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• W2009148545 abstract "CD47 is a unique member of the Ig superfamily with a single extracellular Ig domain followed by a multiply membrane-spanning (MMS) domain with five transmembrane segments, implicated in both integrin-dependent and -independent signaling cascades. Essentially all functions of CD47 require both the Ig and MMS domains, raising the possibility that interaction between the two domains is required for normal function. Conservation of Cys residues among CD47 homologues suggested the existence of a disulfide bond between the Ig and MMS domains that was confirmed by chemical digestion and mapped to Cys33 and Cys263. Subtle changes in CD47 conformation in the absence of the disulfide were suggested by decreased binding of two anti-Ig domain monoclonal antibodies, decreased SIRPα1 binding, and reduced CD47/SIRPα1-mediated cell adhesion. Mutagenesis to prevent formation of this disulfide completely disrupted CD47 signaling independent of effects on ligand binding, as assessed by T cell interleukin-2 secretion and Ca2+responses. Loss of the disulfide did not affect membrane raft localization of CD47 or its association with αvβ3 integrin. Thus, a disulfide bond between the Ig and MMS domains of CD47 is required for normal ligand binding and signal transduction. CD47 is a unique member of the Ig superfamily with a single extracellular Ig domain followed by a multiply membrane-spanning (MMS) domain with five transmembrane segments, implicated in both integrin-dependent and -independent signaling cascades. Essentially all functions of CD47 require both the Ig and MMS domains, raising the possibility that interaction between the two domains is required for normal function. Conservation of Cys residues among CD47 homologues suggested the existence of a disulfide bond between the Ig and MMS domains that was confirmed by chemical digestion and mapped to Cys33 and Cys263. Subtle changes in CD47 conformation in the absence of the disulfide were suggested by decreased binding of two anti-Ig domain monoclonal antibodies, decreased SIRPα1 binding, and reduced CD47/SIRPα1-mediated cell adhesion. Mutagenesis to prevent formation of this disulfide completely disrupted CD47 signaling independent of effects on ligand binding, as assessed by T cell interleukin-2 secretion and Ca2+responses. Loss of the disulfide did not affect membrane raft localization of CD47 or its association with αvβ3 integrin. Thus, a disulfide bond between the Ig and MMS domains of CD47 is required for normal ligand binding and signal transduction. extracellular matrix multiply membrane-spanning T cell receptor signal-regulatory protein methyl-β-cyclodextrin fluorescein isothiocyanate 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine phosphate-buffered saline polyacrylamide gel electrophoresis monoclonal antibody G-protein-coupled receptor 2-(2′-nitrophenylsulfonyl)-3-methyl-3- bromoindolenine A cell responds to its environment through cues arising from binding of soluble mediators of cell-cell communication and from interaction with insoluble molecules in the extracellular matrix (ECM)1 or on adjoining cells. A detailed understanding of how specific plasma membrane molecules transduce information from the extracellular milieu to the cytoplasm and how these are spatially and temporally integrated is required to begin to understand, predict, and potentially regulate these responses in normal and pathologic conditions. Two major models of transmembrane signal transduction in response to ligand binding have been advanced. For many Ig family and growth factor receptors, signaling is initiated by receptor clustering. Other receptors, like the heptaspanin family