αvβ5 Integrin Sustains Growth of Human Pre-B Cells through an RGD-independent Interaction with a Basic Domain of the CD23 Protein
2007; Elsevier BV; Volume: 282; Issue: 37 Linguagem: Inglês
10.1074/jbc.m609335200
ISSN1083-351X
AutoresGillian Borland, Adrienne L. Edkins, Mridu Acharya, Johanne Matheson, Lindsey J. White, Janet M. Allen, Jean‐Yves Bonnefoy, Bradford W. Ozanne, William Cushley,
Tópico(s)Platelet Disorders and Treatments
ResumoCD23 is a type II transmembrane glycoprotein synthesized by hematopoietic cells that has biological activity in both membrane-bound and freely soluble forms, acting via a number of receptors, including integrins. We demonstrate here that soluble CD23 (sCD23) sustains growth of human B cell precursors via an RGD-independent interaction with the αvβ5 integrin. The integrin recognizes a tripeptide motif in a small disulfide-bonded loop at the N terminus of the lectin head region of CD23, centered around Arg172, Lys173, and Cys174 (RKC). This RKC motif is present in all forms of sCD23 with cytokine-like activity, and cytokine activity is independent of the lectin head, an "inverse RGD" motif, and the CD21 and IgE binding sites. RKC-containing peptides derived from this region of CD23 bind αvβ5 and are biologically active. The binding and activity of these peptides is unaffected by inclusion of a short peptide containing the classic RGD sequence recognized by integrins, and, in far-Western analyses, RKC-containing peptides bind to the β subunit of the αvβ5 integrin. The interaction between αvβ5 and sCD23 indicates that integrins deliver to cells important signals initiated by soluble ligands without the requirement for interactions with RGD motifs in their common ligands. This mode of integrin signaling may not be restricted to αvβ5. CD23 is a type II transmembrane glycoprotein synthesized by hematopoietic cells that has biological activity in both membrane-bound and freely soluble forms, acting via a number of receptors, including integrins. We demonstrate here that soluble CD23 (sCD23) sustains growth of human B cell precursors via an RGD-independent interaction with the αvβ5 integrin. The integrin recognizes a tripeptide motif in a small disulfide-bonded loop at the N terminus of the lectin head region of CD23, centered around Arg172, Lys173, and Cys174 (RKC). This RKC motif is present in all forms of sCD23 with cytokine-like activity, and cytokine activity is independent of the lectin head, an "inverse RGD" motif, and the CD21 and IgE binding sites. RKC-containing peptides derived from this region of CD23 bind αvβ5 and are biologically active. The binding and activity of these peptides is unaffected by inclusion of a short peptide containing the classic RGD sequence recognized by integrins, and, in far-Western analyses, RKC-containing peptides bind to the β subunit of the αvβ5 integrin. The interaction between αvβ5 and sCD23 indicates that integrins deliver to cells important signals initiated by soluble ligands without the requirement for interactions with RGD motifs in their common ligands. This mode of integrin signaling may not be restricted to αvβ5. CD23 is a 45-kDa type II transmembrane glycoprotein that functions as the low affinity receptor for IgE and negatively regulates IgE production by B lymphocytes (1Gould H.J. Sutton B.J. Beavil A.J. Beavil R.L. McCloskey N. Coker H.A. Fear D. Smurthwaite L. Annu. Rev. Immunol. 2003; 21: 579-628Crossref PubMed Scopus (539) Google Scholar, 2Bonnefoy J.-Y. Lecoanet-Henchoz S. Gauchat J.F. Graber P. Aubry J.P. Jeannin P. Plater-Zyberk C. Int. Rev. Immunol. 1997; 16: 113-128Crossref PubMed Scopus (68) Google Scholar, 3Delespesse G. Sarfati M. Wu C.Y. Fournier S. Letellier M. Immunol. Rev. 1992; 125: 77-97Crossref PubMed Scopus (152) Google Scholar, 4Fujiwara H. Kikutani H. Suematsu S. Naka T. Yoshida K. Yoshida K. Tanaka T. Suemura M. Matsumoto N. Kojima S. Kishimoto T. Yoshida N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6835-6839Crossref PubMed Scopus (144) Google Scholar, 5Yu P. Kosco-Vilbois M.H. Richards M. Kohler G. Lamers M.C. 