaetinck G. Cell. 1991; 66: 1175-1184Abstract Full Text PDF PubMed Scopus (493) Google Scholar, 16Murata Y. Takaki S. Migita M. Kikuchi Y. Tominaga A. Takatsu K. J. Exp. Med. 1992; 175: 341-351Crossref PubMed Scopus (162) Google Scholar, 18Scibek J.J. Evergren E. Zahn S. Canziani G.A. Van Ryk D. Chaiken I.M. Anal. Biochem. 2002; 307: 258-265Crossref PubMed Scopus (19) Google Scholar). In the GM-CSF and IL-3 ligand-receptor systems, βc binding increases ligand affinity even further. The initial binding of IL-5 to IL-5Rα has a 1:1 stoichiometry, as shown by gel filtration, titration calorimetry, analytical ultracentrifugation, and surface plasmon resonance using soluble receptor molecules or truncated receptor chains (19Devos R. Guisez Y. Cornelis S. Verhee A. Van der Heyden J. Manneberg M. Lahm H.W. Fiers W. Tavernier J. Plaetinck G. J. Biol. Chem. 1993; 268: 6581-6587Abstract Full Text PDF PubMed Google Scholar, 20Quelle F.W. Sato N. Witthuhn B.A. Inhorn R.C. Eder M. Miyajima A. Griffin J.D. Ihle J.N. Mol. Cell Biol. 1994; 14: 4335-4341Crossref PubMed Google Scholar, 21Johanson K. Appelbaum E. Doyle M. Hensley P. Zhao B. Abdel-Meguid S.S. Young P. Cook R. Carr S. Matico R. Cusimano D. Dul E. Angelichio M. Brooks I. Winborne E. McDonnell P. Morton T. Bennett D. Sokoloski T. McNulty D. Rosenberg M. Chaiken I. J. Biol. Chem. 1995; 270: 9459-9471Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Cross-linking and immunoprecipitation studies in the presence of IL-5 demonstrated a close physical association of IL-5Rα and βc (16Murata Y. Takaki S. Migita M. Kikuchi Y. Tominaga A. Takatsu K. J. Exp. Med. 1992; 175: 341-351Crossref PubMed Scopus (162) Google Scholar, 17Tavernier J. Tuypens T. Plaetinck G. Verhee A. Fiers W. Devos R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7041-7045Crossref PubMed Scopus (119) Google Scholar, 22Devos R. Plaetinck G. Van der Heyden J. Cornelis S. Vandekerckhove J. Fiers W. Tavernier J. EMBO J. 1991; 10: 2133-2137Crossref PubMed Scopus (73) Google Scholar, 23Ogata N. Kouro T. Yamada A. Koike M. Hanai N. Ishikawa T. Takatsu K. Blood. 1998; 91: 2264-2271Crossref PubMed Google Scholar). Furthermore, the observation of cytokine-dependent formation of a disulfide linkage between IL-3Rα and βc subunits (24Le F. Stomski F. Woodcock J.M. Lopez A.F. Gonda T.J. J. Biol. Chem. 2000; 275: 5124-5130Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 25Stomski F.C. Woodcock J.M. Zacharakis B. Bagley C.J. Sun Q. Lopez A.F. J. Biol. Chem. 1998; 273: 1192-1199Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 26Stomski F.C. Sun Q. Bagley C.J. Woodcock J. Goodall G. Andrews R.K. Berndt M.C. Lopez A.F. Mol. Cell. Biol. 1996; 16: 3035-3046Crossref PubMed Google Scholar) is consistent with a need for conformational changes in the extracellular domains of the receptor subunits (21Johanson K. Appelbaum E. Doyle M. Hensley P. Zhao B. Abdel-Meguid S.S. Young P. Cook R. Carr S. Matico R. Cusimano D. Dul E. Angelichio M. Brooks I. Winborne E. McDonnell P. Morton T. Bennett D. Sokoloski T. McNulty D. Rosenberg M. Chaiken I. J. Biol. Chem. 1995; 270: 9459-9471Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The reorganization of the intracellular domains upon cytokine binding has been inferred but has not been shown directly. Fluorescence resonance energy transfer (FRET) has been used to directly visualize protein-protein interactions in cells by confocal microscopy. Because energy transfer depends on the inverse 6th power of the distance between donor and acceptor fluorophores, this method allows the detection of energy exchange between these two fluorophores fused to proteins that are in a close spatial proximity (2-10 nm), a distance that correlates with the size of the molecules on the cell surface (27Selvin P.R. Nat. Struct. Biol. 2000; 7: 730-734Crossref PubMed Scopus (866) Google Scholar, 28Hibbs A.R. Confocal Microscopy for Biologists. Kluwer Academic Publishers, Norwell, MA2004Crossref Google Scholar). Using cell FRET and relatively small fluorescent tags that are available, the cellular environment is maintained, conserving transmembrane interactions with minimal perturbation of signaling systems (29Majoul I. Straub M. Duden R. Hell S.W. Soling H.D. J. Biotechnol. 