of heterotrimeric G protein-coupled receptors, seem to signal ligand binding through conformational changes in the membrane-spanning domain that lead ultimately to the activation of effector cascades. CD47 is an Ig superfamily member involved in signaling from both cell-cell and cell-ECM interactions. Coligation of CD47 and the T cell antigen receptor (TCR) is a synergistic signal for activation of T lymphocytes (1Reinhold M.I. Lindberg F.P. Kersh G.J. Allen P.M. Brown E.J. J. Exp. Med. 1997; 185: 1-11Crossref PubMed Scopus (139) Google Scholar, 2Reinhold M.I. Green J.M. Lindberg F.P. Ticchioni M. Brown E.J. International Immunology. 1999; 11: 707-718Crossref PubMed Scopus (48) Google Scholar, 3Rebres R.A. Green J.M. Reinhold M.I. Ticchioni M. Brown E.J. J. Biol. Chem. 2001; 276: 7672-7680Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) that requires CD47-induced association of protein kinase Cθ with cytoskeleton (3Rebres R.A. Green J.M. Reinhold M.I. Ticchioni M. Brown E.J. J. Biol. Chem. 2001; 276: 7672-7680Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In addition, CD47 can exist in a plasma membrane complex with the integrin αvβ3 or α2β1that both modulates integrin function and has signal transduction properties distinct from either CD47 or the integrin in isolation (4Green J.M. Zhelesniak A. Chung J. Lindberg F.P. Sarfati M. Frazier W.A. Brown E.J. J. Cell Biol. 1999; 146: 673-682Crossref PubMed Scopus (155) Google Scholar, 5Lindberg F.P. Gresham H.D. Reinhold M.I. Brown E.J. J. Cell Biol. 1996; 134: 1313-1322Crossref PubMed Scopus (107) Google Scholar, 6Gao A.-G. Lindberg F.P. Dimitry J.M. Brown E.J. Frazier W.A. J. Cell Biol. 1996; 135: 533-544Crossref PubMed Scopus (185) Google Scholar, 7Chung J. Gao A.G. Frazier W.A. J. Biol. Chem. 1997; 272: 14740-14746Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). CD47 binds directly to thrombospondin, a protein of the provisional ECM at sites of inflammation (8Gao A.-G. Lindberg F.P. Finn M.B. Blystone S.D. Brown E.J. Frazier W.A. J. Biol. Chem. 1996; 271: 21-24Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar), and to SIRPα1, a broadly expressed plasma membrane molecule most highly represented on neurons, macrophages, and dendritic cells (9Adams S. van der Laan L.J. Vernon-Wilson E. Renardel d.L. Dopp E.A. Dijkstra C.D. Simmons D.L.,. van den Berg T.K. J. Immunol. 1998; 161: 1853-1859PubMed Google Scholar, 10Seiffert M. Cant C. Chen Z. Rappold I. Brugger W. Kanz L. Brown E.J. Ullrich A. Buhring H.J. Blood. 1999; 94: 3633-3643Crossref PubMed Google Scholar). Interaction with these ligands can lead to cell adhesion to ECM (6Gao A.-G. Lindberg F.P. Dimitry J.M. Brown E.J. Frazier W.A. J. Cell Biol. 1996; 135: 533-544Crossref PubMed Scopus (185) Google Scholar, 7Chung J. Gao A.G. Frazier W.A. J. Biol. Chem. 1997; 272: 14740-14746Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 11Wang X.Q. Frazier W.A. Mol. Biol. Cell. 1998; 9: 865-874Crossref PubMed Scopus (139) Google Scholar), to cell-cell aggregation (12Babic I. Schallhorn A. Lindberg F.P. Jirik F.R. J. Immunol. 2000; 164: 3652-3658Crossref PubMed Scopus (47) Google Scholar, 13Han X. Sterling H. Chen Y. Saginario C. Brown E.J. Frazier W.A. Lindberg F.P. Vignery A. J. Biol. Chem. 2000; 275: 37984-37992Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), or to alterations in cell behavior via heterotrimeric G protein-dependent and -independent mechanisms (3Rebres R.A. Green J.M. Reinhold M.I. Ticchioni M. Brown E.J. J. Biol. Chem. 2001; 276: 7672-7680Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 6Gao A.