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J. 1998; 333: 573-579Crossref PubMed Scopus (39) Google Scholar) to yield soluble CD23 (sCD23) 6The abbreviations used are:sCD23soluble CD23VnRvitronectin receptorVnvitronectinFnfibronectinHIVhuman immunodeficiency virusHRPhorseradish peroxidaseOGPoctyl-β-d-glucopyranosideGSTglutathione S-transferasesiRNAsmall interference RNAPFHMprotein-free hybridoma medium-IILCDlow cell densitymAbmonoclonal antibodyTdRtritiated thymidineSPRsurface plasmon resonanceLPlong peptidePEphycoerythrinFITCfluorescein isothiocyanate proteins with molecular masses ranging from 37 to 16 kDa, all of which retain the capacity to regulate IgE synthesis; human sCD23 proteins exhibit pleiotropic cytokine-like activities (1Gould H.J. Sutton B.J. Beavil A.J. Beavil R.L. McCloskey N. Coker H.A. Fear D. Smurthwaite L. Annu. Rev. Immunol. 2003; 21: 579-628Crossref PubMed Scopus (539) Google Scholar, 2Bonnefoy J.-Y. Lecoanet-Henchoz S. Gauchat J.F. Graber P. Aubry J.P. Jeannin P. Plater-Zyberk C. Int. Rev. 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In association with interleukin-1α, sCD23 promotes differentiation of monocytes and early thymocyte precursors (11Mossalayi M.D. Lecron J.-C. Dalloul A.H. Sarfati M. Bertho J.-M. Hofstetter H. Delespesse G. Drbre P. J. Exp. Med. 1990; 171: 959-964Crossref PubMed Scopus (95) Google Scholar) and, via binding to the αMβ2 (CD11b-CD18), αXβ2 (CD11c-CD18) (12Lecoanet-Henchoz S. Gauchat J.-F. Aubry J.P. Graber P. Life P. Paul-Eugene N. Ferrua B. Corbi A.L. Dugas B. Plater-Zyberk C. Bonnefoy J.-Y. Immunity. 1995; 3: 119-125Abstract Full Text PDF PubMed Scopus (134) Google Scholar), and αvβ3 integrins (13Hermann 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), stimulates tumor necrosis factor-α and interleukin-1α production by monocytes. soluble CD23 vitronectin receptor vitronectin fibronectin human immunodeficiency virus horseradish peroxidase octyl-β-d-glucopyranoside glutathione S-transferase small interference RNA protein-free hybridoma medium-II low cell density monoclonal antibody tritiated thymidine surface plasmon resonance long peptide phycoerythrin fluorescein isothiocyanate The structures of the derCD23 protein, a naturally occurring sCD23 fragment generated by action of the derp1 protease from Dermatophagoides pteronyssinus, and of a 25-kDa sCD23 (residues 150-321), have recently been solved by heteronuclear nuclear magnetic resonance spectroscopy (14Hibbert R.G. Teriete P. Grundy G.J. Beavil R.L. Reljic R. Holers V.M. Hannan J.P. Sutton B.J. Gould H.J. McDonnell J.M. J. Exp. Med. 2005; 202: 751-760Crossref PubMed Scopus (118) Google Scholar) and x-ray crystallography (15Wurzburg B.A. Tarchevskaya S.S. Jardetzky T.S. Structure. 2006; 14: 1049-1058Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), respectively. Although there are pronounced differences between the structures derived by the two methods, both show a generally consistent overall structure for the C-type lectin head domain comprising eight β sheets and two α helices (14Hibbert R.G. Teriete P. Grundy G.J. Beavil R.L. Reljic R. Holers V.M. Hannan J.P. Sutton B.J. Gould H.J. McDonnell J.M. J. Exp. Med. 2005; 202: 751-760Crossref PubMed Scopus (118) Google Scholar, 15Wurzburg B.A. Tarchevskaya S.S. Jardetzky T.S. Structure. 2006; 14: 1049-1058Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The NMR structure reveals a striking distribution of acidic and basic residues on opposites faces of the lectin head domain and demonstrates unequivocally that the interaction surfaces for IgE and CD21 binding are spatially distinct (14Hibbert R.G. Teriete P. Grundy G.J. Beavil R.L. Reljic R. Holers V.M. Hannan J.P. Sutton B.J. Gould H.J. McDonnell J.M. J. Exp. Med. 2005; 202: 751-760Crossref PubMed Scopus (118) Google Scholar); the data also support unequivocally earlier studies that suggested that these binding sites and the structures responsible for expression of cytokine-like activities are distinct (16Mossalayi M.D. Arock M. Delespesse G. Hofstetter H. Bettler B. Dalloul A.H. Killcher E. Quaaz F. Debré P. Sarfati M. EMBO J. 1992; 11: 4323-4328Crossref PubMed Scopus (30) Google Scholar). The crystal structure confirms that CD23 contains only a single calcium binding site in the lectin head and that conformational changes in the CD23 structure accompany calcium coordination in this site (15Wurzburg B.A. Tarchevskaya S.S. Jardetzky T.S. Structure. 2006; 14: 1049-1058Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The binding site for integrins was not identified in either study, and, as reported here, this resides at the N-terminal region of the C-type lectin domain. The αvβ5 integrin is a member of the vitronectin receptor (VnR) family. VnRs are heterodimers of the αv integrin chain in association with any one of five β subunits (17van der Flier A. Sonnenberg A. Cell Tissue Res. 2001; 305: 285-298Crossref PubMed Scopus (818) Google Scholar) and have well documented roles in cell attachment, spreading, and migration, rescue from apoptosis, and angiogenesis (18Hood J.D. Cheresh D.A. Nat. Rev. Cancer. 2002; 2: 91-100Crossref PubMed Scopus (1495) Google Scholar, 19Kumar C.C. Oncogene. 1998; 17: 1365-1373Crossref PubMed Scopus (240) Google Scholar, 20Stupack D.W. Cheresh D.A. J. Cell Sci. 2002; 115: 3729-3738Crossref PubMed Scopus (514) Google Scholar, 21Marshall J.F. Hart I.R. Semin. Cancer Biol. 1996; 7: 129-138Crossref PubMed Scopus (94) Google Scholar). VnRs bind to the extracellular matrix proteins vitronectin (Vn) and fibronectin (Fn) by recognition of an Arg-Lys-Asp (RGD) motif in target proteins (22Ruoslahti E. Annu. Rev. Cell Biol. 1996; 12: 697-715Crossref Scopus (2588) Google Scholar); both the α and β integrin subunits contribute to ligand binding (23Xiong J.-P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S.L. Arnout M.A. Science. 2001; 294: 339-345Crossref PubMed Scopus (1118) Google Scholar, 24Xiong J.-P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S.L. Arnout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1410) Google Scholar). Integrins are known to bind to proteins via other peptide motifs (22Ruoslahti E. Annu. Rev. Cell Biol. 1996; 12: 697-715Crossref Scopus (2588) Google Scholar), usually resulting in enhancement of the outcome of occupation of the RGD site. In particular, αvβ5 binds to a basic domain on the HIV Tat protein (25Vogel B.E. Lee S.J. Hildebrand A. Craig W. Pierschbecher M.D. Wong-Staal F. Ruoslahti E. J. Cell Biol. 1993; 121: 461-468Crossref PubMed Scopus (232) Google Scholar), although no functional consequence for this interaction has been established. However, the greater affinity of αvβ5 for the Tat basic domain compared with an equivalent Vn domain (25Vogel B.E. Lee S.J. Hildebrand A. Craig W. Pierschbecher M.D. Wong-Staal F. Ruoslahti E. J. Cell Biol. 1993; 121: 461-468Crossref PubMed Scopus (232) Google Scholar) suggests that ligands other than Vn could interact with the αvβ5 basic domain binding site and could have distinct signaling functions. This report demonstrates that the αvβ5 integrin interacts with sCD23 using a site distinct from the RGD binding pocket and that recognition of a basic motif on CD23 that is distinct from its interaction sites for IgE, CD21, or itself, enhances survival of human pre-B cells. Materials—Anti-CD47 (BRIC 126, IgG2b), anti-αvβ3 (LM609, IgG1), anti-αvβ5 (P1F6, IgG1; 15F11, IgG2a), biotinyl-murine IgG1, rabbit polyclonal anti-peptide antibodies specific for integrin αv and β5 subunits and purified αvβ5 protein were obtained from Chemicon, UK. Anti-αv/CD51 (AMF7, IgG1) was obtained from Beckman Coulter, High Wycombe, UK; BU38 anti-CD23 was purchased from AMS Biotechnology, Abingdon, UK. Fluorescein isothiocyanate (FITC)-anti-mouse IgG1 and FITC- and phycoerythrin (PE)-labeled murine IgG1 proteins were supplied by DAKO Ltd., Denmark. Radiochemicals and materials for enhanced chemiluminescence (ECL) were obtained from Amersham International plc, Amersham, England, and fine chemicals, including streptavidin-Quantum Red, Fn, HRP-coupled protein A, and octyl-β-d-glucopyranoside (OGP), were supplied