2002; 82: 267-277PubMed Google Scholar, 30Li M. Reddy L.G. Bennett R. Silva Jr., N.D. Jones L.R. Thomas D.D. Biophys. J. 1999; 76: 2587-2599Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 31Karasawa A. Tsuboi Y. Inoue H. Kinoshita R. Nakamura N. Kanazawa H. J. Biol. Chem. 2005; 280: 41900-41911Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Recent studies have applied FRET to different cytokine receptor systems, including those of IL-2 (32Damjanovich S. Bene L. Matko J. Alileche A. Goldman C.K. Sharrow S. Waldmann T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13134-13139Crossref PubMed Scopus (120) Google Scholar), IL-10 (33Krause C.D. Mei E. Mirochnitchenko O. Lavnikova N. Xie J. Jia Y. Hochstrasser R.M. Pestka S. Biochem. Biophys. Res. Commun. 2006; 340: 377-385Crossref PubMed Scopus (25) Google Scholar), IL-13 (34Yang X. Capotosto R. DiBlasio E. Hu Z. Kriz R. Lorenzo M. Malakian K. Wolfrom S. Wilhelm J. Wolf S.F. Anal. Biochem. 2006; 351: 158-160Crossref PubMed Scopus (2) Google Scholar), IL-17 (35Kramer J.M. Yi L. Shen F. Maitra A. Jiao X. Jin T. Gaffen S.L. J. Immunol. 2006; 176: 711-715Crossref PubMed Scopus (83) Google Scholar), leptin (36Couturier C. Jockers R. J. Biol. Chem. 2003; 278: 26604-26611Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), and growth hormone (1Brown R.J. Adams J.J. Pelekanos R.A. Wan Y. McKinstry W.J. Palethorpe K. Seeber R.M. Monks T.A. Eidne K.A. Parker M.W. Waters M.J. Nat. Struct. Mol. Biol. 2005; 12: 814-821Crossref PubMed Scopus (295) Google Scholar). In the current study, we used cell FRET to assess assembled states of IL-5Rα and βc and the impact of IL-5 on the assembly process. In order to test whether IL-5 receptor subunits multimerize at the plasma membrane and to measure their dependence on IL-5, we expressed the receptor subunits as full-length fusion proteins linked at their carboxyl termini to either cyan or yellow fluorescent proteins. We assessed the assembly of IL-5 receptors and the effect of IL-5 by FRET after acceptor photobleaching. Our data demonstrate that the IL-5Rα subunit is present as a monomer on the cell surface, whereas βc exists as a dimer or a higher order homo-oligomer. We found that IL-5 binding induces IL-5Rα·βc assembly. Interestingly, we also found that IL-5 binding leads to organizational changes within the intracellular domains of βc·βc multimers in the presence of IL-5·IL-5Rα. The results demonstrate a hitherto unknown structural rearrangement in the intracellular domain of βc subunit that could be important in switching on cytokine signal transduction. Protein Expression and Purification—The human IL-5 protein was expressed and purified as previously described (18Scibek J.J. Evergren E. Zahn S. Canziani G.A. Van Ryk D. Chaiken I.M. Anal. Biochem. 2002; 307: 258-265Crossref PubMed Scopus (19) Google Scholar, 37Ishino T. Urbina C. Bhattacharya M. Panarello D. Chaiken I. J. Biol. Chem. 2005; 280: 22951-22961Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). AF17121 peptide was synthesized and purified as before (33Krause C.D. Mei E. Mirochnitchenko O. Lavnikova N. Xie J. Jia Y. Hochstrasser R.M. Pestka S. Biochem. Biophys. Res. Commun. 2006; 340: 377-385Crossref PubMed Scopus (25) Google Scholar, 42Shanafelt A.B. Miyajima A. Kitamura T. Kastelein R.A. EMBO J. 1991; 10: 4105-4112Crossref PubMed Scopus (76) Google Scholar). Reagents—All enzymes were purchased from New England Biolabs Inc. (Beverly, MA). The Phoenix cell line was purchased from ATCC (Manassas, VA). DNA oligonucleotide primers were purchased from Invitrogen. Dulbecco's modified Eagle's medium was from Invitrogen. pEYFP-N1 and pECFP-N1 were from Clontech (Palo Alto, CA). Opti-MEM was from Invitrogen. Fetal bovine serum was from Sigma. FuGENE 6 was from Roche Applied Science. Vectashield