-G. Lindberg F.P. Dimitry J.M. Brown E.J. Frazier W.A. J. Cell Biol. 1996; 135: 533-544Crossref PubMed Scopus (185) Google Scholar, 14Frazier W.A. Gao A.G. Dimitry J. Chung J. Brown E.J. Lindberg F.P. Linder M.E. J. Biol. Chem. 1999; 274: 8554-8560Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). CD47-deficient mice or cells show a variety of abnormalities consistent with a significant role for this molecule in modulating cell responses to adhesive stimuli. Since CD47 is an Ig family member that can in at least some circumstances signal via heterotrimeric G proteins, the paradigm for signal transduction through this interesting molecule is uncertain. CD47 is an unusual member of the Ig superfamily because, in addition to a single Ig domain, it has a highly hydrophobic, multiply membrane-spanning (MMS) domain that is thought to contain five transmembrane segments (15Lindberg F.P. Gresham H.D. Schwarz E. Brown E.J. J. Cell Biol. 1993; 123: 485-496Crossref PubMed Scopus (302) Google Scholar). Structure-function studies have demonstrated that both the Ig domain and the MMS domain of CD47 are essential for its role in signal transduction as well as for localization of CD47 to the cholesterol-rich plasma membrane domains known as glycosphingolipid-enriched membranes (gems) or rafts (1Reinhold M.I. Lindberg F.P. Kersh G.J. Allen P.M. Brown E.J. J. Exp. Med. 1997; 185: 1-11Crossref PubMed Scopus (139) Google Scholar, 2Reinhold M.I. Green J.M. Lindberg F.P. Ticchioni M. Brown E.J. International Immunology. 1999; 11: 707-718Crossref PubMed Scopus (48) Google Scholar,4Green J.M. Zhelesniak A. Chung J. Lindberg F.P. Sarfati M. Frazier W.A. Brown E.J. J. Cell Biol. 1999; 146: 673-682Crossref PubMed Scopus (155) Google Scholar). Chimeric molecules in which the CD8 Ig domain or the FLAG epitope was substituted for CD47's Ig were mislocalized and nonfunctional, even when the new extracellular domain was ligated by appropriate antibodies (3Rebres R.A. Green J.M. Reinhold M.I. Ticchioni M. Brown E.J. J. Biol. Chem. 2001; 276: 7672-7680Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). This requirement for its specific Ig domain is unlike those of more conventional Ig superfamily signaling molecules (16Kolanus W. Romeo C. Seed B. Cell. 1993; 74: 171-183Abstract Full Text PDF PubMed Scopus (304) Google Scholar) and suggests that CD47 aggregation is insufficient to initiate signaling. Thus, the Ig domain plays a fundamental role in CD47 signal transduction in addition to its binding of the ligands thrombospondin and SIRPα1, which suggests the possibility that the CD47 Ig domain is required for a signaling-competent conformation of the molecule. The purpose of the present study was to determine the reason for the requirement for the CD47 Ig domain in its signaling function. We have found that there is a long range disulfide bond between Cys33 and Cys263 in human CD47 that is required for signal transduction as well as for normal SIRPα1 binding. Moreover, the cysteines involved in this long range disulfide are conserved in all species' CD47 paralogs and in the poxvirus molecules of unknown function with structural homology to CD47. The unusual requirement for the CD47 Ig domain in signaling function can be explained at least in part by its interaction with the MMS domain, leading to appropriate conformation not only for ligand binding but for association with intracellular signaling cascades as well. Jurkat cells and the CD47-deficient Jurkat clone JinB8 (2Reinhold M.I. Green J.M. Lindberg F.P. Ticchioni M. Brown E.J. International Immunology. 