Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M609335200 on May 31, 2007

J. Biol. Chem., Vol. 282, Issue 37, 27315-27326, September 14, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/37/27315    most recent
M609335200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Borland, G.
Right arrow Articles by Cushley, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Borland, G.
Right arrow Articles by Cushley, W.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

{alpha}vbeta5 Integrin Sustains Growth of Human Pre-B Cells through an RGD-independent Interaction with a Basic Domain of the CD23 Protein*

Gillian Borland{ddagger}1, Adrienne L. Edkins{ddagger}2, Mridu Acharya{ddagger}2, Johanne Matheson{ddagger}, Lindsey J. White{ddagger}3, Janet M. Allen§, Jean-Yves Bonnefoy, Bradford W. Ozanne||4, and William Cushley{ddagger}5

From the {ddagger}Division of Biochemistry & Molecular Biology, Institute of Biomedical & Life Sciences, University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, United Kingdom, the ||Cancer Research UK Beatson Laboratories, Switchback Road, Glasgow G61 1BD, Scotland, United Kingdom, §Conway Institute, University College Dublin, Dublin 4, Ireland, and Transgene, 67028 Strasbourg, France

Received for publication, October 2, 2006 , and in revised form, May 30, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha}vbeta5 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 {alpha}vbeta5 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 beta subunit of the {alpha}vbeta5 integrin. The interaction between {alpha}vbeta5 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 {alpha}vbeta5.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (1-5). CD23 is cleaved by membrane-associated metalloproteases (6, 7) to yield soluble CD23 (sCD23)6 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 (1-3). In the B cell compartment, sCD23 inhibits apoptosis of germinal center centrocytes and promotes their differentiation into plasmablasts (8), at least in part by binding to CD21 (9), and sCD23 also inhibits apoptosis in pre-B cell lines (10) through, as we report here, an interaction with the {alpha}vbeta5 integrin. In association with interleukin-1{alpha}, sCD23 promotes differentiation of monocytes and early thymocyte precursors (11) and, via binding to the {alpha}Mbeta2 (CD11b-CD18), {alpha}Xbeta2 (CD11c-CD18) (12), and {alpha}vbeta3 integrins (13), stimulates tumor necrosis factor-{alpha} and interleukin-1{alpha} production by monocytes.

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 (14) and x-ray crystallography (15), 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 beta sheets and two {alpha} helices (14, 15). 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 (14); 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 (16). 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 (15). 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 {alpha}vbeta5 integrin is a member of the vitronectin receptor (VnR) family. VnRs are heterodimers of the {alpha}v integrin chain in association with any one of five beta subunits (17) and have well documented roles in cell attachment, spreading, and migration, rescue from apoptosis, and angiogenesis (18-21). VnRs bind to the extracellular matrix proteins vitronectin (Vn) and fibronectin (Fn) by recognition of an Arg-Lys-Asp (RGD) motif in target proteins (22); both the {alpha} and beta integrin subunits contribute to ligand binding (23, 24). Integrins are known to bind to proteins via other peptide motifs (22), usually resulting in enhancement of the outcome of occupation of the RGD site. In particular, {alpha}vbeta5 binds to a basic domain on the HIV Tat protein (25), although no functional consequence for this interaction has been established. However, the greater affinity of {alpha}vbeta5 for the Tat basic domain compared with an equivalent Vn domain (25) suggests that ligands other than Vn could interact with the {alpha}vbeta5 basic domain binding site and could have distinct signaling functions.

This report demonstrates that the {alpha}vbeta5 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Anti-CD47 (BRIC 126, IgG2b), anti-{alpha}vbeta3 (LM609, IgG1), anti-{alpha}vbeta5 (P1F6, IgG1; 15F11, IgG2a), biotinyl-murine IgG1, rabbit polyclonal anti-peptide antibodies specific for integrin {alpha}v and beta5 subunits and purified {alpha}vbeta5 protein were obtained from Chemicon, UK. Anti-{alpha}v/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-beta-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 (26); 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 (27). 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 x 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 (10). 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 {alpha}v 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 x 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-{alpha}v integrin mAbs.

For flow cytometry, 5 x 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-{alpha}vbeta5, 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 x g for 10 min at 4 °C, and the resulting supernatant further was centrifuged at 35,000 x 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 beta5 bands were scanned, and identical peak widths were selected for calculation of areas under the selected beta5 peaks.


Figure 1
View larger version (38K):
[in this window]
[in a new window]

  		FIGURE 1.
The {alpha}vbeta5 Integrin is a CD23 receptor in SMS-SB cells. A, low cell density (LCD, 2,500 cells/100 µl or 25,000 cells/ml) cultures of SMS-SB in PFHM were established with the indicated concentrations of recombinant 25-kDa sCD23 (panel i) or other cytokines (panel ii) and cellular proliferation (panel i) or ratio of viable to apoptotic cells (panel ii) determined. Apoptosis was determined by staining harvested cells with propidium iodide and Hoechst 33342 for 1 min prior to analysis on a Coulter Elite flow cytometer as previously described (10). Normal cell density cultures (2.5-5 x 105 cells/ml) served as a control. B, extracts of SMS-SB cells in OGP buffer were passed over a bovine serum albumin-Affi-Gel column, and the flow-through was collected and subsequently incubated on CD23-Affi-Gel; aliquots of flow-through and eluted fractions from both columns were electrophoresed under reducing conditions. Proteins were transferred to nitrocellulose and probed with anti-{alpha}v (panel i) or anti-beta5 (panel ii) anti-peptide antibodies, and binding was visualized with protein A-HRP and ECL. C, SMS-SB cells were stained (solid lines) with mAbs specific for {alpha}vbeta5, CD47, or {alpha}vbeta3 and visualized with the appropriate secondary reagent; isotype control staining is shown in the shaded area.