by Sigma. Vn was obtained from Invitrogen. Recombinant 25-kDa sCD23, encompassing residues Met151-Ser231 with an N-terminal His6 tag, was expressed in Escherichia coli and affinity-purified by nickel chelate chromatography, or was purchased from R&D Systems. The derCD23 protein was a generous gift from Dr. J. McDonnell, University of Oxford, UK, a CD23-GST fusion protein preparation comprising residues Asp48-Gly248 of CD23 (referred to as sCD2348-248) was obtained from Bio-supplies Ltd., Bradford, UK. C1 sensor chips were obtained from BIAcore SpA, Uppsala, Sweden, and siRNA constructs were purchased from Qiagen. Cell Culture, siRNA Procedures, and Flow Cytometry—The SMS-SB cell line was derived from a female patient presenting with acute lymphoblastic leukemia (26Smith R.G. Cancer. 1984; 54: 471-476Crossref PubMed Scopus (13) Google Scholar); Nalm-6 and Blin-1 cell lines were from laboratory stocks. Cell lines were maintained in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mm fresh glutamine, and penicillin and streptomycin, at 37 °C in a 5% CO2 in air in a humidified atmosphere. Human telomerized fibroblasts were cultured as described previously (27Scott L.A. Vass J.K. Parkinson E.K. Gillespie D.A. Winnie J.N. Ozanne B.W. Mol. Cell. Biol. 2004; 24: 1540-1559Crossref PubMed Scopus (32) Google Scholar). Cytokines, obtained from R&D Laboratories, were used at 5-10 ng/ml, but had no effect over a wide dose-response range. SMS-SB cells were also propagated in protein-free hybridoma medium-II (PFHM, Invitrogen), at 2.5-5 × 105 cells/ml ("normal cell density"). In stimulation experiments, SMS-SB cells were cultured at 2500 cells/100-μl culture (low cell density (LCD)) a seeding density at which the cells are prone to apoptosis (10White L.J. Ozanne B.W. Graber P. Aubry J.-P. Bonnefoy J.-Y. Cushley W. Blood. 1997; 90: 234-243Crossref PubMed Google Scholar). NALM-6 and Blin-1 cells were washed extensively in PFHM prior to culture at 2500 cells/100-μl culture. Cultures were propagated in the presence or absence of cytokines, mAbs or peptides, at 37 °C for 72 h followed by addition of 0.3 μCi/well tritiated thymidine ([3H]TdR) for 18 h prior to harvest; incorporation was determined by liquid scintillation spectrometry. Integrin αv knockdown was achieved using siRNA targeting the sequence, 5′-AGCAACTTTATTATAGATTTA-3′ (Hs_ITGAV_1_HP siRNA), whereas the control non-silencing siRNA (cat. no. 1022076) targeted the sequence, 5′-AATTCTCCGAACGTGTCACGT-3′.89 μl of 2 μm siRNA was diluted in 2.9 ml of Dulbecco's modified Eagle's medium (serum-free) then 44.5 μl of HiPerFect (Qiagen) was added prior to 10-min incubation at room temperature. This solution was added to a 10-cm plate that had been seeded 2 h previously with 6 × 105 cells in 12 ml of Dulbecco's modified Eagle's medium containing 20% (v/v) fetal calf serum. The medium was replaced 48 h later, and further control and silencing siRNA molecules were added as above. Cells were harvested after a further 24 h and analyzed for binding of CD23, peptides, and anti-αv integrin mAbs. For flow cytometry, 5 × 105 cells were stained with either FITC-conjugated or unlabeled primary mAb for 30-60 min; unlabeled primary antibody was visualized using a secondary FITC-conjugated anti-mouse IgG or, in the case of biotinylated anti-αvβ5, using streptavidin-Quantum Red. Cells were analyzed on a FACScan flow cytometer, using CellQuest software. Affinity Isolation and Analysis of Cellular Proteins—108 SMS-SB cells were harvested, washed twice in ice-cold phosphate-buffered saline, suspended in 1.5 ml of ice-cold OGP extraction buffer (1% (w/v) OGP in 50 mm HEPES/KOH, pH 7.4, 5 mm CaCl2, 140 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 1 mm aprotinin, 1 mm leupeptin), and lysed with 40 strokes of a chilled glass homogenizer. The homogenate was centrifuged at 1,000 × g for 10 min at 4 °C, and the resulting supernatant further was centrifuged at 35,000 × g for 45 min at 4 °C. Cellular extracts were added to bovine serum albumin-Affi-Gel pre-equilibrated with OGP extraction buffer and incubated at 4 °C for 6 h. The matrix was pelleted, and unbound proteins were recovered, added to pre-equilibrated sCD23-Affi-Gel, and incubated at 4 °C overnight. The sCD23-Affi-Gel was pelleted, and the unbound fraction was retained. Both matrices were exhaustively washed, and specifically bound material eluted by boiling in sample buffer and subjected to SDS-PAGE under reducing conditions on a 10% (w/v) acrylamide gel. Eluates were transferred to nitrocellulose membranes and probed with anti-VnR-component antibodies, followed by an HRP-labeled secondary antibody and ECL. For far-Western analysis, purified integrin proteins were subjected to SDS-PAGE under non-reducing conditions and transferred to nitrocellulose membranes. The blots were probed with biotinylated CD23-derived peptides, in the presence or absence of competing peptides, and binding was visualized using HRP-conjugated streptavidin and ECL. Areas of the peaks visualized by ECL/autoradiography were determined using ImageJ software. Lanes containing β5 bands were scanned, and identical peak widths were selected for calculation of areas under the selected β5 peaks. Peptide Biochemistry and Surface Plasmon Resonance Studies—A library of 83 overlapping nonapeptides encompassing residues 151-321 of the 25-kDa sCD23 sequence was synthesized by Mimotopes Inc. (Chester, UK); biotinylated and non-biotinylated peptides and peptides with specific substitutions were synthesized by the same firm. For screening experiments, each peptide was synthesized as tridecapeptide comprising a unique CD23-derived nonapeptide sequence, plus a common N-terminal tetrapeptide extension (SGSG) to which a biotin moiety was attached (i.e. biotin-SGSG-X9 where X9 is the specific CD23-derived nonapeptide sequence). The C-terminal residue was amidated. Each unique nonapeptide sequence had a two-residue C-terminal offset relative to its immediate neighbor. The specific nonapeptide sequences were #9, KWINFQRKC; #10, INFQRKCYY; #11, FQRKCYYFG; #12, RKCYYFGKG; #9AA, KWINFQAAC; and #12AA, AACYYFGKG; the LP long peptide sequence are KWINFQRKCYYFGKG. Biotinylated peptides were captured on 96-well streptavidin-coated enzyme-linked immunosorbent assay trays and blocked with 1% (w/v) casein. Purified αvβ5 integrin (0.2 μg) was added to each well, and binding was quantitated by use of P1F6 mAb and HRP-anti-mouse IgG followed by tetramethylbenzidine as substrate. Binding of biotinylated peptides to cells was visualized with fluorochrome-conjugated streptavidin and flow cytometry; mean fluorescence intensity data were derived using the CellQuest program. In cell stimulation experiments, peptides were used in the 10 nm to 6 μm concentration range, and appropriate solvent vehicle controls (e.g. acetonitrile and Me2SO) were always performed. Biotinylated and non-biotinylated peptides provoked identical responses in functional assays. Interactions between CD23-derived peptides and αvβ5 integrin were assessed at 25 °C using a BIA-core 2000 instrument (Biacore AB). The αvβ5 integrin was immobilized at ∼4000 response units on a C1 chip, and an underivatized reference cell was employed as a control surface. CD23-derived peptides in HBS-EP buffer (0.01 m HEPES-KOH, pH 7.4, 0.15 m NaCl, 3 mm EDTA, 0.005% (v/v) Surfactant P20) were injected over the test and control chip surfaces at a flow rate of 40 μl·min-1 with a 3-min association phase followed by a 6-min dissociation phase. In all experiments, peptides bound exclusively to the test cell, there was no nonspecific binding to the control reference cell, and regeneration of a stable baseline (using 0.2 m glycine-HCl, pH 2.5, where necessary) was readily achieved. Data were standardized according to standard double referencing data subtraction methods (28Myszka D.G. J. Mol. Recognit. 1999; 12: 279-284Crossref PubMed Scopus (656) Google Scholar), using control and blank injections, prior to kinetic analysis using BIAevaluation 3.0.2 software. The αvβ5 Integrin Is a CD23-binding Protein—Recombinant sCD23 rescues cultures of an acute lymphoblastic leukemia-derived human pre-B cell-like cell line, SMS-SB (26Smith R.G. Cancer. 