1999; 11: 707-718Crossref PubMed Scopus (48) Google Scholar) were maintained in RPMI with 10% FBS, nonessential amino acids, 2 mm glutamine, 50 μmβ-mercaptoethanol, and 50 μg/ml gentamicin. OV10 cells expressing human β3 integrin were maintained in Iscove's modified Dulbecco's medium with 10% fetal bovine serum, 10 μg/ml ciprofloxicin, and 100 μg/ml hygromycin. The following mAbs were employed: anti-CD47 Ig domain 2D3, 2E11, 1F7, B6H12, 2B7, 3G3, and 410 (all IgG) and 10G2 (IgM) (17Brown E.J. Hooper L. Ho T. Gresham H.D. J. Cell Biol. 1990; 111: 2785-2794Crossref PubMed Scopus (315) Google Scholar, 18Oldenborg P.A. Zheleznyak A. Fang Y.F. Lagenaur C.F. Gresham H.D. Lindberg F.P. Science. 2000; 288: 2051-2054Crossref PubMed Scopus (1280) Google Scholar, 19Hermann P. Armant M. Brown E. Rubio M. Ishihara H. Ulrich D. Caspary R.G. Lindberg F.P. Armitage R. Maliszewski C. Delespesse G. Sarfati M. J. Cell Biol. 1999; 144: 767-775Crossref PubMed Scopus (72) Google Scholar); anti-CD47 C-terminal peptide (NQKTIQPPRNN) mAb 131 and mAb 151 (distinct epitopes) (15Lindberg F.P. Gresham H.D. Schwarz E. Brown E.J. J. Cell Biol. 1993; 123: 485-496Crossref PubMed Scopus (302) Google Scholar); anti-FLAG epitope (M2; Sigma); myeloma IgG1 (MOPC-21; Sigma); anti-CD8α (53-6.7; Pharmingen), anti-CD28 (15E8; Caltag); anti-CD3 (OKT3); anti-mouse IL-2 (JES6-1A12; Pharmingen), biotin-conjugated anti-mouse IL-2 (JES6-5H4; Pharmingen). Secondary antibodies goat anti-mouse IgG (ICN/Cappel, Durham, NC), rabbit anti-mouse IgG (+/−) FITC (Sigma), and goat anti-human IgG Fc (+/−) FITC (Sigma) were commercially available. Streptavidin-horseradish peroxidase was purchased from Sigma. Methyl-β-cyclodextrin was from Aldrich. Magentic beads for cell sorting were from Dynal Biotech (Lake Success, NY) or Miltenyi Biotec (Auburn, CA). Chimeric forms of CD47 were made with standard molecular techniques and have been described previously (1Reinhold M.I. Lindberg F.P. Kersh G.J. Allen P.M. Brown E.J. J. Exp. Med. 1997; 185: 1-11Crossref PubMed Scopus (139) Google Scholar,2Reinhold M.I. Green J.M. Lindberg F.P. Ticchioni M. Brown E.J. International Immunology. 1999; 11: 707-718Crossref PubMed Scopus (48) Google Scholar). Briefly, CD47/CD7 is composed of the Ig domain of CD47 linked to the transmembrane domain of CD7. CD47/GPI contains the CD47 Ig domain with a glycosylphosphatidylinositol anchor. FLAG-MMS and CD8-MMS include a FLAG epitope tag or murine CD8α Ig domain, respectively, linked to the MMS domain of CD47. Fig. 1 A shows a schematic model of CD47 indicating the sites of designed MMS domain mutations. All mutations were constructed using standard molecular techniques. All products from PCR were confirmed by sequencing in both directions. The mutant, termed first ICL (1 st ICL), replaces163KTLKYRSGGMDEK175 (all amino acid numbering based on immature CD47) with KAAAAAKAAAAAK. The second ICL mutation (2 nd ICL) replaces234LTS236 with KKK. The third transmembrane mutation (3 rd TM) replaces224HYY226 with AAA. The Cys/Ser mutants replace Cys residues at 33, 259, and/or 263 with Ser, as indicated. cDNA encoding the extracellular three Ig domains of human SIRPα1 (SHPS-1) was obtained as IMAGE clone number 2017171. It was modified and transferred by standard molecular techniques into pCDM8 containing the coding sequence of the hinge and constant regions of the heavy chain of human IgG1 (20Aruffo A. Stamenkovic I. Melnick M. Underhill C.B. Seed B. Cell. 1990; 61: 1303-1313Abstract Full Text PDF PubMed Scopus (2155) Google Scholar). SIRPα1-Fc fusion protein was produced by transient transfection of COS cells or stable transfection of 293 cells using lipofectamine/PLUS reagent (Life Technologies, Inc.). Protein was harvested from culture supernatants using Protein A-Sepharose and confirmed to exist as a dimer by Western blot with anti-human IgG-Fc (apparent molecular mass of ∼130 kDa). SIRPα1/CD7 was constructed from the modified IMAGE clone and pIAP323, which contains the CD7 transmembrane domain (5Lindberg