 
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 {alpha}vbeta5 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 {alpha}vbeta5 integrin were assessed at 25 °C using a BIA-core 2000 instrument (Biacore AB). The {alpha}vbeta5 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 (28), using control and blank injections, prior to kinetic analysis using BIAevaluation 3.0.2 software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The {alpha}vbeta5 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 (26), from low cell density-induced apoptosis in a dose-dependent manner (10) (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 (10) (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 {alpha}v and beta5 integrin proteins, respectively (data not shown). The interpretation that these two species were indeed {alpha}v and beta5 was confirmed by performing CD23-Affi-Gel affinity chromatography on lysates of unlabeled cell extracts followed by Western blotting. Both {alpha}v and beta5 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-{alpha}v antiserum used in this experiment (Fig. 1B, panel i) binds to a C-terminal epitope on {alpha}v that is located on the 25-kDa fragment generated during biosynthetic maturation of {alpha}v (29). No beta3 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 {alpha}vbeta5 heterodimer (30), and anti-CD47 VnR-associated protein mAbs, but do not stain with the {alpha}vbeta3-specific LM609 mAb (30) (Fig. 1C). Reverser transcription-PCR analysis of SMS-SB RNA yielded amplicons of the expected sizes for CD47, CD51/{alpha}v, beta1 and beta5, but no correctly sized PCR products were detected for beta3, beta6, or beta8 coding sequences (data not shown).


Figure 2
View larger version (21K):
[in this window]
[in a new window]

  		FIGURE 2.
Ligand-selective responses regulated by{alpha}vbeta5. LCD cultures (2500 cells/well) of SMS-SB (A) or Nalm-6 cells (B) were propagated with the indicated concentrations of AMF7 anti-{alpha}v mAb (black bar) or IgG1 isotype control mAb (gray bar); proliferation was measured by [3H]thymidine incorporation. In B, AMF7 and IgG1 were used at 5 µg/ml. C, effect of assembled heterodimer-specific anti-{alpha}vbeta5 mAbs P1F6 (dark gray bar) and 15F11 (light gray bar) anti-{alpha}vbeta5 mAbs with corresponding IgG1 and IgG2a isotype controls on SMS-SB cellular proliferation. D illustrates the effect of culture with bovine serum albumin (white bar) Vn (dark gray bar) or Fn (light gray bar). All experiments were performed a minimum of three times.

 
The demonstration that SMS-SB cells express {alpha}vbeta5 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 {alpha}vbeta5-specific monoclonal antibodies (mAb) or with Vn and Fn, the normal ligands for {alpha}vbeta5. Incubation with the {alpha}v-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 {alpha}vbeta5 heterodimers but does not impede {alpha}vbeta5 binding to Vn (31), stimulated SMS-SB proliferation (Fig. 2C). However, the P1F6 mAb, which recognizes a distinct epitope on assembled {alpha}vbeta5 heterodimers and does inhibit {alpha}vbeta5 binding to Vn (30), failed to stimulate proliferation (Fig. 2C). These data are reminiscent of those of Hermann and colleagues (13) who demonstrated that an anti-{alpha}vbeta3 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 {alpha}vbeta5 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 {alpha}vbeta5 with a basic motif of the HIV Tat protein (25).


Figure 3
View larger version (35K):
[in this window]
[in a new window]

  		FIGURE 3.
Definition of CD23 sequences bound by purified{alpha}vbeta5 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 {alpha}vbeta5 integrin. The black lines denote the RKC-containing peptides #9-#12 that bind the {alpha}vbeta5 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 {alpha}vbeta5 integrin resides. Purified {alpha}vbeta5 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 {alpha}vbeta5 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.

 
{alpha}vbeta5 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 {alpha}vbeta5 to identify binding sites for the integrin. Purified {alpha}vbeta5 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 (14, 15) 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 {alpha}vbeta5 was assessed by surface plasmon resonance (SPR) analysis. This analysis demonstrates that the four CD23-derived, RKC-containing peptides bind to immobilized {alpha}vbeta5 integrin in a rank order of #11 > #12 > #9 > #10, whereas peptide #58, which does not have an RKC motif, failed to bind to the integrin (Fig. 3C). A longer RKC-containing, CD23-derived pentadecapeptide (the "long peptide" or LP), spanning all 15 amino acids encompassed by peptides #9 to #12, displayed greater binding to {alpha}vbeta5 than the RKC nonapeptides (Fig. 3D). The slow dissociation of the LP suggests that, although the RKC motif may be essential for the CD23-{alpha}v integrin interaction, the complete binding site may extend well beyond the small common RKC tripeptide motif. Fig. 3E illustrates a representative sensorgram for the binding of derCD23 to immobilized {alpha}vbeta5 integrin; the affinity of this interaction (6.04 x 10-6 M) is of the same order of magnitude as those for binding of derCD23 to CD21 (8.7 x 10-7 M) and to the Fc region of human IgE (1.3 x 10-6 M), respectively (14). Fig. 3F states the affinities of each of peptides #9 to #12 and long peptide for immobilized {alpha}vbeta5 integrin. LP had the highest affinity, consistent with the sensorgrams shown in Fig. 3 (C and D), whereas the remaining peptides had affinities for the integrin similar to that of derCD23. There was no consistent correlation between affinity of a peptide for {alpha}vbeta5 and its biological activity. A similar trend of data was observed when purified {alpha}vbeta3 integrin was immobilized on the sensor surface (data not shown). The SPR data demonstrate that CD23-derived, RKC-containing peptides bind to {alpha}vbeta5 integrin. Fig. 3G demonstrates that the binding of CD23 to SMS-SB cells was greatly attenuated by inclusion of excess peptide #11 but not by a variant of peptide #11 where the arginine and lysine have been replaced by alanines (peptide #11AA). Excess LP also inhibited binding of CD23 to the cells (data not shown). Fig. 3H shows the reciprocal experiment, where excess CD23 reduced the binding of peptide #11 to background levels. These data indicate that CD23 and peptides derived from it compete for binding to the same sites on SMS-SB cells.