mounting medium was from Vector Laboratories (Burlingame, CA). Poly-d-lysine coverslips were from BD Biosciences. The CD4-YFP cDNA was a gift from Dr. Tian Jin (Laboratory of Immunogenetics, Twin-brook II Facility, NIAID, National Institutes of Health, Rockville, MD). cDNA, Mutagenesis, Cell Lines, and Transfections—A 1300-base pair fragment encoding the full-length human IL-5Rα was amplified by PCR using forward (5′-TATAGAATTCATGATCATCGTGGCGCATGTATTAC-3′; EcoRI site underlined) and reverse (5′-TATAACCGGTCCAAACACAGAATCCTCCAGGG-3′; AgeI site underlined) oligonucleotides. A 2700-base pair fragment encoding the full-length human βc receptor was amplified by PCR using forward (5′-TATAGAATTCATGGTGCTGGCCCAGGGGCTGC-3′; EcoRI site underlined) and reverse (5′-TATAACCGGTCCACACACCTCCCCAGGC-3′; AgeI site underlined) oligonucleotides. These fragments were digested by restriction enzyme, purified from agarose gel, ligated into pECFP-N1 (CFP) and pEYFP-N1 (YFP), and then transformed using DH5α-competent cells. The βc-pEYFP-N1 mutant constructs (Y15S, F79A, Y347Q or Y347S, and Y390S) were generated from the corresponding wild type vector by site-directed mutagenesis using QuikChange (Stratagene). Nontagged IL-5Rα was generated by introducing an additional AgeI restriction enzyme site at the end of the receptor coding sequence using site-directed mutagenesis, followed by removal of the Y/CFP DNA and religation into the original vectors. Phoenix cells, a derivative of human embryonic kidney 293 cells, were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin (Sigma), and 2 mml-glutamine at 37 °C in 5% CO2. Exponentially growing cells were dispersed with trypsin (Sigma). For immunoblotting analysis, cells were seeded at a density of 2 × 105/well in 6-well plates and transiently transfected with a combination of tagged IL-5Rα and βc chains using FuGENE 6 (Roche Applied Science) according to the manufacturer's recommendations and challenged with IL-5 or IL-5/AF17121 mixture for the times and doses indicated. For fixed cell imaging and acceptor photobleaching assays, cells were seeded at a density of 1 × 105/well on poly-d-lysine-treated coverslips in 6-well plates and transiently co-transfected with combinations of IL-5Rα-CFP and βc-YFP chains, IL-5Rα-CFP and IL-5Rα-YFP, or βc-CFP and βc-YFP chains. Forty-eight hours post-transfection, cells were challenged with IL-5 or IL-5/AF17121 mixture for the times and doses indicated and then fixed with 4% paraformaldehyde and washed with phosphate-buffered saline containing Ca2+ and Mg2+, and the coverslips were mounted onto Superfrost microscope slides (Fisher) using Vectashield (Vector Laboratories). Western Blotting—Phoenix cells expressing either the wild type human IL-5Rα and wild type human βc or wild type human IL-5Rα and human βc mutants were washed with cold phosphate-buffered saline and then lysed on ice using radioimmune precipitation buffer (Sigma). After centrifugation at 10,000 × g for 15 min, soluble proteins were separated by 4-20% SDS-PAGE and electroblotted onto nitrocellulose membranes. The membranes were blocked with 5% milk in Tris-buffered saline with 0.05% Tween 20 (TBST) and probed with the specific primary antibodies against IL-5Rα (SC-673; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), βc (SC-676; Santa Cruz Biotechnology), C/YFP (monoclonal antibody JL-8; Clontech), or actin (SC-1616; Santa Cruz Biotechnology) for 1 h at room temperature in 5% milk with TBST. Washed membranes were then incubated with a 1:5000 dilution of the appropriate secondary horseradish peroxidase-conjugated anti-rabbit or mouse-IgG (GE Healthcare) for 1 h at room temperature and developed using ECL+, an enhanced chemiluminescence detection kit (GE Healthcare). Acceptor Photobleaching FRET Assay by Confocal Microscopy—Phoenix cells transiently transfected and co-expressing combinations of IL-5Rα-CFP, βc-YFP, IL-5Rα-YFP, and/or βc-CFP were examined with a ×40 objective lens on a Leica TCS SP2 laser-scanning confocal microscope