F.P. Gresham H.D. Reinhold M.I. Brown E.J. J. Cell Biol. 1996; 134: 1313-1322Crossref PubMed Scopus (107) Google Scholar). It consists of the three extracellular Ig domains of SIRPα1 linked to the transmembrane domain of CD7. Jurkat and JinB8 cells were transfected by electroporation at 280 V, 1000 microfarads, and 25 °C in RPMI and immediately placed on ice. After 10 min, cells were placed in nonselective medium for 24 h and then transferred to medium containing 1.5 mg/ml Geneticin. OV10 cells were transfected using LipofectAMINE reagent. After 24 h, cells were passed into medium containing 400 μg/ml Geneticin. None of the mutations prevented surface expression, and all cells were sorted by fluorescence-activated cell sorting or magnetic bead methods to isolate positive populations of equal expression level (Fig.1 B). CD47 was isolated from human placenta as previously described (17Brown E.J. Hooper L. Ho T. Gresham H.D. J. Cell Biol. 1990; 111: 2785-2794Crossref PubMed Scopus (315) Google Scholar). Briefly, tissue was minced and homogenized in 50 mm Tris, 0.25 msucrose, 5 mm iodoacetamide, 2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin (pH 7.4). Membrane fractions were prepared from the cleared homogenate, dissolved in homogenization buffer with 80 mm β-d-octyl glucoside, and incubated with anti-CD47 Sepharose. Anti-CD47-Sepharose was washed with low and high salt CHAPS buffers (150 or 500 mm NaCl, 25 mm Hepes, 10 mm CHAPS, 3 mmCa2+, 1 mm Mg2+, 25 μm paranitrophenyl paraguanidinobenzoate, pH 7.4), and bound material was eluted with 10 mm CHAPS in 500 mm NaCl, 100 mm glycine, pH 3.3, and neutralized with one-tenth volume of 1 m Tris (pH 9.0). Eluted material was then dialyzed against low salt CHAPS buffer, reapplied to the anti-CD47 Sepharose, eluted, and dialyzed as described. Aliquots of isolated CD47 were lyophilized and redissolved in 1% acetic acid. Three volumes of 1 mg/ml BNPS-skatole in glacial acetic acid were added, and the mixture was incubated in the dark at 25 °C for 24 h. The digest was diluted 1:1 with water and centrifuged at 10,000 × g for 5 min to remove precipitated BNPS-skatole. The supernatant was concentrated in a Speedvac evaporator (Savant) to reduce volume and remove residual acetic acid and treated with Tricine sample buffer (0.1 mTris, 24% glycerol, 8% SDS, 0.02% Coomassie Blue G-250) with or without 200 mm dithiothreitol for 40 min at 40 °C. Samples were run on Tris-Tricine gels, blotted to Immobilon-P-SQ membranes (Millipore Corp.), and probed with mAb 131. A similar method of affinity isolation of JinB8 transfectant CD47 was performed for endoproteinase Arg-C digests. Briefly, cells were treated with 20 mm iodoacetamide in PBS in the dark at 0 °C for 45 min and then lysed in 10 mm CHAPS, 10 mmiodoacetamide, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride in PBS (pH 7.4) at 4 °C for 30 min. Lysates were cleared by centrifugation at 14,000 ×g, and CD47 protein was immunoprecipitated with anti-CD47-Sepharose, washed, eluted, and dialyzed as described (17Brown E.J. Hooper L. Ho T. Gresham H.D. J. Cell Biol. 1990; 111: 2785-2794Crossref PubMed Scopus (315) Google Scholar). Digests were performed in 10 mm CHAPS, 0.1 mHepes (pH 8.2), with 20 μg/ml protein sequencing grade endoproteinase Arg-C (Sigma) for 1 h at 37 °C. Samples were then treated with Laemmli sample buffer with or without β-mercaptoethanol at 60 °C for 15 min, run on 4–20% gradient SDS-PAGE gels, blotted to polyvinylidene difluoride, and probed with mAb 151. For evaluation of the trypsin sensitivity of the CD47 mutants, cells were pretreated with 10 mm iodoacetamide in PBS for 15 min on ice, lysed in 1% Triton X-100, 20 mm Tris, 140 mm NaCl, 10 mm iodoacetamide, 2 mmEDTA, pH 8.2, for 30 min and centrifuged at 14,000 × gfor 5 min. The cleared whole cell lysates were then digested with trypsin at 2.5 mg/ml for 30 min at 37 °C, and the reaction was stopped by the addition of Laemmli sample buffer and incubation at 60 °C for 15 min. Samples were then Western blotted with anti-CD47 Ig domain-specific mAb B6H12. For assessment of binding of SIRPα1-Fc protein, cells were incubated with SIRPα1-Fc fusion protein, anti-CD47, or control mAb in PBS with 1% bovine serum albumin for 30 min on ice, washed, and labeled with anti-human IgG-Fc-FITC or anti-mouse IgG-FITC. Samples were then analyzed on a flow cytometer, and the mean fluorescence was determined, from which a ratio of SIRPα1/CD47 fluorescence was calculated. For examination of mAb 10G2 binding, cells were pretreated with 100 ng/ml phorbol 12-myristate 13-acetate plus 2 μmionomycin for 18 h prior to analysis, and the percentage of positive staining was calculated. For treatment with methyl-β-cyclodextrin (MβCD), cells were harvested in PBS with 5 mm EDTA, washed once with PBS and once with Iscove's modified Dulbecco's medium plus 0.1% fatty acid-free bovine serum albumin. Cells were then resuspended at 2.5 × 105cells/ml with or without 10 mm MβCD, incubated at 37 °C for 10–15 min, and washed with Iscove's modified Dulbecco's medium plus fatty acid-free bovine serum albumin. Samples were then processed for flow cytometry as described. JinB8 cells transfected with CD47 mutants were loaded with carboxy-SNARF-1 AM (Molecular Probes, Inc., Eugene, OR), and JinB8 cells transfected with SIRPα1/CD7 or vector were loaded with CellTracker Green CMFDA (5-chloromethylfluorescein diacetate) (Molecular Probes). Cells expressing normal CD47 or mutants were next incubated with anti-CD47 (2D3)-coated 4.5 μm magnetic beads (Dynal), and 1 × 105 of these cells were added to a 24-well plate well along with 1 × 105SIRPα1/CD7 cells in 1 ml of RPMI medium. After incubation for 1–8 h, cells were transferred to Eppendorf tubes, and bead-bound cells were separated with a magnet. Approximately 75% of SNARF-1-labeled (CD47+) cells were isolated by this procedure. Magnet-associated cells were lysed in 1% Nonidet P-40, 10 mm Tris (pH 7.4), 145 mm NaCl, and cleared lysates were evaluated in a fluorescence plate reader at SNARF-1 and CellTracker Green wavelengths versus standards of known cell number. Adhesion indices were calculated as the ratios of green (SIRPα1/CD7-expressing) to red (CD47-expressing) cells adherent to the magnet after correction for background (SIRPα1-independent adhesion) by subtracting the adhesion index for SIRPα1/CD7-deficient cells (typically ∼10% of the adhesion of the SIRPα1/CD7-expressing cells). A typical adhesion index for cells expressing wild type CD47 was 0.5, which represented 1 SIRPα1/CD7+ cell pulled down per 2 CD47+ cells. All experimental points were assayed in triplicate. Human CD47-transfected murine 3.L2 T cells were stimulated by murine CH27 B cells presenting peptide antigen at varied doses as previously described (1Reinhold M.I. Lindberg F.P. Kersh G.J. Allen P.M. Brown E.J. J. Exp. Med. 1997; 185: 1-11Crossref PubMed Scopus (139) Google Scholar). Briefly, 1 × 1053.L2 cells, 1 × 104 CH27 cells, and 0.03–1 μm antigenic peptide (amino acids 64–76 of hemoglobind (21Evavold B.D. Williams S.G. Hsu B.L. Buus S. Allen P.M. J. Immunol. 1992; 148: 347-353PubMed Google Scholar)) were added in RPMI medium to the wells of 96-well plates and incubated for 18–24 h at 37 °C. An IL-2 enzyme-linked immunosorbent assay was performed on harvested supernatants according to the manufacturer's instructions (Pharmingen). Duplicate samples were assayed, each in at least three replicates of each experiment. Jurkat or JinB8 cells at 2 × 107cells/ml in RPMI complete medium were incubated with 3 μm fura-2-AM at 37 °C for 20 min and then diluted 10-fold and incubated an additional 20 min. Cells were then washed and incubated with the indicated concentrations of mAb on ice for 20 min. After labeling, cells were washed once with RPMI medium and twice with calcium buffer (25 mm Hepes, 125 mm NaCl, 5 mm KCl, 1 mm Na2HPO4, 0.5 mm MgCl2, 1 mmCaCl2, pH 7.4) and resuspended at 2.5 × 106 cells/ml in calcium buffer on ice. Fluorescence changes of a 2-ml stirred cell suspension warmed to 37 °C were monitored with a F-2000 or F-4500 spectrofluorimeter (Hitachi Instruments, Danbury, CT) using 340- and 380-nm excitation wavelengths and 510-nm emission wavelength following the addition of 10 μg/ml secondary antibody. Calcium concentrations were calculated as described by Grynkiewicz et al. (22Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Calcium flux from CD47 cross-linking was identical when cells were coated with 1 or 10 μg/ml anti-CD47 mAb, but no flux occurred when 0.1 μg/ml anti-CD47 was used or when the cross-linking antibody was omitted, confirming that CD47 aggregation was required for the rise in [Ca2+]i. The addition of 2 μm ionomycin served as a positive control for Ca2+ flux. The location of cell surface proteins in sucrose density gradients was evaluated using tracer125I-labeled antibodies as described (4Green J.M. Zhelesniak A. Chung J. Lindberg F.P. Sarfati M. Frazier W.A. Brown E.J. J. Cell Biol. 1999; 146: 673-682Crossref PubMed Scopus (155) Google Scholar). Cells were incubated with 5 μg/ml 125I-mAb in growth medium at 4 °C; washed; and lysed in 20 mm Tris-HCl, pH 8.2, 140 mm NaCl, 2 mm EDTA, 25 μg/ml aprotinin, 25 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, 0.1% Brij 58. Sucrose solution was added to a final concentration of 40% using a stock of 60% sucrose in 20 mm Tris-HCl, pH 8.2, 140 mm NaCl, 2 mm EDTA, and this mixture was layered over a volume of 60% sucrose. 25 and 5% sucrose layers were added to form a step gradient, which was centrifuged at 170,000 × g for 18 h at 4 °C. Fractions of 0.5 ml were collected from the top of the gradient, and radioactivity in each fraction as well as the pellet was assessed. Complexes were isolated as described (4Green J.M. Zhelesniak A. Chung J. Lindberg F.P. Sarfati M. Frazier W.A. Brown E.J. J. Cell Biol. 1999; 146: 673-682Crossref PubMed Scopus (155) Google Scholar). Briefly, OV10 cells expressing CD47 or mutants were mixed slowly in HBSS with anti-β3 integrin-coated magnetic beads for 15 min at 37 °C. Adherent cells were separated with a magnet and lysed in CHAPS buffer with mixing for 10 min. Beads were reisolated, bead-associated protein complexes were eluted with Laemmli sample buffer, and β3-associated CD47 was quantitated as described (4Green J.M. Zhelesniak A. Chung J. Lindberg F.P. Sarfati M. Frazier W.A. Brown E.J. J. Cell Biol. 1999; 146: 673-682Crossref PubMed Scopus (155) Google Scholar). Values are expressed in relative units based on densitometry of chemiluminscence-exposed bands on x-ray films. All experiments were repeated at least three times. Error bars in graphs depict S.E. The statistical significance of each set of results was evaluated by performing a one-way analysis of variance followed by Dunnett or individualt tests as appropriate. A p value of <0.05 was considered significant. Previous studies have indicated a requirement for both Ig and MMS domains of CD47 to mediate virtually all described functions of CD47 and for its efficient localization to membrane rafts. This suggests that an interaction between these domains may be important for function and subcellular localization. The Ig domain contains