Figure 4
View larger version (31K):
[in this window]
[in a new window]

  		FIGURE 4.
RKC-containing, CD23-derived peptides bind to pre-B cells. Binding of biotinylated peptides to pre-B cell lines was detected by addition of PE-streptavidin and flow cytometry; mean fluorescence index data were derived using CellQuest software. The flow cytometric plots for binding of peptides #9 (black line), #11 (gray line), and #57 (shaded area) are illustrated (A). Mean fluorescence index data for binding of the indicated peptides to SMS-SB cells are shown together with the unique nine amino acid sequence of each biotinylated tridecapeptide; the RKC motif shared by {alpha}vbeta5 binding peptides is displayed in bold (B). C, panel i illustrates competition of binding of biotinylated peptide #9 to SMS-SB cells; negative control (biotinyl-peptide #76 in this case) is shown in the filled area, and binding of biotinylated peptide #9 in the absence (black line) or presence of a 10-fold excess of named competitor peptide (gray line) shown for peptides #8, #10, and #12. Panels ii and iii show percentage inhibition of binding of biotinylated peptide #9 to SMS-SB and NALM-6 cells, respectively. D, binding of the P1F6 mAb (1 µg/ml) to Blin-1 cells in the presence (shaded area) or absence (thick black line) of 1 µg/ml LP. Isotype-control staining is shown by the dotted line. E, illustrates the binding of the indicated mAbs (panels i-iii) and biotinylated CD23 (panel iv) or peptides #9 or #41 (panels v and vi) derived from CD23 to telomerized human fibroblasts cultured for 96 h with either control siRNA (gray-filled area) or siRNA {alpha}v (solid line) prior to staining; note that the dotted line in panel iv represents staining with streptavidin-PE alone.

 
CD23-derived, RKC-containing Peptides Bind to Cells and Are Biologically Active—The data of Fig. 3 indicate that CD23-derived RKC-containing peptides bind to {alpha}vbeta5 in vitro. The binding of biotinylated CD23-derived peptides to SMS-SB cells was detected by flow cytometry, and all four RKC-containing peptides bound to SMS-SB cells (Fig. 4, A and B), and to other {alpha}vbeta5+ pre-B cell lines such as NALM-6 (data not shown). CD23-derived peptides immediately adjacent to peptides #9 and #12, namely #8 and #13, or peptide #15, that had similar net charge to the peptides that bound to {alpha}vbeta5, or a randomly chosen peptide, #57, all failed to bind to {alpha}vbeta5 or to cells (Fig. 4B). Peptides #61-63, which have an RKL tripeptide sequence, or peptides #78-80 that have the inverse RGD (DGR) sequence, also did not bind to {alpha}vbeta5 or to cells (data not shown). Binding of biotinylated peptide #9 to SMS-SB cells was significantly inhibited by the presence of excess non-biotinylated peptides #10 and #12, but not peptide #8 (Fig. 4C, panels i and ii): a similar result was obtained for NALM-6 cells (data not shown). The LP inhibited binding of the {alpha}vbeta5 heterodimer-specific mAb P1F6 to Blin1 pre-B cells (Fig. 4D), indicating that binding of this peptide to the integrin on the cell surface perturbs the conformation of the integrin such that the integrity of the epitope recognized by the antibody is compromised. Finally, the contribution of {alpha}vbeta5 to binding of peptides and CD23 was investigated by using an RNA interference-based approach. It did not prove possible to deliver siRNA constructs to SMS-SB or other pre-B cell lines to achieve a reduction in integrin RNA or protein expression in these cells (data not shown). As an alternative, siRNA specific for the {alpha}v subunit (siRNA {alpha}v) was introduced into telomerized human fibroblasts, and the effects on expression of {alpha}v integrins and binding of CD23 and CD23-derived peptides to the cells was determined. The {alpha}v subunit was targeted because successful attenuation of {alpha}v expression will reduce expression of all {alpha}v-containing integrins due to the requirement for {alpha} and beta chains to assemble in the endoplasmic reticulum prior to transport to the cell surface. Moreover, because the beta5 subunit pairs only with {alpha}v (17), a successful knockdown of {alpha}v expression will also diminish {alpha}vbeta5 cell surface levels. Fig. 4E shows that exposure of telomerized fibroblasts to siRNA {alpha}v, but not the control siRNA, significantly reduced staining by the AMF7 and P1F6 reagents (Fig. 4E, panels i and ii, respectively), showing both total {alpha}v and {alpha}vbeta5 expression at the cell surface were reduced; the background level of staining with the isotype control IgG1 was unaffected by either siRNA (Fig. 4E, panel iii). Similarly, binding of biotinylated CD23 (Fig. 4E, panel iv) or peptide #9 (Fig. 4E, panel v) to siRNA {alpha}v-treated cells was reduced by a substantial amount, whereas the binding of a negative control peptide (#41, Fig. 4E, panel vi) was unaffected. These data indicate that loss of {alpha}v integrin expression in general, and {alpha}vbeta5in particular, correlates with a dramatic reduction of binding of CD23 and RKC-containing CD23-derived peptides to the cells, confirming that {alpha}v integrins are the target for CD23 binding. Because the fibroblasts also express beta1 and beta2 integrin subunits, the expression of which will be unaffected by siRNA {alpha}v, it is clear that none of these integrins, and in particular the beta1 integrin subunit, can substitute for {alpha}v in binding CD23. These data are entirely consistent with the SPR data of Fig. 3, which show that CD23 binds the {alpha}vbeta5 integrin.