at the Imaging Facility, Drexel University College of Medicine. Cells transfected with IL-5Rα-CFP or βc-YFP alone were used to calibrate laser intensity to prevent bleed-through. For each slide, cells were viewed using the differential interface contrast function, and the field of cells was scanned to find healthy cells. Following this, cells were digitally imaged using the acceptor photobleaching module of the Leica Confocal Software (version 2.61.1537), and a region including cells expressing both fused receptors was defined as the region of interest (ROI). Images of the donor were acquired by exciting with a 458-nm laser and reading emitted spectra between 467 and 505 nm. Images of the acceptor (YFP) were acquired by exciting with a 514-nm laser and reading emitted spectra between 520 and 575 nm. These provided reference images for the donor and acceptor expression levels before photobleaching. The Leica Confocal Software was configured to achieve 80% photobleaching of YFP in the selected ROI, using maximum power of the 514-nm line. This led to nearly complete loss of YFP fluorescence when visualized by microscopy. Images of the donor and acceptor fluorescence after photobleaching were obtained by exciting at 458 nm and at 514 nm, respectively. These were generated simultaneously as soon as 80% photobleaching was achieved. Data were collected from 15-50 different cells in different fields from the same slide, and 25 ROIs were measured per slide in each experiment. Each experiment was performed at least three times. After photobleaching, additional ROIs were chosen at cell membrane locations on the cells within the photobleached area (4-5 ROIs/cell), and the mean donor fluorescence before and after photobleaching was obtained using the acceptor photobleaching application of the Leica Confocal Software. The FRET efficiency between CFP (donor) and YFP (acceptor) was quantified with the acceptor photobleaching method (38Karpova T.S. Baumann C.T. He L. Wu X. Grammer A. Lipsky P. Hager G.L. McNally J.G. J. Microsc. 2003; 209: 56-70Crossref PubMed Scopus (259) Google Scholar) using the equation FRET efficiency (E) = ((Dpost - Dpre)/Dpost) and calculated with the Leica Confocal Software acceptor photobleaching application for each ROI. Here, Dpost is the fluorescence intensity of the CFP (D for "donor") after photobleaching, and Dpre is the fluorescence intensity of the CFP before photobleaching. Statistical Analysis—Data are presented as mean ± S.E. The significance of differences between the means of various treatment conditions was determined by Student's t test assuming equal variance. For each treatment, at least 25 ROIs were chosen per experiment. Differences were considered to be significant at p < 0.05 (not corrected for multiple comparisons). Each experiment was performed at least three times (unless otherwise indicated), carried out on different days and with different cell preparations. IL-5Rα-C/YFP and βc-C/YFP Expression in Transfected Phoenix Cells—To assess IL-5 receptor subunit assembly in cells by FRET, full-length human IL-5Rα and βc were fused to either CFP or YFP at their carboxyl termini to prepare four distinct tagged receptor constructs (Fig. 1A). These were then transiently transfected into Phoenix cells. Cells were co-transfected with the following combination of vectors: 1) IL-5Rα-CFP and βc-YFP, 2) IL-5Rα-CFP and IL-5Rα-YFP, and 3) βc-CFP and βc-YFP. CFP and YFP were chosen because they have excitation and emission wavelengths favorable for FRET, coupled with suitable extinction coefficients and quantum yields (39Zacharias D.A. Violin J.D. Newton A.C. Tsien R.Y. Science. 