1984; 54: 471-476Crossref PubMed Scopus (13) Google Scholar), from low cell density-induced apoptosis in a dose-dependent manner (10White L.J. Ozanne B.W. Graber P. Aubry J.-P. Bonnefoy J.-Y. Cushley W. Blood. 1997; 90: 234-243Crossref PubMed Google Scholar) (Fig. 1A, panel i); sCD23 is the only cytokine tested that rescues SMS-SB cells from apoptosis (Fig. 1A, panel ii). SMS-SB cells do not express CD23 itself or any of its known receptors (10White L.J. Ozanne B.W. Graber P. Aubry J.-P. Bonnefoy J.-Y. Cushley W. Blood. 1997; 90: 234-243Crossref PubMed Google Scholar) (Fig. 1C, and data not shown), and this suggested that the interaction of sCD23 with acute lymphoblastic leukemia-derived pre-B cells was mediated via a hitherto undefined receptor. CD23-Affi-Gel affinity chromatography of lysates of [35S]methionine-labeled SMS-SB cells enriched two proteins of ∼120 and 80 kDa, molecular masses that are consistent with those of mature αv and β5 integrin proteins, respectively (data not shown). The interpretation that these two species were indeed αv and β5 was confirmed by performing CD23-Affi-Gel affinity chromatography on lysates of unlabeled cell extracts followed by Western blotting. Both αv and β5 proteins were detected in the eluate from the CD23 matrix but not in the eluate from the bovine serum albumin column (Fig. 1B). The polyclonal anti-αv antiserum used in this experiment (Fig. 1B, panel i) binds to a C-terminal epitope on αv that is located on the 25-kDa fragment generated during biosynthetic maturation of αv (29Suzuki S. Argraves W.S. Pytela R. Arai H. Krusius T. Pierschbacher M.D. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8614-8618Crossref PubMed Scopus (146) Google Scholar). No β3 protein was detected in the eluate from the CD23-Affi-Gel column or in whole cell lysates (data not shown). SMS-SB cells stain with both the P1F6 mAb that recognizes the assembled αvβ5 heterodimer (30Wayner E.A. Orlando R.A. Cheresh D.A. J. Cell Biol. 1991; 113: 919-929Crossref PubMed Scopus (298) Google Scholar), and anti-CD47 VnR-associated protein mAbs, but do not stain with the αvβ3-specific LM609 mAb (30Wayner E.A. Orlando R.A. Cheresh D.A. J. Cell Biol. 1991; 113: 919-929Crossref PubMed Scopus (298) Google Scholar) (Fig. 1C). Reverser transcription-PCR analysis of SMS-SB RNA yielded amplicons of the expected sizes for CD47, CD51/αv, β1 and β5, but no correctly sized PCR products were detected for β3, β6, or β8 coding sequences (data not shown). The demonstration that SMS-SB cells express αvβ5 and that it interacts with CD23 suggests that this integrin is linked to pre-B cell growth and survival. To test this proposal, SMS-SB cells were incubated with αvβ5-specific monoclonal antibodies (mAb) or with Vn and Fn, the normal ligands for αvβ5. Incubation with the αv-specific mAb AMF7 stimulated proliferation of SMS-SB cells in response to increasing concentrations of mAb, whereas an isotype-matched control mAb was without effect (Fig. 2A). Importantly, a second pre-B cell line, NALM-6, also showed enhanced proliferation when stimulated by the AMF7 mAb (Fig. 2B). The 15F11 mAb that recognizes fully assembled αvβ5 heterodimers but does not impede αvβ5 binding to Vn (31Stuiver I. Smith J.W. Hybridoma. 1995; 14: 545-550Crossref PubMed Scopus (4) Google Scholar), stimulated SMS-SB proliferation (Fig. 2C). However, the P1F6 mAb, which recognizes a distinct epitope on assembled αvβ5 heterodimers and does inhibit αvβ5 binding to Vn (30Wayner E.A. Orlando R.A. Cheresh D.A. J. Cell Biol. 1991; 113: 919-929Crossref PubMed Scopus (298) Google Scholar), failed to stimulate proliferation (Fig. 2C). These data are reminiscent of those of Hermann and colleagues (13Hermann 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) who demonstrated that an anti-αvβ3 reagent directed to the RGD binding site could not impede CD23-driven production of cytokine release by monocytic cells. Moreover, finding that neither Vn nor Fn stimulated proliferation of SMS-SB cells (Fig. 2D) supports the contention that the binding sites for RGD-containing ligands and sCD23 are distinct. These data show that αvβ5 regulates a pro-survival response in pre-B cell lines and that this response seems to be both ligand-selective and potentially independent of the integrin binding site for RGD. The lack of an RGD sequence in sCD23 suggests that the motif on CD23 recognized by the integrin is not an RGD-related sequence (see Fig. 3B; there is an inverse RGD sequence of DGR but this is, of course, stereochemically different to RGD). One precedent for such recognition is the non-RGD-dependent interaction of αvβ5 with a basic motif of the HIV Tat protein (25Vogel B.E. Lee S.J. Hildebrand A. Craig W. Pierschbecher M.D. Wong-Staal F. Ruoslahti E. J. Cell Biol. 1993; 121: 461-468Crossref PubMed Scopus (232) Google Scholar).FIGURE 3Definition of CD23 sequences bound by purifiedαvβ5 integrin in vitro. A, illustrates the sequence of 25-kDa sCD23, from Met151 to Ser321, used as the parent sequence for construction of the library of 83 synthetic biotinylated tridecapeptides, of the form biotinyl-SGSG-X9, where X9 is the unique nonapeptide sequence based on the CD23 sequence that was probed with purified αvβ5 integrin. The black lines denote the RKC-containing peptides #9-#12 that bind the αvβ5 integrin. A schematic diagram of the CD23 protein is illustrated in B showing the lectin head and stalk domains; the bolded loop represents the region of the protein where the RKC motif recognized by the αvβ5 integrin resides. Purified αvβ5 integrin was immobilized on a CM1 sensor chip, and different concentrations of the indicated peptides were injected at room temperature. C, binding sensorgrams for peptides #9-#12 and, as a control, peptide #58; D, illustrates the binding of the pentadecapeptide LP in comparison with peptides #11 and #58; the traces shown are for injections of 20 μm peptide. E, sensorgram for binding of derCD23, at the indicated concentrations; F, measured affinities (±S.D.), assuming a simple linear 1:1 association model, for derCD23 and each of peptides #9-#12 and LP for immobilized αvβ5 integrin. G, illustrates binding of CD23 to SMS-SB cells in the absence (gray-filled area) or presence of an excess of peptide #11 (solid line) or peptide #11AA (dotted line). H, binding of biotinylated peptide #11 in the absence (gray-filled area) or presence (solid line) of excess sCD23; binding of streptavidin-PE alone is shown in the dotted line.View Large Image Figure ViewerDownload Hi-res image Download (PPT) αvβ5 Binds a Basic Motif in CD23—The sequence of 25-kDa human CD23 (Met151 to Ser321) is shown in Fig. 3A together with a schematic representation of CD23 showing the major functional regions of the protein (Fig. 3B). A library of 83 biotinylated overlapping nonapeptides was constructed based on the 25-kDa sCD23 sequence (Fig. 3A) and probed with purified αvβ5 to identify binding sites for the integrin. Purified αvβ5 binds specifically and consistently to a series of four adjacent peptides (peptides #9 -#12) encompassing residues Lys166-Gly180 (data not shown), and the sole feature common to these four peptides is a tri-peptide of sequence Arg-Lys-Cys (RKC) beginning at Arg172 in the CD23 sequence. The structural data for sCD23 (14Hibbert R.G. Teriete P. Grundy G.J. Beavil R.L. Reljic R. Holers V.M. Hannan J.P. Sutton B.J. Gould H.J. McDonnell J.M. J. Exp. Med. 2005; 202: 751-760Crossref PubMed Scopus (118) Google Scholar, 15Wurzburg B.A. Tarchevskaya S.S. Jardetzky T.S. Structure. 2006; 14: 1049-1058Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) demonstrate that the RKC motif resides in a small disulfide-bonded loop at the N-terminal end of the C-type lectin domain. The interaction between the four CD23-derived RKC-containing peptides and αvβ5 was assessed by surface plasmon resonance (SPR) analysis. This analysis demonstrates that the four CD23-derived, RKC-containing peptides bind to immobilized αvβ5 integrin in a rank order of #11 > #12 > #9 > #10, where
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