The biological activities of CD23-derived peptides were assessed in standard low cell density proliferation assays. Peptides #9 and #12 stimulated thymidine incorporation by SMS-SB cells, demonstrating that CD23-derived peptides that bind {alpha}vbeta5 via their RKC motif can mimic the effect of CD23 (Fig. 5A), but peptides #10 and #11, which bound to SMS-SB cells, did not stimulate proliferation, raising the possibility that these peptides might act as inhibitors of the stimulatory activity of peptides #9 and #12 (Fig. 5A). Accordingly, SMS-SB cells were incubated with an excess of peptides #10 or #11 and then challenged with either peptide #9 or #12. For both peptide #9 and #12, peptides #10 and #11 suppressed proliferation, whereas the irrelevant peptide #57 did not (Fig. 5B, panels i and ii). These data indicate that peptides #10 and #11 can indeed inhibit the action of stimulatory peptides #9 and #12. However, in striking contrast, a tetrapeptide of sequence RGDS failed to inhibit either binding of CD23-derived RKC-containing peptides to SMS-SB cells or stimulation of proliferation of the cells by peptides #9 and #12 (Fig. 5C, panels i and ii). These data demonstrate that the integrin does not use the RGD binding site to capture sCD23.

sCD23 Cytokine Activity Is Independent of the IgE and CD21 Binding Sites—The hypothesis that {alpha}vbeta5 integrin recognizes the dibasic peptide region in CD23 was tested by substituting Arg172 and Lys173 in peptides #9 and #12 with alanine (peptides #9AA and #12AA, respectively). Peptide #9AA shows a severely impaired ability to sustain growth of SMS-SB cells compared with wild-type peptide #9, being comparable to that achieved with peptide #58 (Fig. 6A, panel i). Biotinylated peptide #9AA displayed greatly reduced binding to SMS-SB cells (Fig. 6A, panel ii), and SPR analysis demonstrated clearly that essentially all peptide #9AA binding to immobilized {alpha}vbeta5 integrin was lost when the two basic residues of peptide #9 were substituted with alanine (Fig. 6A, panel iii). Similar data were obtained for peptide #12AA, which induced SMS-SB proliferation at levels identical to those seen with control peptide #58 (Fig. 6B), and which also shows reduced binding to SMS-SB cells (data not shown). Peptide #11 showed the highest level of binding to immobilized {alpha}vbeta5 (see Fig. 3D) and to cells (see Fig. 4B), and the peptide #11AA double-alanine mutant failed to completely bind to immobilized {alpha}vbeta5 as determined by SPR analysis (Fig. 6C). Finally, a CD23-GST fusion protein comprising residues 48-248 of CD23 (sCD2348-248), which contains the RKC motif but lacks the inverse RGD motif, the CD21 and IgE interaction surfaces, and all four protein residues that participate in coordination of calcium binding in the lectin head (15), sustained growth of SMS-SB cells in a dose-dependent manner (Fig. 6D). The approximate EC50 for the sCD2348-248 construct, in the 5-10 nM range, is broadly similar to that observed for 25-kDa sCD23 (10) (Fig. 1A, panel i). The LP, comprising the RKC motif flanked by six residues on either side, also promoted growth of SMS-SB cells (Fig. 6E) supporting the conclusion that the RKC motif is necessary and sufficient for expression of sCD23 cytokine activity in SMS-SB cells. The data are consistent with the interpretation that the cytokine activity of CD23 mediated via the {alpha}vbeta5 integrin in pre-B cells does not require any contribution from the inverse-RGD motif or from the IgE and CD21 binding sites.


Figure 5
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 5.
CD23-derived peptides containing the RKC motif are biologically active. A, LCD cultures of SMS-SB cells (2500 cells/well) were established in the presence of the indicated concentrations of peptides #9 -#12 (black bars), and pulsed for 16 h with [3H]TdR after 72 of culture. (*, note that the "vehicle" control (Veh) is shown only for peptide #9; all other vehicle controls for different solvents also gave incorporation values in the range of 3000 to 5000 cpm, which is comparable to untreated controls (white bars).) B, LCD cultures of SMS-SB cells were propagated in the presence of a 10-fold molar excess of either peptide #10, #11, or #57, before addition of either peptide #9 (panel i) or peptide #12 or, as a negative control, peptide #57 (panel ii); [3H]TdR uptake was assessed after 72 h. In each case, the effect of stimulatory peptide alone is shown in the dark gray bar, and the effect of excess individual competitor shown as light gray bars; the effects of excess peptides alone on SMS-SB cells are shown in panel i (white bars). C, peptide binding experiments were established as in Fig. 4A. Panel i shows mean fluorescence index values for peptide binding determined in the absence (dark gray bar) or presence (light gray bar) of 10 µg/assay of RGDS. LCD cultures were established with no stimulus (white bar), with 10 µg/ml RGDS peptide (light gray bar), peptide #9 or #12 alone (dark gray bars), or either peptide #9 or #12 together with 10 µg/ml RGDS (mid-gray bars); [3H]TdR uptake was assessed as noted in Fig. 2.

 
RKC-containing Peptides Derived from CD23 Bind Integrin beta Chains—The data of Fig. 6 indicate that the RKC-containing CD23-derived peptides do not bind to the RGD binding site of the {alpha}v integrins, and this is fully supported by the x-ray crystallographic data for {alpha}vbeta3 in association with an RGD-containing cyclic pentapeptide that suggest that the lysine is too large to be accommodated in the RGD binding site (24). As a first step in seeking the binding site for RKC-containing peptides in the {alpha}vbeta5 integrins, a far-Western approach was adopted in which purified {alpha}vbeta5 and {alpha}vbeta3 integrins were electrophoresed under non-reducing conditions and the separated subunits were transferred to nitrocellulose and probed with biotinylated RKC-containing peptides. Fig. 7A (lanes 1 and 2) illustrates that peptide #9 bound strongly to the beta5 and beta3 chain but only very weakly, if at all, to the {alpha}v subunit; treatment of identical blots with an irrelevant biotinylated peptide (#76), gave no signal (Fig. 7A, lanes 3 and 4). Fig. 7B (panel i) demonstrates that binding of peptide #9 to the beta5 subunit is not impeded by the presence of excess, negative control peptide #58 (lane 2), where no reduction of peak areas of the beta5 bands was evident (Fig. 7B, panel ii); inclusion of excess peptide #10 (lane 3) inhibited binding of biotinylated peptide #9 to the separated beta5 subunit by ~90% (Fig. 7B, panel ii). These data are completely consistent with those of Fig. 5 showing that peptide #10 inhibited peptide #9-driven proliferation of SMS-SB cells. The data of Fig. 7 indicate that CD23-derived, RKC-containing peptides bind to a structure present on the beta chains of the {alpha}v integrins.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study identifies the {alpha}vbeta5 integrin as a CD23 receptor, defines the recognition site for the integrin on the CD23 protein, and demonstrates a new role for CD23 in regulation of human B cell precursor growth. The data demonstrate that survival signaling via the integrin is ligand-selective and is mediated via a structure on the integrin that is distinct from the RGD binding site and recognizes a basic domain on CD23; this second site appears to reside on the beta subunit. Importantly, given the protein-free conditions of the proliferation assays and the sizes of the ligands used, the data demonstrate that integrins send important signals to cells without the need for ligation of the RGD binding site. This property may not be limited to {alpha}vbeta5. The data illustrate further the complex roles of CD23 in regulating human B cell function and show clearly that the pleiotropic functions of CD23 are controlled by discrete structural motifs on the protein.