2002; 296: 913-916Crossref PubMed Scopus (1750) Google Scholar). Cells were initially analyzed by Western blotting to confirm protein expression. Fig. 1B (left) shows the expression of full-length IL-5Rα-YFP (αY) and IL-5Rα-CFP (αC) in cells transfected with each or co-transfected with βc(αC/βY). Fig. 1B (right) shows the expression of full length βc-YFP (βY) and βc-CFP (βC) in cells transfected with a single vector or co-transfected with both IL-5Rα-CFP and βc-YFP (αC/βY). Transfected proteins were detected with anti-IL-5Rα or anti-βc antibodies, respectively. The 154 kDa band corresponds to βc-C/YFP fusion protein, whereas the lower band at 82 kDa corresponds to the IL-5Rα-C/YFP. Fig. S1 shows the expression and co-expression of these fusion proteins as detected with anti-C/YFP monoclonal antibody JL-8 raised against GFP and cross-reactive with both CFP and YFP. IL-5-induced Oligomerization of IL-5Rα and βc Subunits—A variety of studies have indicated that IL-5Rα associates with βc in the presence of ligand (16Murata Y. Takaki S. Migita M. Kikuchi Y. Tominaga A. Takatsu K. J. Exp. Med. 1992; 175: 341-351Crossref PubMed Scopus (162) Google Scholar, 17Tavernier J. Tuypens T. Plaetinck G. Verhee A. Fiers W. Devos R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7041-7045Crossref PubMed Scopus (119) Google Scholar, 22Devos R. Plaetinck G. Van der Heyden J. Cornelis S. Vandekerckhove J. Fiers W. Tavernier J. EMBO J. 1991; 10: 2133-2137Crossref PubMed Scopus (73) Google Scholar, 40Migita M. Yamaguchi N. Mita S. Higuchi S. Hitoshi Y. Yoshida Y. Tomonaga M. Matsuda I. Tominaga A. Takatsu K. Cell Immunol. 1991; 133: 484-497Crossref PubMed Scopus (65) Google Scholar, 41Lopez A.F. Shannon M.F. Hercus T. Nicola N.A. Cambareri B. Dottore M. Layton M.J. Eglinton L. Vadas M.A. EMBO J. 1992; 11: 909-916Crossref PubMed Scopus (88) Google Scholar, 42Shanafelt A.B. Miyajima A. Kitamura T. Kastelein R.A. EMBO J. 1991; 10: 4105-4112Crossref PubMed Scopus (76) Google Scholar). We examined whether the addition of IL-5 ligand affected receptor subunit hetero-oligomerization. This was done by analyzing the effect of IL-5 on FRET between IL-5Rα and βc subunits (Fig. 2A). We first assessed IL-5Rα-CFP and βc-YFP expression and localization by capturing images of cells using the acceptor photobleaching mode of the confocal microscope (see "Experimental Procedures"), recording signals simultaneously in the CFP and YFP channels. CFP or YFP alone was expressed mainly in the cytoplasm (data not shown). In contrast, the tagged receptors were expressed predominantly at the cell membrane (Figs. 2B and S2), along with some accumulation of the tagged receptors in the cytoplasm. This type of distribution has been seen with other cytokine receptors expressed ectopically (35Kramer J.M. Yi L. Shen F. Maitra A. Jiao X. Jin T. Gaffen S.L. J. Immunol. 2006; 176: 711-715Crossref PubMed Scopus (83) Google Scholar). These data showed that co-transfected cells expressed both IL-5 receptor subunits appropriately on the cell surface. We next assessed the possible presence of FRET between IL-5Rα and βc. Full-length receptors IL-5Rα-CFP and βc-YFP were co-expressed in Phoenix cells, which were then treated with IL-5 or with a combination of IL-5 and AF17121 and subjected to FRET analysis after photobleaching YFP to 20% of basal levels. The addition of IL-5 for 30 min to cells coexpressing IL-5Rα-CFP and βc-YFP had no effect on basal surface expression levels of either IL-5Rα or βc (Fig. 2B). When IL-5 was added to cells co-expressing IL-5Rα-CFP and βc-YFP, an increase in the fluorescence of the donor was observed after photobleaching (Fig. 2B, compare data in the first and third panels of the second row). This visible increase in fluorescence of donor after photobleaching suggested that FRET was occurring between the tagged IL-5Rα and βc subunits in the presence of IL-5. This response was inhibited by AF17121, a peptide antagonist of IL-5Rα (37Ishino T. Urbina C. Bhattacharya M. Panarello D. Chaiken I. J. Biol. Chem. 2005; 280: 22951-22961Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 43England B.P. Balasubramanian P. Uings I. Bethell S. Chen M.J. Schatz P.J. Yin Q. Chen Y.F. Whitehorn E.A. Tsavaler A. Martens C.L. Barrett R.W. McKinnon M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6862-6867Crossref PubMed Scopu

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