conserved cysteine residues at positions 41 and 114 that are necessary for the Ig domain formation as well as a potentially free Cys33 (Fig.1,A and C). Analysis of the structurally unusual MMS domain predicts a membrane topology with five membrane-spanning segments and residues Cys259 and Cys263 on the ectoplasmic face of the membrane (15Lindberg F.P. Gresham H.D. Schwarz E. Brown E.J. J. Cell Biol. 1993; 123: 485-496Crossref PubMed Scopus (302) Google Scholar) (Fig. 1 A). We postulated that one of these cysteines could be involved in a disulfide bridge between the Ig and MMS domains potentially important for an Ig/MMS interaction. We isolated CD47 from human placenta (17Brown E.J. Hooper L. Ho T. Gresham H.D. J. Cell Biol. 1990; 111: 2785-2794Crossref PubMed Scopus (315) Google Scholar) or JinB8 transfectants and performed chemical and enzymatic digests to evaluate disulfide linkage of the Ig and MMS domains. Attempts to determine the presence of a long range disulfide bond via classic methods of enzymatic digestion, high pressure liquid chromatography peptide separation, and mass spectrometry yielded only Ig domain peptides, as in previous studies using Edman degradation instead of mass spectrometry (17Brown E.J. Hooper L. Ho T. Gresham H.D. J. Cell Biol. 1990; 111: 2785-2794Crossref PubMed Scopus (315) Google Scholar). It is likely that MMS domain peptides were lost during processing due to their hydrophobic character. Instead, digestion with either BNPS-skatole or endoproteinase Arg-C followed by Western blotting with a mAb that recognizes the COOH terminus of CD47 was used to identify the fragmentation pattern with or without reduction. BNPS-skatole, which cleaves carboxyl-terminal to tryptophan residues, could determine the presence or absence of the putative long range disulfide, because an ∼15-kDa carboxyl-terminal fragment resulting from cleavage after Trp157 in the first transmembrane segment would be present without reduction only in the absence of a disulfide between the Ig and MMS domains (Fig.2 A). As shown in Fig.2 B, the 15.5-kDa fragment was not observed on Western blotting of nonreduced samples of placental CD47 after treatment with BNPS-skatole but was easily detected in digested samples that had been reduced prior to separation on SDS-PAGE. The smaller 5-kDa band seen in reduced digested samples must represent cleavage by BNPS at another residue. Since secondary reaction sites for BNPS are Cys and Met, the most likely site for this secondary BNPS cleavage is Cys259(yielding a 4.9-kDa fragment). If Cys259 was involved in a disulfide bond with the Ig domain and the cleavage site was Cys263, BNPS cleavage would have released the C terminus even without sample reduction. The closest potential methionyl cleavage site (Met172) would have yielded a significantly larger fragment (14 kDa) and is therefore unlikely to account for this small fragment. Whatever the identity of the cleavage site, it is within 5 kDa of the C terminus yet is covalently linked to the amino terminus of the protein in the absence of reduction, demonstrating that a cysteine carboxyl-terminal to this cleavage site is involved in a disulfide bond. CD47 point mutants C33S, C259S, and C263S were transfected into the CD47-deficient Jurkat clone JinB8 (2Reinhold M.I. Green J.M. Lindberg F.P. Ticchioni M. Brown E.J. International Immunology. 1999; 11: 707-718Crossref PubMed Scopus (48) Google Scholar) to identify the location of the cysteines involved in disulfide bonding. Unfortunately, it proved impossible" @default.
• W2009148545 created "2016-06-24" @default.
• W2009148545 creator A5054268751 @default.
• W2009148545 creator A5061070012 @default.
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