Figure 6
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 6.
Cytokine activity of sCD23 is independent of the IgE and CD21 binding sites. A, proliferation, cell binding, and SPR experiments were established as noted in previous figures. LCD SMS-SB cultures were stimulated with the indicated peptides at 10 µg/ml for 72 h prior to addition of [3H]TdR and determination of proliferation (A, panel i). Binding of wild-type peptide #9, the double-alanine mutant #9AA and irrelevant peptide (#58) to cells (A, panel ii) and to immobilized purified {alpha}vbeta5 integrin (A, panel iii) are illustrated. The capacity of wild-type peptides #12, #12AA, and #58 to promote SMS-SB proliferation is shown in B, whereas binding of peptides #11, #11AA, and #58 to immobilized {alpha}vbeta5 is illustrated in C. The effects of sCD2348-248 and of LP on SMS-SB cell proliferation are illustrated in D and E, respectively.

 
The RKC motif recognized by the {alpha}vbeta5 integrin is located at positions 172-174 of the human CD23 protein and is preserved in all forms of sCD23 that possess cytokine activity (1). Fig. 8A places the RKC motif in the context of its general position in the CD23 protein and demonstrates that the RKC-centered integrin binding site is completely distinct from those used to capture either IgE or CD21 (14). It is also separate from the sequence in the stalk region reported to bind to major histo-compatibility complex class II molecules (33). The finer detail provided by the NMR structure of derCD23 (14) and crystal structure of sCD23 (15) locates the RKC in a small disulfide-bonded loop near the N-terminal end of the lectin domain at a solvent-exposed tight turn between the beta0 and beta1 strands, with the side chain of Cys174 participating in formation of a disulfide bond with Cys163 (Figs. 3B and 8). The data of Fig. 6, showing loss of binding and activity of peptide #9 when alanine groups replace the basic arginine and lysine residues, are consistent with a role for these solvent-available basic side chains on the corner of the loop in binding to the integrin. The failure of peptide #11AA to competitively inhibit binding of sCD23 to SMS-SB cells further supports this interpretation. Comparing 20 NMR structures for derCD23 with respect to the RKC motif (Fig. 8C), it is clear that Cys174 occupies relatively few positions, presumably by virtue of disulfide bond formation with Cys163, and Lys173 is also found in relatively few distinct conformations. However, Arg172, exposed on the corner of the tight bend, samples a wide range of orientations. The crystal structure of sCD23 and the NMR structure of derCD23 do show striking differences, and in the specific case of the RKC motif, both Arg172 and Lys173 are clearly exposed to the solvent in the derCD23 structure (Fig. 8D, gray lines), whereas the sCD23 crystal structure shows Lys173 occupying a less solvent-available position close to the {alpha}1 helix and the arginine in a different but still exposed position (Fig. 8D, blue lines, and Fig. 8B, panels ii and iv). Finally, although binding of calcium causes notable conformational changes in the structure of sCD23 as a whole (15), there is relatively little effect on the orientations and relative positions of the RKC residues in the calcium-free and bound states (Fig. 8E). The NMR structure can accommodate a direct binding model for the {alpha}vbeta5-CD23 interaction, based on having both Arg172 and Lys173 available for binding, but the crystal structure does not preclude an induced-fit model in which Arg172 provides an initial point of contact that allows the two protein structures to alter such that a larger interaction surface becomes available. A comparison of the human and murine sequences in the context of the {alpha}vbeta5 binding site defined in this report offers a partial explanation for the apparent lack of cytokine-like activity in murine CD23 mediated via the {alpha}v integrins. The human CD23 sequence between residues 169 and 181 is NFQRKCYYFGKGT while the equivalent murine sequence is HFQQKCYYFKGS; importantly, the RKC motif that may be the primary determinant of binding and activity is altered to QKC in the murine CD23 sequence. In contrast to the large, mobile, and basic arginine side chain, glutamine may lack both sufficient length and flexibility to enter a binding cleft and/or may lack the charged termini required to bind securely to the target integrin in a conformation capable of signaling to the cell interior. Although Arg172 alone is unlikely to be the sole determinant of cytokine activity, it is clearly a key residue for binding and, by extension, biological activity.


Figure 7
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 7.
RKC-containing peptides bind to integrin beta chains. A, purified {alpha}vbeta3 and {alpha}vbeta5 integrins (0. 5 µg) were electrophoresed under non-reducing conditions, and separated subunits were transferred to nitrocellulose filters and probed with 4.5 µg/ml biotinylated peptides #9 (lanes 1 and 2) or #76 (lanes 3 and 4). Peptide binding was visualized with streptavidin-HRP and ECL. Blots of separated {alpha}vbeta5 integrin chains, generated as described above, were probed with biotinylated peptide #9 (lane 1) in the presence of a 2-fold molar excess of peptide #58 (lane 2) or #10 (lane 3) (B, panel i). Peak area analysis was performed using ImageJ software (B, panel ii).

 
The data of Fig. 2 demonstrate that different {alpha}vbeta5 ligands (CD23, Vn, and Fn) and mAbs directed to different epitopes of {alpha}vbeta5 itself elicit characteristic responses in pre-B cell lines by acting via distinct, RGD-dependent and RGD-independent, binding sites on {alpha}vbeta5. There are precedents for ligand-selective signaling via {alpha}vbeta3 in monocytes where CD23 promotes pro-inflammatory cytokine synthesis, while Vn drives cell spreading but no cytokine production (13). The importance of adhesion interactions between pre-B cells or acute lymphoblastic leukemia cells with stromal cells for survival is widely appreciated (e.g. the VLA-4-VCAM-1 interaction (34-36)), and, in the bone marrow environment where several {alpha}v ligands are present (i.e. Fn and Vn (37) and CD23 (38)), the availability of ligand-selective responses, mediated via two distinct binding sites on the same integrin, may be important. If engagement of the RGD binding site was linked to cell survival, then Vn and Fn in the bone marrow would sustain growth of precursor cells in a non-selective manner, resulting in failure to eliminate redundant or malignant precursors. However, the {alpha}vbeta5 integrin may allow B cell precursors to adhere to the stromal matrix via Vn in an interaction that is neutral with respect to cell survival. Signals for inhibition of apoptosis and promotion of cell growth would then be delivered via the second, non-RGD binding site on the {alpha}vbeta5 integrin. Whether the {alpha}vbeta5 integrin delivers a growth-sustaining signal to cell types other than lymphoid precursors remains to be established.

In our system, RGD-containing peptides do not sustain cell growth themselves, and fail to inhibit either peptide #9 or #12 binding to cells or their ability to sustain cell growth (Fig. 5), and far-Western data (Fig. 7) suggest that RKC-containing peptides bind separated beta3 and beta5 chains. The x-ray crystallographic model of {alpha}vbeta3 integrin in association with an RGD-containing cyclic pentapeptide fully supports the interpretation that {alpha}vbeta5 binds CD23 using a structure distinct from the RGD binding site. The RGD-peptide arginine is secured by a bidentate salt link with Asp218 and a second such link with Asp150 from the {alpha}v chain (24), whereas the aspartate side chain makes contacts with Tyr122, Arg214, and Asn215 from the beta3 chain, plus a Mn2+ ion (24). The peptide glycine residue resides at the interface between the {alpha}v and beta3 domains making multiple hydrophobic interactions, including a dominant contact with the carbonyl oxygen of Arg216 of {alpha}v (24). The long side chain of the RKC peptide lysine would collide with this carbonyl moiety and so preclude stable insertion of the RKC motif into the RGD binding site.


Figure 8
View larger version (56K):
[in this window]
[in a new window]

  		FIGURE 8.
Structural contexts of the CD23 RKC motif. Structural data files for the NMR structure of derCD23 (1T8C (14)) and the crystal structures of calcium-free and calcium bound sCD23 (2H2R and 2H2T, respectively (15)) were acquired from the Brookhaven protein data base, and images were generated using the PyMOL software suite (W. L. DeLano (2002) PyMOL, DeLano Scientific, San Carlos, CA). A, the position of the IgE (blue), CD21 (red), and {alpha}vbeta5 (yellow) binding sites on the 1T8C NMR derCD23 structure (14) as a surface model. B, compares the derCD23 and 2H2R Ca2+-free sCD23 structures (15), with Arg172 (red) and Lys173 (blue) high-lighted and the positions of the extended side chains shown in panels ii and iv. C, compares as a ribbon diagram twenty individual NMR structures close to the RKC motif (14); the individual orientations of Arg172, Lys173, and Cys174 are illustrated as sticks. D, RKC motif in detail, superimposing the derCD23 (1T8C) structure as gray sticks with the Ca2+-free sCD23 structure (2H2R) as blue sticks. E, orientation of the RKC motif in sCD23 structures with calcium either bound (yellow sticks) or free (blue sticks) (15).

 
Integrins recognize sequences other than RGD (22). Thus, the {alpha}2beta1 integrin binds a basic tetrapeptide sequence (RKKH) in the snake venom metalloprotease, jararhagin, a potent inhibitor of platelet binding to collagen (39), and the I-domain of the integrin is known to be the binding site. However, it is becoming increasingly apparent that small groups of basic residues, such as described in this report, are often targets for integrin binding, and in many cases recognition of such motifs by integrins is linked to, or cooperates with, binding to the RGD binding site in facilitating adhesion reactions. For example, both binding of the angiogenic factor CCN3 to {alpha}vbeta5 and CCN3-induced migration of fibroblasts is inhibited by RGD-containing peptides even though the protein does not possess an RGD, or related, sequence (40). The C-terminal domain of the closely related CCN1 protein has two distinct regions, each containing a pair of adjacent lysine residues, that bind the {alpha}Mbeta2 integrin and support monocyte adhesion (41). CCN3 has equivalent sequences in its C-terminal domain, although whether these are bound by {alpha}vbeta5 remains to be formally proven. The binding of the non-RGD-containing cobra venom cardiotoxin A5 to {alpha}vbeta3 and its ability to inhibit osteoclastogenesis is inhibited by RGD-containing peptides (42). Interestingly, the related A6 toxin binds only weakly to {alpha}vbeta3 and is not cardiotoxic, and this is explained by replacement of Lys28 and Lys29 of the A5 molecule with valine and alanine, respectively (42). The {alpha}vbeta5 integrin recognizes the HIV Tat protein via a non-RGD motif that is basic in character (25). Studies of binding of {alpha}IIbbeta3 to peptide fragments of fibrinogen demonstrated that the integrin recognizes a cluster of basic residues located between residues 370 and 381 of the {gamma}C domain that is devoid of an RGD sequence (43). Intriguingly, the structure of the {gamma}C domain (32) reveals two pairs of basic residues on exposed surfaces of the protein that are crucial for the interaction with the integrin. In particular, substitution of either Lys380 or Lys381 of {gamma}C peptides with alanine reduces peptide binding to {alpha}IIbbeta3, and a double substitution completely abolishes binding (43). The data from other systems are completely consistent with our data showing loss of binding and activity of CD23-derived peptides when Arg172 and Lys173 are substituted with alanine.

A range of integrin-mediated responses may be driven by recognition of basic domains on other extracellular proteins, and such responses may be independent of occupation of the RGD binding site or may show cross-talk with RGD-specific binding events. Indeed, the cellular response initiated following ligation of the basic domain recognition site on {alpha}v or other integrins may also be dependent on the integrin heterodimer being engaged. Definition of the precise basic domain binding structure on the beta chain of the {alpha}v integrins will help address these issues.


   FOOTNOTES
 
* This work was supported in part by project grants from the Biotechnology & Biological Sciences Research Council (BBSRC), the Leukemia Research Fund, and the Arthritis Research Campaign. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by a Wellcome Trust Value in People award. Back

2 Supported by the Wellcome Trust PhD program, Molecular Functions in Disease. Back

3 Supported by a post-graduate scholarship from the BBSRC. Back

4 Supported by Cancer Research UK. Back

5 To whom correspondence should be addressed. Tel.: 44-141-330-5261; Fax: 44-141-330-4620; E-mail: W.Cushley{at}bio.gla.ac.uk.

6 The abbreviations used are: sCD23, soluble CD23; VnR, vitronectin receptor; Vn, vitronectin; Fn, fibronectin; HIV, human immunodeficiency virus; HRP, horseradish peroxidase; OGP, octyl-beta-D-glucopyranoside; GST, glutathione S-transferase; siRNA, small interference RNA; PFHM, protein-free hybridoma medium-II; LCD, low cell density; mAb, monoclonal antibody; TdR, tritiated thymidine; SPR, surface plasmon resonance; LP, long peptide; PE, phycoerythrin; FITC, fluorescein isothiocyanate. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Jim McDonnell of the University of Oxford for the generous gift of recombinant human derCD23 protein, Dr. Sharon Kelly for expert assistance with the SPR analysis, and Dr. Lynn McGarry of the Beatson Institute for expert guidance with the siRNA study.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gould, H. J., Sutton, B. J., Beavil, A. J., Beavil, R. L., McCloskey, N., Coker, H. A., Fear, D., and Smurthwaite, L. (2003) Annu. Rev. Immunol. 21, 579-628[CrossRef][Medline] [Order article via Infotrieve]
  2. Bonnefoy, J.-Y., Lecoanet-Henchoz, S., Gauchat, J. F., Graber, P., Aubry, J. P., Jeannin, P., and Plater-Zyberk, C. (1997) Int. Rev. Immunol. 16, 113-128[Medline] [Order article via Infotrieve]
  3. Delespesse, G., Sarfati, M., Wu, C. Y., Fournier, S., and Letellier, M. (1992) Immunol. Rev. 125, 77-97[CrossRef][Medline] [Order article via Infotrieve]
  4. Fujiwara, H., Kikutani, H., Suematsu, S., Naka, T., Yoshida, K., Yoshida, K., Tanaka, T., Suemura, M., Matsumoto, N., Kojima, S., Kishimoto, T., and Yoshida, N. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6835-6839[Abstract/Free Full Text]
  5. Yu, P., Kosco-Vilbois, M. H., Richards, M., Kohler, G., and Lamers, M. C. (1994) Nature 369, 753-756[CrossRef][Medline] [Order article via Infotrieve]
  6. Bailey, S., Bolognese, B., Buckle, D. R., Faller, A., Jackson, J., Flamberg, P. L., McCord, M., Mayer, R. J., Marshall, L. A., and Smith, D. G. (1998) Bioorg. Med. Chem. Lett. 8, 23-28[CrossRef][Medline] [Order article via Infotrieve]
  7. Marolewski, A. E., Buckle, D. R., Christie, G., Earnshaw, D. L., Flamberg, P. L., Marshall, L. A., Smith, D. G., and Mayer, R. J. (1998) Biochem. J. 333, 573-579[Medline] [Order article via Infotrieve]
  8. Liu, Y.-J., Mason, D. Y., Johnson, G. D., Abbot, S., Gregory, C. D., Hardie, D. L., Gordon, J., and MacLennan, I. C. M. (1991) Eur. J. Immunol. 21, 1905-1910[Medline] [Order article via Infotrieve]
  9. Bonnefoy, J.-Y., Henchoz, S., Hardie, D., Holder, M. J., and Gordon, J. (1993) Eur. J. Immunol. 23, 969-972[Medline] [Order article via Infotrieve]
  10. White, L. J., Ozanne, B. W., Graber, P., Aubry, J.-P., Bonnefoy, J.-Y., and Cushley, W. (1997) Blood 90, 234-243[Abstract/Free Full Text]
  11. Mossalayi, M. D., Lecron, J.-C., Dalloul, A. H., Sarfati, M., Bertho, J.-M., Hofstetter, H., Delespesse, G., and Drbre, P. (1990) J. Exp. Med. 171, 959-964[Abstract/Free Full Text]
  12. Lecoanet-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., and Bonnefoy, J.-Y. (1995) Immunity 3, 119-125[CrossRef][Medline] [Order article via Infotrieve]
  13. Hermann, P., Armant, M., Brown, E., Rubio, M., Ishihara, H., Ulrich, D., Caspary, R. G., Lindberg, F. P., Armitage, R., Maliszewski, C., Delespesse, G., and Sarfati, M. (1999) J. Cell Biol. 144, 767-775[Abstract/Free Full Text]
  14. Hibbert, R. G., Teriete, P., Grundy, G. J., Beavil, R. L., Reljic, R., Holers, V. M., Hannan, J. P., Sutton, B. J., Gould, H. J., and McDonnell, J. M. (2005) J. Exp. Med. 202, 751-760[Abstract/Free Full Text]
  15. Wurzburg, B. A., Tarchevskaya, S. S., and Jardetzky, T. S. (2006) Structure 14, 1049-1058[Medline] [Order article via Infotrieve]
  16. Mossalayi, M. D., Arock, M., Delespesse, G., Hofstetter, H., Bettler, B., Dalloul, A. H., Killcher, E., Quaaz, F., Debré, P., and Sarfati, M. (1992) EMBO J. 11, 4323-4328[Medline] [Order article via Infotrieve]
  17. van der Flier, A., and Sonnenberg, A. (2001) Cell Tissue Res. 305, 285-298[CrossRef][Medline] [Order article via Infotrieve]
  18. Hood, J. D., and Cheresh, D. A. (2002) Nat. Rev. Cancer 2, 91-100[CrossRef][Medline] [Order article via Infotrieve]
  19. Kumar, C. C. (1998) Oncogene 17, 1365-1373[CrossRef][Medline] [Order article via Infotrieve]
  20. Stupack, D. W., and Cheresh, D. A. (2002) J. Cell Sci. 115, 3729-3738[Abstract/Free Full Text]
  21. Marshall, J. F., and Hart, I. R. (1996) Semin. Cancer Biol. 7, 129-138[CrossRef][Medline] [Order article via Infotrieve]
  22. Ruoslahti, E. (1996) Annu. Rev. Cell Biol. 12, 697-715[CrossRef]
  23. Xiong, J.-P., Stehle, T., Diefenbach, B., Zhang, R., Dunker, R., Scott, D. L., Joachimiak, A., Goodman, S. L., and Arnout, M. A. (2001) Science 294, 339-345[Abstract/Free Full Text]
  24. Xiong, J.-P., Stehle, T., Zhang, R., Joachimiak, A., Frech, M., Goodman, S. L., and Arnout, M. A. (2002) Science 296, 151-155[Abstract/Free Full Text]
  25. Vogel, B. E., Lee, S. J., Hildebrand, A., Craig, W., Pierschbecher, M. D., Wong-Staal, F., and Ruoslahti, E. (1993) J. Cell Biol. 121, 461-468[Abstract/Free Full Text]
  26. Smith, R. G. (1984) Cancer 54, 471-476[CrossRef][Medline] [Order article via Infotrieve]
  27. Scott, L. A., Vass, J. K., Parkinson, E. K., Gillespie, D. A., Winnie, J. N., and Ozanne, B. W. (2004) Mol. Cell. Biol. 24, 1540-1559[Abstract/Free Full Text]
  28. Myszka, D. G. (1999) J. Mol. Recognit. 12, 279-284[CrossRef][Medline] [Order article via Infotrieve]
  29. Suzuki, S., Argraves, W. S., Pytela, R., Arai, H., Krusius, T., Pierschbacher, M. D., and Ruoslahti, E. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8614-8618[Abstract/Free Full Text]
  30. Wayner, E. A., Orlando, R. A., and Cheresh, D. A. (1991) J. Cell Biol. 113, 919-929[Abstract/Free Full Text]
  31. Stuiver, I., and Smith, J. W. (1995) Hybridoma 14, 545-550[Medline] [Order article via Infotrieve]
  32. Yee, V. C., Pratt, K. P., Cote, H. C., Trong, I. L., Chung, D. W., Davie, E. W., Stenkamp, R. E., and Teller, D. C. (1997) Structure 5, 125-138[Medline] [Order article via Infotrieve]
  33. Kijimoto, O. S., and Noguchi, A. (2000) Biochem. Biophys. Res. Commun. 267, 686-691[CrossRef][Medline] [Order article via Infotrieve]
  34. Hewson, J., Bianchi, A., Bradstock, K., Makrynikola, V., and Gottlieb, D. (1996) Br. J. Haematol. 92, 77-87[CrossRef][Medline] [Order article via Infotrieve]
  35. Makrynikola, V., Bianchi, A., Bradstock, K., Gottlieb, D., and Hewson, J. (1994) Leukemia 8, 1734-1743[Medline] [Order article via Infotrieve]
  36. Makrynikola, V., and Bradstock, K. F. (1993) Leukemia 7, 86-92[Medline] [Order article via Infotrieve]
  37. Gronthos, S., Simmons, P. J., Graves, S. E., and Robey, P. G. (2001) Bone 28, 174-181[Medline] [Order article via Infotrieve]
  38. Fourcade, C., Arock, M., Ktorza, S., Quaaz, F., Merle-B'ral, H., Mentz, F., Kilchherr, E., Debré, P., and Mossalayi, M. D. (1992) Eur. Cytokine Net. 3, 539-543[Medline] [Order article via Infotrieve]
  39. Ivaska, J., Käpylä, J., Pentikäinen, O., Hoffrén, A.-M., Hermonen, J., Huttenen, P., Johnson, M. S., and Heino, J. (1999) J. Biol. Chem. 274, 3513-3521[Abstract/Free Full Text]
  40. Lin, C. G., Chen, C. C., Leu, S. J., Grzeszkiewicz, T. M., and Lau, L. F. (2005) J. Biol. Chem. 280, 8229-8237[Abstract/Free Full Text]
  41. Schober, J. M., Lau, L. F., Ugarova, T. P., and Lam, S. C. (2003) J. Biol. Chem. 278, 25808-25815[Abstract/Free Full Text]
  42. Wu, P. L., Lee, S. C., Chuang, C. C., Mori, S., Akakura, N., Wu, W. G., and Takada, Y. (2006) J. Biol. Chem. 281, 7937-7945[Abstract/Free Full Text]
  43. Podolnikova, N. P., Gorkun, O. V., Loreth, R. M., Yee, V. C., Lord, S. T., and Ugarova, T. P. (2005) Biochemistry 44, 16920-16930[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/37/27315    most recent
M609335200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Borland, G.
Right arrow Articles by Cushley, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Borland, G.
Right arrow Articles by Cushley, W.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement