THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry ’ and Molecular Biology, Inc.

Vol. 268, No. 27, Issue of September 25, pp. 20352-20359,1993 Printed in U. S.A.

A Novel Cyclic Pentapeptide Inhibits a401 and a501 Integrin-mediated Cell Adhesion*

(Received for publication, April 6, 1993, and in revised form, May 13, 1993)

Dawn M. Nowlin, Frank Gorcsan, Mary Moscinski, Shiu-Lan Chiang, Tom J. Lobl, and Pina M. CardarelliS From the Tanabe Research Laboratories, San Diego, California 92121

Lymphocytes and monocytes initiate and modulate inflammatory and immune responses for host defense. This process is dependent upon extravasation of leu- kocytes from the circulation to sites of antigenic chal- lenge and is controlled, in part, by various integrins, including a481 and ~ ~ 5 8 1 . A small cyclic pentapeptide that inhibits, in vitro, both a481 and a5B1 activity is described. This peptide, Arg-Cys-Asp-Thioproline-Cys (RC*D[ThioP]C*), is cyclized by a disulfide bond through the cysteine residues (the asterisks denote cy- clizing residues). RC*D(ThioP)C* inhibits aSj3l-me- diated leukocyte adhesion to the 120-kDa Arg-Gly- Asp (RGD)-containing binding site of fibronectin. Two different adhesion activities of a481 are also inhibited: a4j3l-mediated cell adhesion to the alternatively spliced CS-1 site of fibronectin and the a4j31-depend- ent binding of leukocytes to cytokine-activated endo- thelial cells. Both a481 and a581 can be purified by affinity chromatography using the immobilized pen- tapeptide. The peptide does not inhibit adhesion to other extracellular matrix proteins including laminin and vitronectin. The specificity of the RC*D(ThioP)C* peptide for a481 and a681 suggests potential therapeu- tic utility for inhibiting inflammatory disease.

Specific cellular adhesion to extracellular matrix compo- nents and other cells are fundamental to many different biological responses. In inflammatory reactions, leukocytes adhere to the blood vessel wall at sites of immunologic chal- lenge, a crucial first step in mounting an effective immune response. Proteins that mediate these adhesive reactions be- long to a supergene family called integrins and consist of heterodimeric complexes of non-covalently associated alpha (a) and beta (p) subunits (for reviews, see Refs. 1-3). Integrins of the 81 subfamily (4), also know as VLA (very late activa- tion) (3), bind extracellular matrix proteins collagen, laminin, and fibronectin (FN)’ (4-6). Lymphocytes and monocytes interact with FN at two spatially distinct binding domains on the molecule and through two distinct integrins, a5pl and

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$To whom correspondence should be addressed. Tel.: 619-558- 9211; Fax: 619-558-9383.

’ The abbreviations used are: FN, fibronectin; EC, endothelial cells; ICAM-I, intercellular adhesion molecule-1; PAGE, polyacrylamide gel electrophoresis; RC*D(ThioP)C*, Arg-Cys-Asp-Thioproline-Cys peptide cyclized (*) through the side chain sulfhydryls to a disulfide; ThioP, thiazolidine-4-carboxcylic acid; SPDP, 3-(2-pyridyldi- thio)propionic acid N-hydroxysuccinimide ester; VCAM-1, vascular cell adhesion molecule-1; PBS, phosphate-buffered saline.

a4pl (7). The central cell-binding domain of FN, containing the amino acid sequence Arg-Gly-Asp (RGD), has been iden- tified as the site mediating cell attachment (8-10) by a5pl (l l) , avpl(l2), a301 (13), and aIIb @3 (14). The a401 integrin mediates cell adhesion to the alternatively spliced region of the FN A chain through recognition of a 10 amino acid sequence (GPEILDVPST) within the CS-1 peptide (7, 15, 16). Cells which express a high avidity form of a4Pl are capable of recognizing a 5-6 amino acid epitope that contains the requisite Leu-Asp-Val (LDV) sequence (17). The a4pl integrin also mediates lymphocyte adhesion to cytokine-acti- vated endothelial cells (EC) through recognition of vascular cell adhesion molecule-1 (VCAM-1) (3,16, 18-20). The inter- action of lymphocytes with EC is an important mechanism allowing for transmigration of circulating lymphocytes into lymphoid tissue or to sites of inflammation (21).

The RGD sequence of FN is a common motif (9) that is also part of the recognition site for several extracellular matrix proteins including fibrinogen, von Willebrand factor (22-24), vitronectin (25), and recent evidence confirms RGD-depend- ent binding of a201 to collagen (26). Soluble RGDS peptides have been shown to abrogate aIIbp3-mediated platelet aggre- gation (23), and cellular attachment to vitronectin (25) and FN (8). Modification of the primary amino acid sequence of the RGDS peptide, such as enantiomeric substitution of one of the residues, alters the inhibitory activity of the tetrapep- tide in adhesion assays, as well as the specificity toward the different adhesion receptors (14), while cyclization of RGD peptides enhances their potency (14, 26-28). In addition to antagonist function, affinity chromatography using RGDS peptides has been successfully used to purify vitronectin receptors and aIIbp3 (10, 25). However, specific binding of a5pl to a peptide-affinity matrix was achieved only with much larger polypeptides (10).

Strategies were employed to examine the minimum ring size of cyclic RGD peptides that retained the requisite charge distribution conferred by the Arg and Asp residues while maintaining potent antagonist activity. A disulfide cyclic Arg- Cys-Asp-Thioproline-Cys peptide, RC*D(ThioP)C*, was de- veloped and found to be a potent inhibitor of leukocyte cell adhesion to FN. The activity of this novel peptide was further characterized by examining a501- and a4pl-mediated integrin functions. The combined a4pl and a5B1 modulating proper- ties of the RC*D(ThioP)C* peptide suggest therapeutic po- tential as an anti-inflammatory agent.

EXPERIMENTAL PROCEDURES

Materials-FN was purified on gelatin Sepharose as described (29). Collagen type I, was purchased from Collaborative Research Labo- ratories (Bedford, MA). Laminin and the 120-kDa (central cell- binding domain) chymotryptic fragment of FN were purchased from Telios Pharmaceuticals (San Diego, CA). All other chemicals were

20352

RCD Peptide Inhibits a4pl and a5pl Integrin Function 20353

from Sigma unless otherwise indicated. Peptide Synthesis-Amino acid precursor were purchased from

BACHEM (Torrance, CA). Synthetic peptides were prepared on a System 990 automated peptide synthesizer (Beckman Instruments, Inc., Palo Alto, CA) using t-butoxycarbonyl methodology following the standardized cycle (30). Biotin (Aldrich) was coupled to the peptide resin in the presence of BOP Reagent (Richelieu Biotechnol- ogies, St. Hyacinthe Quebec, Canada). Cyclized peptides were formed by the iodine method (31). Peptides were purified on a Waters Delta Prep 3000 high performance liquid chromatography system with a C18 column (RPC18 Ultrasphere, 5 pm 4.6 X 150 mm inner diameter) and sent to Beckman Research Institute of the City of Hope (Duarte, CA) for FAB mass spectral analysis. Table I lists the peptides used in these studies.

Monoclonal Antibodies-Monoclonal antibodies to a5 (PlD6), a2 (PlE6), a3 (PlB5), and av (VNR147) integrin subunits were pur- chased from Telios Pharmaceuticals. Antibody to the a4 subunit, HP2/1, was from AMAC (Westbrook, ME), and the p l subunit antibody, 4B4, was obtained from Coulter Clone (Hialeah, FL). Polyclonal peptide antiserum to the cytoplasmic domain of av was kindly provided by Bruce Vogel of the La Jolla Cancer Research Foundation. Intercellular adhesion molecule-1 (ICAM-1) antibody (BBA4) was purchased from British Biotechnology Ltd (Cowley Oxford, United Kingdom). Sam Wright of Rockerfeller University kindly provided antibody to the 02 integrin subunit CD18 (IB4). Flow cytometry utilizing secondary fluorescein isothiocyanate-conjugated sheep-anti-mouse antibody (Organon Teknika Corp., West Chester, PA) was performed on a Becton Dickinson FACS Star.

Cell Lines-Jurkat T lymphoblastoid and U937 myelomonocytic cells were obtained from American Type Culture Collection (Rock- ville, MD) and maintained in RPMI-1640 containing 10% fetal bovine serum and 1 mM glutamine. MG63 osteosarcoma cells (ATCC) were grown in Dulbecco's modified Eagle's medium, 10% fetal bovine serum, and 1 mM glutamine and passaged by trypsinization. Human umbilical vein EC were purchased from Clonetics Corp. (San Diego, CA) and subcultured in EGM-UV medium containing growth factors as described (32).

Cell Adhesion to Extracellular Matrix Proteins-The cell adhesion assay was conducted as reported previously (33). Briefly, matrix proteins or their fragments were coated onto Falcon, flat bottom, 96- well plates (Becton and Dickinson, Lincoln Park, NJ) at suboptimal concentrations. Jurkat or U937 cells were suspended in Dulbecco's modified Eagle's medium with 0.25% bovine serum albumin at a density of 3 X lo6 cells/ml and were incubated at 37 "C for 60 min on substrate-coated plates in the presence or absence of peptide or antibody competitors. For MG63 cells, a concentration of 2.5 X lo5 cells/ml were used and the analysis completed after a 45-min incu- bation. Adherent cells were fixed with paraformaldehyde prior to staining by 0.5% toluidine blue then solubilized with SDS for absorb- ance measurements at 590 nm in a Molecular Devices microplate reader. Typically, 10-30% of the cell population bound to immobilized substrate.

Cell Adhesion to CS-I Peptide-coated Plates-The CS-1-derived peptide CLHPGEILDVPST and scrambled sequence CLHGPIELV- SDPT were immobilized onto microtiter plates using the heterobi- functional cross-linker 3-(2-pyridyldithio)propionic acid N-hydroxy- succinimide ester (SPDP) as reported (34). Microtiter plates were

TABLE I Synthetic peptides used for these studies

Peptide sequence is by single letter amino acid coding. Sequence

GRGDSP RC*D(ThioP)C*".* K(Anc)RC*D(ThioP)C* RC*(ThioP)DC* LHGPEILDVPST LHPGIELVSDPT CLHGPEILDVPST CLHPGIELVSDPT Biotinvl(Anc)RC*D(ThioP)C"'

The asterisk denotes the cysteine residues that participate in the

ThioP is an abbreviation for thiazolidine-4-carboxcylic acid. Anc designates the inclusion of 6-aminocaproic acid during pep-

formation of a disulfide bond to form a cyclic peptide.

tide synthesis.

coated with 20 pg/ml human serum albumin then derivatized with 10 pg/ml SPDP. After washing, the cystein containing peptides were added and allowed to cross-link to the plates overnight at 4 "C.

Cell ,Adhesion to RC*D(ThioP)C*-coated Plates-The bioti- nyl(Anc)RC*D(ThioP)C* peptide was immobilized for adhesion as- says by incubating excess (34 p ~ ) peptide in phosphate-buffered saline (PBS) with microwells previously coated with different con- centrations of anti-biotin antibody (Sigma). The peptide concentra- tion required to obtain submaximal binding was determined in a second experiment with serial dilutions of biotinyl(Anc)- RC*D(ThioP)C*. For this assay, plates were precoated with the suboptimal concentration of anti-biotin antibody (0.1 p ~ ) determined in the initial experiment.

Heterotypic Cell Adhesion-EC cultured in 96-well plates were stimulated with 0.1 ng/ml (50 units/ml) IL-1 (Amgen, Thousand Oaks, CA) for 4 h at 37 "C (35). Jurkat cells were labeled with fluorescein diacetate (18), suspended in Dulbecco's modified Eagle's medium, 0.25% human serum albumin containing peptide or anti- body, and then added (3 X lo5 cells/well) to the cytokine stimulated EC monolayer for a 20 min, 37 "C incubation. Non adherent lympho- cytes were removed by vacuum aspiration and washing with PBS cation buffer (PBS, 1 mM CaC12, 1 mM MgC12). Fluorescence in each well was measured by a Pandex Fluorescence Concentration Analyzer. For some experiments, EC were pretreated with peptides for 20 min at 37 "C then washed with PBS cation buffer prior to the addition of Jurkat cells.

Affinity Chromatography-The K(Anc)RC*D(ThioP)C* peptide was cross-linked to cyanogen bromide-activated Sepharose 4B as per the manufacturer's instructions. The method of affinity chromatog- raphy was adapted from Pytela et al. (25). U937 or MG63 cell suspensions (108/ml) in PBS were radiolabeled with 1 mCi '251-sodium iodide (ICN Biomedicals, Costa Mesa, CA) using iodobeads (Pierce Chemical Co.), then solubilized with buffer A (Tris-buffered saline, pH 7.5, 1 mM MnC12, 1 mM MgC12, 1 mM phenylmethylsulfonyl fluoride, 1 pg/ml each of pepstatin, leupeptin, and aprotinin), and 100 mM n-octyl glucopyranoside. Solubilized membranes were incu- bated on the column at 4 "C overnight. Unbound material was eluted with 10 bed volumes of buffer B (50 mM n-octyl glucopyranoside in buffer A) followed by elution of bound material with 3 column volumes of buffer B containing the indicated peptides or 10 mM EDTA. 100 pl from individual 1-ml fractions were analyzed by SDS-polyacryl- amide gel electrophoresis (PAGE) under non-reducing conditions while the remainder of the peak fractions from each eluate were pooled for immunoprecipitation with integrin-specific antibodies and anti-mouse Ig-agarose. The samples were subjected to SDS-PAGE and dried gels were exposed to X-Omat AR film for autoradiography.

RESULTS

Flow Cytometric Analysis-Flow cytometry of cells surface labeled with specific antibodies was done to establish an integrin expression profile for the cell types used in this study (Table 11). Both U937 and Jurkat cells expressed high levels of a4 and a5, but low levels of av and a 3 integrins. In contrast, MG63 cells expressed abundant levels of a5 and a 3 integrins and moderate levels of a4 and av receptors. The majority of the MG63 cells expressed a2 receptors (78% positive) at a low

TABLE I1 Surface expression of integrin subunits determined

by flow cytometric fluorescence analysis (FACS) FACS analysis of three different cell types in suspension was done

with the indicated primary antibody followed by reaction with fluo- rescein isothiocyanate-conjugated anti-mouse Ig secondary antibody.

U937 Antibody

Jurkat MG63

%+" MFI* %+ MFI %+ MFI

a4 (HP2/1) 100 128 96 91 54 26 a5 (PlD6) 99 170 99 41 99 58 av (VNR147) 29 14 4 7 58 24 a3 (PlB5) 33 12 16 21 98 45 a2 (PlE6) ND' ND 78 18 %+ is the percentage of cells that stained positive for the antigen. MFI indicates mean fluorescence intensity.

E ND, not determined.

20354 RCD Peptide Inhibits a4pl and a5pl Integrin Function

receptor density (18% mean fluorescent intensity units). Cell Adhesion to FN-The U937 cells bound to FN in a

concentration-dependent manner reaching a plateau at a plat- ing concentration of 5 pg/ml. This concentration was used to assess the inhibitory activities of novel RC*D(ThioP)C* pep- tides (see Table I for peptide abbreviations). As shown in Fig. la, RC*D(ThioP)C* was much more potent at inhibiting cell binding to FN than the linear peptide GRGDSP with IC50 values of 5 p M and 400 pM, respectively. In contrast, the scrambled control peptide RC*(ThioP)DC* was not inhibitory at all concentrations tested. Binding of Jurkat cells to FN- coated wells was also inhibited by RC*D(ThioP)C* (Fig. l b ) much to the same degree as that observed for the U937 cell line, although the peptide GRGDSP was not active toward the Jurkat cells at the concentrations tested (2 mM).

Cell Adhesion to FN Fragments-In order to test whether RC*D(ThioP)C* blocked cell adhesion mediated by a5p1, assays were conducted using the 120-kDa chymotryptic frag- ment of FN that contains the RGD cell-binding domain and lacks the CS-1 cell attachment site. RC*D(ThioP)C* effec- tively blocked U937 and Jurkat cell adhesion to the 120-kDa fragment, exhibiting nearly equivalent ICs0 values for binding to intact FN, i.e. 2 and 5 p ~ , respectively (Fig. 2, a and b) . An increased sensitivity toward GRGDSP inhibition was demonstrated in both the U937 and Jurkat cell lines, partic- ularly for the Jurkat cells, as evidenced by an IC50 value of 100 p~ on the 120-kDa fragment compared to >2 mM on intact FN. As with intact FN, RC*D(ThioP)C* was more than 70 times as potent as GRGDSP at inhibiting U937 cell adhe- sion to the 120-kDa fragment.

Cell Adhesion to Immobilized CS-1 Peptides-To determine if RC*D(ThioP)C* inhibited a4pl-mediated binding to FN through an RGD-independent mechanism, we utilized a pep-

a. U937

150

0 ,."""" 0 2 1 0 1 0 0 '00.0 1000 0

Peptide toncent ro t lon (&q/,">I)

b. Jurkat

*:i..,...."- 0.04 0.10 1 00 10 00 l*. 100 00 ~ - Pepllde concentrotion (pg/ml)

FIG. 1. Cell adhesion to intact FN. U937 ( a ) or Jurkat ( b ) cells (3 X lo6 cells/well) were added to FN-coated (5 pg/ml) plates in the absence or presence of duplicate, serial dilutions of peptides GRGDSP (closed squares), RC*D(ThioP)C* (closed circles), K ( A n c ) R C * D (ThioP)C* (open squares), or RC*(ThioP)DC* (open circles). After a 60-min incubation at 37 "C, plates were washed, cells were stained, and absorbance at 590 nm was determined for SDS-solubilized ad- herent cells. Absorbance due to cell adhesion to bovine serum albu- min-coated wells has been subtracted and the data reported as the percentage of the control (adhesion in the absence of antibody or peptide). One representative (n = 5) experiment is shown. Standard deviation values were less than 10%. Curves were generated by 4th order polynomial regression.

a. U937 s 1 .o 10.0 100.0 1000.0

Pcptrde Concentrotion (pg/ml)

b. Jurkat

4 50 x

25

0 0 1 1 0 100 100 0 1000.0

Peptlde Concentrollon (pg/ml)

FIG. 2. Cell adhesion to the 120-kDa fragment of FN. Mi- crotiter plates were coated with 15 or 1.5 pg/well of the 120-kDa fragment for U937 (a) or Jurkat ( b ) cell adhesion, respectively. Experimental methods and symbols for the various peptides are as indicated in Fig. 1 (n = 3).

0 5 1 0 10 0 100 0

FIG. 3. Jurkat cell adhesion to CLHPGEILDVPST peptide- coated plates. a, serial dilutions of CLHPGEILDVPST (open cir- cles) or scrambled CLHGPIELVSDPT (open squares) were cross- linked by the reagent SPDP to microtiter wells preadsorbed with 20 pg/ml human serum albumin (open triangles). Cell adhesion to the peptide coated plates is as described in Fig. 1. b, adhesion of cells to peptide CLHPGEILDVPST cross-linked at a concentration of 100 pg/ml (0.7 mM) to human serum albumin was monitored in the absence or presence of RC*D(ThioP)C* (closed circles), RC*(ThioP) DC* (open circles), LHGPEILDVPST (open triangles), or LHGPIELVSDPT (closed triangles) peptides.

tide sequence from the CS-1 domain, CLHPGEILDVPST, as an adhesion substrate. The CS-1 peptide supported Jurkat cell adhesion in a concentration-dependent manner (Fig. 3a). In contrast, U937 cells were unable to bind to the CLHPGEILDVPST peptide, consistent with reports by Way- ner et al. (17). Binding of Jurkat cells to the scrambled peptide,

RCD Peptide Inhibits a4pl and a5pl Integrin Function 20355

CLHGPIELVSDPT, remained at background levels compa- rable to human serum albumin cross-linked in the absence of peptide (Fig. 3a). Maximal cell binding was achieved at pep- tide coating concentrations of 200 pg/ml and a coating con- centration of 100 pg/ml was chosen as the suboptimal concen- tration for all subsequent experiments. RC*D(ThioP)C* was nearly 20 times more potent (IC5o = 3 ELM) than LHGPEILDVPST (I& = 60 p ~ ) at inhibiting adhesion to the CLHPGEILDVPST substrate (Fig. 3b). Two different scrambled peptides, RC*(ThioP)DC* and LHGPIELVSDPT, showed no significant inhibitory activity in this assay.

Cell Adhesion to Immobilized RC*D(ThioP)C*-The above studies implicate RC*D(ThioP)C* as an inhibitor of a5pl and a4pl-mediated cell adhesion to FN. Whether this peptide could support cell adhesion in a specific manner when im- mobilized onto microtiter wells was next pursued. Since the biotinyl(Anc)RC*D(ThioP)C* peptide inhibited U937 and Jurkat cell adhesion to FN (data not shown), a biotin to anti- biotin antibody interaction was selected to immobilize the peptide onto microtiter wells. A concentration-dependent in- crease in cell adhesion was observed that paralleled the levels of antibody when a fixed concentration of bioti- nyl(Anc)RC*D(ThioP)C* was bound to immobilized anti- biotin antibody (Fig. 4a, closed diamonds). Loss of cell adhe- sion occurred with limiting concentrations of antibody, and

a

b

FIG. 4. Cell adhesion to RC*D(ThioP)C*-coated plates. Op- timal concentrations of anti-biotin antibody and bioti- nyl(Anc)RC*D(ThioP)C* were determined by titration experiments (n = 3). a, anti-biotin antibody was adsorbed to microtiter plates then incubated with (closed diamonds) or without (open diamonds) 34 p~ biotinyl(Anc)RC*D(ThioP)C*. In a separate experiment, anti- biotin antibody (0.1 p ~ ) was preadsorbed to the plate and bioti- nyl(Anc)RC*D(ThioP)C* added at different Concentrations (closed triangles). Unbound peptide was removed followed by Jurkat cell adhesion as described in Fig. 1. b, anti-biotin antibody and bioti- nyl(Anc)RC*D(ThioP)C* were conjugated at suboptimal concentra- tions (0.1 and 0.02 p ~ , respectively) to form an adhesion matrix. Jurkat cell adhesion was determined in the presence of buffer (con- trol), 200 pM RC*(ThioP)DC*, 10 pM RC*D(ThioP)C*, 200 pM GRGDSP, 200 PM LHPGIELVSDPT, 200 PM LHGPEILDVPST, 10 pg/ml anti-ICAM-1 (BBA4), 1:lOOO anti-a5 (PlD6), 10 pg/ml anti- a4 (HP2/1), or 1 pg/ml anti-01 (4B4). Values shown are the percent of control and S.E. (n = 5 ) .

few cells adhered to the anti-biotin antibody in the absence of peptide (Fig. 4a, open diamonds). The minimal concentra- tion of biotinyl(Anc)RC*D(ThioP)C* required to maintain maximal cell binding was determined by titration of the peptide with a fixed concentration of the anti-biotin antibody (Fig. 4a, closed triangles). Cell adhesion to the biotinylated peptide was concentration dependent with maximum levels achieved at 0.02 pM.

Utilizing the suboptimal concentrations of anti-biotin an- tibody (0.1 p ~ ) and biotinyl(Anc)RC*D(ThioP)C* (0.02 pM) established in the previous experiments, competition assays with peptides and antibodies were next examined (Fig. 4b). RC*D(ThioP)C* completely abolished cell adhesion to the sandwich substrate at a concentration of 10 pM. LHGPEILDVPST exhibited modest inhibition, but required much higher concentrations (200 WM) to achieve suppression to a level of 27% of control. GRGDSP and the scrambled peptide RC*(ThioP)DC* did not perturb Jurkat cell binding to the substrate. Antibodies directed toward the a4 and p l integrin subunits inhibited cell adhesion to the bioti- nyl(Anc)RC*D(ThioP)C* peptide, while antibodies for the a5 subunit and ICAM-1 had no effect.

RC*D(ThioP)C* Inhibits Lymphocyte-EC Interactions- The a401 integrin has multiple functions, one of which is to mediate binding to VCAM-1 molecules present on the surface of cytokine (IL-1) stimulated EC (3, 36). Several different antibodies known to have in vitro blocking capability were tested to verify that Jurkat-EC interactions in our assay utilize a4pl. In these experiments an increase in Jurkat cell adhesion of 3-5-fold was observed for IL-1-stimulated EC over unstimulated cells (Fig. 5a). The addition of a4 antibod- ies reduced Jurkat cell adhesion to the IL-1-stimulated EC to background levels observed for non IL-1-treated EC (35 and 26% of the IL-1 stimulated control, respectively). Antibodies to p l also significantly reduced (57% suppression) cell adhe- sion levels, while antibodies specific for a5, ICAM-1, or CD18 had no effect.

Peptide competition experiments were done to determine if RC*D(ThioP)C* could inhibit the binding of lymphocytes to IL-1-activated EC. The peptide was a very potent inhibitor (ICso = 5 p ~ ) of Jurkat-EC adhesion while the scrambled peptide, RC*(ThioP)DC*, was ineffective (Fig. 5b). In this assay system, lymphocytes, EC, or EC-substrate adhesion could potentially be affected by peptide antagonists. To elim- inate the possibility that the apparent loss of Jurkat cell adhesion was due to detachment of EC from the microwells, the cytokine-stimulated EC monolayer was treated with 100 p~ RC*D(ThioP)C* or RC*(ThioP)DC* peptide solutions for 20 min, the time span of our adhesion assay, and then the peptides were removed. The relative number of Jurkat cells that adhered to peptide pretreated EC was equivalent to EC treated with buffer alone (Fig. 5c). Furthermore, visual in- spection of the plates indicated no detachment of the EC monolayer. However, the addition of 100 ~ L M RC*DthioPC* together with Jurkat and ECs reduced lymphocyte adhesion to background or to the level of unstimulated EC (Fig. 5, b and c).

Specificity of RC*D(ThioP)C* Inhibition-Because resting U937 or Jurkat cells preferentially bind the extracellular matrix protein FN and not laminin, vitronectin, or collagen, the MG63 cell line was used to explore the specificity of RC*D(ThioP)C* with other extracellular matrix molecules and to test whether all 01 integrins are inhibited by this peptide. MG63 cells express high levels of av, a3, and a2 integrins (see Table 11) that form receptors for multiple extracellular matrix proteins such as vitronectin, collagen,

20356 RCD Peptide Inhibits a4Pl and a5Pl Integrin Function a

1L.l: 0 1 + f + +

T I

b.

C.

Dcelrealea Unlrealed 91-

u a u u

FIG. 5. Heterotypic lymphocyte-EC adhesion. EC were un- treated (0) or stimulated with IL-1 (+). Fluorescently labeled Jurkat cells were then added in the presence of antibodies ( a ) ICAM-1 (BBA4), 10 pg/ml; CD18 (IB4), 10 pg/ml; a5 (PlD6), diluted 1:lOOO; a4 (HP2/1), 10 pg/ml; and 81 (4B4), 10 pg/ml; or peptides ( b ) RC*D(ThioP)C* (closed circles), or RC*(ThioP)DC* (open circles). Relative mean fluorescence of adherent cells for each well was con- verted to percent of control (IL-1 stimulated, without antibody or peptide treatment) and S.E. ( n = 6). c, prior to the addition of Jurkat cells, IL-1-stimulated EC were incubated with buffer, 100 p~ RC*D(ThioP)C*, or RC*(ThioP)DC* for 20 min, 37 "C. Peptides were removed, followed by cell adhesion as above (Pretreated). Bind- ing of Jurkat cells in the presence of 100 p~ RC*D(ThioP)C* or RC*(ThioP)DC* is also shown (Untreated).

laminin, and including FN (1-3). In contrast to the FN-coated substrates, adhesion of MG63 cells to laminin was not affected by RC*D(ThioP)C*, while some inhibitory activity for colla- gen binding was observed (Fig. 6). The high IC50 value (>1.6 mM), however, would indicate that RC*D(ThioP)C* is not a potent antagonist of collagen binding. Importantly, RC*D(ThioP)C* did not perturb binding to vitronectin, an adhesion mechanism sensitive to RGD inhibition (25). As expected, RC*D(ThioP)C* inhibited adhesion of MG63 cells to FN (IC60 = 50 p ~ ) while scrambled RC*(ThioP)DC* did not (Fig. 6). Binding of the MG63 cells to the FN substrate was inhibited by GRGDSP only at concentrations greater than 1 mg/ml (data not shown). Since MG63 cells have multiple receptors that can interact with the RGD cell adhe- sion site of FN, we cannot rule out the possibility of RC*D(ThioP)C* inhibition of more than one integrin (e.g. cuvfil or 4 3 3 ) by this experiment.

125

IO0

E 75

9 x 50

25

0 2 I O 100 1000

I O , woo Peptide C o n ~ ~ n l r o l i o n (pq/ml)

FIG. 6. MG63 cell adhesion to extracellular matrix pro- teins. Microtiter plates were coated with 5 pg/ml each of FN (closed circles), collagen type I (open triangles), laminin (open squares), or vitronectin (closed diamonds). 2.5 X IO4 cells/well were allowed to adhere to the proteins indicated in the presence of RC*D(ThioP)C*. The relative absorbance obtained for adherent cells in the absence of peptide was defined as 100%.

200-

69-

200- . . I-

91-

R - 69-

FIG. 7. Affinity chromatography on an RC*D(ThioP)C* peptide matrix. Detergent lysates of radioiodinated cells were in- cubated with K(Anc)RC*D(ThioP)C* coupled to Sepharose. Eluted material was analyzed by SDS-PAGE under non-reducing conditions, and fractions were pooled for immunoprecipitation. a, chromatogra- phy of U937 cellular proteins eluted sequentially with 500 pM RC*(ThioP)DC* (lanes 2-6), 500 p~ RC*D(ThioP)C* (lanes 8-16), and 10 mM EDTA (lanes 18-26). The fraction number (1 ml volume) of the eluted material is indicated above the gel lane. b, immunopre- cipitation with a 4 antibody HP2/1 (lanes I , 3, and 51, or a5 antibody P1D6 (lanes 2, 4, and 6). Molecular weight markers are "C-methyl- ated-myosin (200 kDa), -phosphorylase b (97.4 kDa), and -bovine serum albumin (69 kDa).

Affinity Chromatography-A peptide affinity matrix was generated to more directly assess specific integrin binding to RC*D(ThioP)C*. As shown in Fig. la, K(Anc)RC*D(ThioP) C* inhibited adhesion of U937 cells to FN (ICso = 30 pM uersw 5 p~ for RC*D(ThioP)C*). The K(Anc)RC*D(ThioP) C* affinity matrix was incubated with a detergent lysate of radiolabeled U937 cells. Elution of the column with 500 PM of the scrambled peptide, RC * ( T h i o P ) D C * , afforded no significant release of radioactivity (Fig. 7a, lanes 2-6), indi- cating that proteins were bound in a specific manner. Signif- icant levels of protein were eluted by 500 p~ RC*D(ThioP)C* (Fig. 7a, lanes 8-16), while a subsequent elution with 10 mM EDTA released additional material (Fig. 7a, lanes 18-26). The major proteins eluted had molecular mass of -100 kDa and

RCD Peptide Inhibits a4@1 and a5pl Integrin Function 20357

140-155 kDa non-reduced, a characteristic pattern for inte- grins. Additional, as yet unidentified, proteins were observed in the EDTA eluate with molecular mass of 220 and 46 kDa (not visible in figure). Pooled column fractions were subjected to immunoprecipitation to identify the integrins that bound specifically to the column. Material eluted with RC*D(ThioP)C* contained both a4 and a5 subunits, as well as, the pl subunit which coprecipitated (Fig. 7b, lanes 3 and 4 ) . Proteolytic products of the a4 subunit with molecular mass of 70-80 kDa (37, 38) were also immunoprecipitated. Minor levels of a4pl (Fig. 7b, lane 5 ) and trace amounts of a5pl (Fig. 7b, lane 6 ) were eluted from the column with EDTA, while neither a4pl or a5pl were present in the eluate of the scrambled peptide, RC*(ThioP)DC* (Fig. 7b, lanes 1 and 2).

To address whether the a.5/31 and a4B1 integrins could be released from the column independently of each other, elution was done with peptides derived from the RGD- or CS-1- binding domains of FN, respectively. U937 cell extracts were eluted sequentially from the affinity column by 1 mM GRGDSP, 1 mM LHPGEILDVPST, and 1 mM RC*D(Thio P)C*. A distinguishable peak of radioactivity was observed for each peptide elution (data not shown). Immunoprecipita- tion determined that the a5p1 integrin was present in the GRGDSP eluate with trace amounts present in the RC*D(ThioP)C* eluate (Fig. 8, lanes 2 and 6). The majority of bound a4pl eluted with RC*D(ThioP)C*, while a modest amount of a4pl was released by LHPGEILDVPST (Fig. 8, lanes 3 and 5 ) . There were no detectable levels of a4pl in the GRGDSP wash, nor was a501 present in the LHPGEILDVPST eluate (Fig. 8, lanes 1 and 4, respectively).

Since MG63 cells express a variety of p l integrins (see Table 11), these cells were used to address specificity of integrin binding to the RC*D(ThioP)C* peptide. MG63 cell lysate bound to the column and eluted by 500 I.LM GRGDSP, followed by 500 I.LM RC*D(ThioP)C* and 10 mM EDTA, yielded distinct peaks of integrin-like proteins from each solution (data not shown). Immunoprecipitation demon- strated that a5pl was released in the GRGDSP eluate (Fig. 9, lane 2), while a4pl remained bound to the column until elution by RC*D(ThioP)C* and EDTA (Fig. 9, lane 4 and 7, respectively). The molecular weight range of eluted material would preclude the possibility of alp1 binding to the column and immunoprecipitation yielded no detectable a3 (data not shown) or a2 integrins in peptide or EDTA washes (Fig. 9, lanes 3, 6, and 9). Integrins of the av family bound to the RC*D(ThioP)C* column, but only avpl was released by

200-

- m BO-

FIG. 8. Affinity chromatography of U937 cellular proteins. Experiments were done essentially as in Fig. 7. Specifically bound material was eluted sequentially by 1 mM GRGDSP (lanes I and 2 ) , 1 mM LHGPEILDVPST (lanes 3 and 4 ) , and 1 mM RC*D(ThioP)C* (lanes 5 and 6 ) followed by immunoprecipitation with a4 (lanes 1,3, and 5) or a5 antibodies (lanes 2,4, and 6 ) .

100-

ff-

BO-

FIG. 9. Affinity chromatography of MG63 cellular proteins. Experiments were done essentially as in Fig. 7. Specifically bound material was eluted sequentially with 500 p~ GRGDSP (lanes 1-3), 500 p M RC*D(ThioP)C* (lanes 4-6), and 10 mM EDTA (lanes 7-9). Results for immunoprecipitation by antibodies to a4 (lanes I, 4, and 7), a5 (lanes 2 ,5 , and 8), or a2 antibody P1E6 (lanes 3,6, and 9) are shown.

RC*D(ThioP)C*, suggesting a specific interaction, while in- tegrins avp3 and cw@5 were only eluted by EDTA?

DISCUSSION

Activity of a small cyclic peptide that displays limited specificity to receptors of the D l integrin subfamily has been described. The RC*D(ThioP)C* peptide modulates cell adhe- sion to FN in a concentration-dependent manner and is more potent than GRGDSP which is the traditional inhibitor for this type of adhesion mechanism. Moreover, cell adhesion to the 120-kDa chymotryptic fragment of FN, which contains the RGD central cell-binding domain, was also inhibited by RC*D(ThioP)C*. These results suggest that RC*D(ThioP)C* interacts with integrins that utilize an RGD-dependent mech- anism for recognition of FN. Although several integrins are capable of binding to FN in this region (8, 9, 39), RC*D(ThioP)C* most likely acts upon a5pl in our assay, since this integrin is predominantly expressed on both lym- phocytes and monocytes (Table 11), while other FN-binding integrins, excluding a4p1, are less abundant. This conclusion is supported by our data that Jurkat and U937 cell attachment to the 120-kDa FN fragment is completely abolished by treat- ment with a5-specific antibodies (data not shown).

RC*D(ThioP)C* antagonist activity not only modulates cell adhesion to the RGD central cell-binding domain of FN, but also inhibits cell attachment to the CS-1 region of this protein. Only one 81 integrin, a4p1, has been identified as the receptor for the second cell-binding domain of FN (15-17, 36). Jurkat cell adhesion to immobilized CS-1 or RC*D(ThioP)C* pep- tides was a401 dependent and was inhibited by both LHPGEILDVPST and RC*D(ThioP)C* peptides. RC*D(Th ioP)C* was an order of magnitude more potent for each of the two different peptide substrates than were peptides derived from the CS-1 region of FN. This leads us to postulate that the affinity of a4pl is higher for RC*D(ThioP)C* than for CS-1 sequence-containing peptides. Support for this hypoth- esis is evident in affinity chromatography experiments where high concentrations of LHPGEILDVPST elute only a pro- portion of bound a4pl from the RC*D(ThioP)C* peptide column (Fig. 8).

One mechanism for heterotypic cell adhesion between T lymphocytes and EC activated by specific cytokines is a4B1 binding to VCAM-1 (3,36). a4pl-dependent adhesion to EC was confirmed in our experiments by abrogation of cell-cell binding by a4 and 81 antibodies. Inhibition of binding by 81

* D. M. Nowlin, unpublished results.

20358 RCD Peptide Inhibits a4Pl

antibodies was not complete, however, suggesting that a4 may also be complexed to an additional p subunit (40), such as p7 (41). In this assay there was no evidence that the lymphocyte- EC adhesion was attributed to CD18/ICAM-1 or (~5p1/FN interactions. RC*D(ThioP)C* was a very potent inhibitor of the heterotypic cell adhesion which was not due to perturba- tions of the EC monolayer, suggesting that the site of action of RC*D(ThioP)C* is on lymphocyte (~481. Currently, there is no direct evidence that the counter receptor on EC is VCAM-1, yet based on published data, we assume this is the case (16,18-20). Alternatively, a4pl could be interacting with EC-associated FN (42,43) or other unidentified ligands (44). Studies are underway to address this more definitively.

More direct evidence that RC*D(ThioP)C* binds to both a4p1 and a581 integrins was established by affinity chroma- tography with immobilized peptide. Both receptors bound to the column and could be eluted by RC*D(ThioP)C* or by peptides taken from the sequence of each integrins' native ligand. These data suggest that for each integrin, binding of the native ligand is to a region of the protein that overlaps with, or is identical to the site of RC*D(ThioP)C* binding, although we cannot rule out the possibility of allosteric effects. Epitope mapping with 04-specific monoclonal antibodies lends support for this hypothesis because VCAM-1 and FN bind to a4pl at distinct, yet overlapping sites on the integrin (45). Our peptide may bind within this hypothetical region, thus inhibiting both FN and VCAM-1 interactions. It is not known whether the RC*D(ThioP)C* peptide would also in- hibit a4-dependent cytolytic T lymphocyte/B lymphocyte target interactions (46, 47), however, preliminary evidence* indicates that the peptide abrogates a4-dependent homotypic aggregation (45, 48, 49).

The affinity of RC*D(ThioP)C* for either a5pl or a4pl cannot be assessed quantitatively by our experimental meth- ods. Initial analyses would suggest that the peptide preferen- tially binds to ~ ~ 4 8 1 , since only this integrin was found to mediate cell attachment to biotinyl(Anc)RC*D(ThioP)C* coated onto microtiter plates. This is consistent with previous reports that a581 does not bind small RGD-containing pep- tide substrates (50). However, a significant level of a5pl in detergent micelles bound to the RC*D(ThioP)C* affinity col- umn. To our knowledge, this is the first report of a cyclic non- RGD peptide antagonist for the a581 receptor. Additionally, this is the first demonstration that a5Pl can be purified by affinity chromatography using a small immobilized peptide.

Cell adhesion to laminin or vitronectin was not inhibited by RC*D(ThioP)C*, and adhesion to collagen type I was modestly effected, suggesting that not all Dl-mediated adhe- sion events are modulated by this peptide. Integrins of the 83 and p5 subfamily and some pl integrins bind to their sub- strates through RGD-dependent mechanisms. However, RC*D(ThioP)C* may not be an RGD peptide mimic based on several observations. First, RGD peptides are potent inhibi- tors of vitronectin adhesion (25), yet RC*D(ThioP)C* did not antagonize cell adhesion to this substrate. Second, a conserv- ative substitution of aspartic acid to glutamic acid in the RGD series of peptides results in a loss of RGD-dependent binding activity (8), while this same substitution, RC*E(ThioP)C*, does not affect the potency of our peptide for a5pl-FN- or a4pl-mediated adhesion events3 Finally, short RGD peptides are not recognized by the a5pl receptor when immobilized to an affinity matrix (lo), while an affinity matrix of RC*D(ThioP)C* can be used to purify this integrin.

One might speculate that RC*D(ThioP)C* mimics the LDV

3 D . M. Nowlin, F. Gorscan, M. Moscinski, S.-L. Chiang, T. J. Lobl, and P. M. Cardarelli, unpublished results.

and a5pl Integrin Function

epitope required for a4pl binding to FN, but this does not explain why a581 binding to FN is also affected by low concentrations of RC*D(ThioP)C*. Furthermore, LDV-con- taining peptides do not inhibit adhesion of cells to the 120- kDa fragment of FN (data not shown). Studies show that GRGDS and GRGES inhibited melanoma cell adhesion to CS-1 peptides (42), and it is therefore possible that RC*D(ThioP)C* disrupts a481 adhesion to CS-1 peptides by an analogous mechanism. However, GRGDSPC did not alter lymphocyte adhesion to FN fragments containing the CS-1 domain (51), and GRGDSP was unable to elute a4pl from the RC*D(ThioP)C* affinity column (Figs. 8 and 9). The multiple inhibitory activities observed for RC*D(ThioP)C* suggest that the peptide-binding site in a481 and a5pl defines a region of shared homology or topography between these two integrins. It is conceivable that RC*D(ThioP)C* binds to integrins at multiple contact sites as previously postulated (52,53). Further probing of the RC*D(ThioP)C*-binding site may provide the information required to support this hypoth- esis.

A large number of proteins contain the sequence RCD. Of interest are two RCD sequences found in the fibrin I1 site of FN (54-61), however, there is no evidence that this region promotes cell adhesion in intact FN. Also of interest, six of the eight integrin p subunits contain the RCD sequence in the sixth conserved cysteine position (62), and the RCD sequence is found adjacent to the YIGSR sequence in the laminin B1 chain. The probability of finding the same 3 amino acid sequence in several proteins with related function may be statistically probable. However, the expression of the RCD sequence in a conserved region suggests functional utility and warrants further investigation.

In summary, a novel peptide has been identified that dis- plays limited specificity to receptors of the 81 integrin subfam- ily. The antagonist, RC*D(ThioP)C*, modulates a5pl func- tion by binding to a5pl receptors, thus preventing interaction with the RGD sequence of FN. In addition, RC*D(ThioP)C* inhibits multiple a4pl functions, specifically, (~481-mediated adhesion to the CS-1 domain of FN, and a4pl-dependent T- lymphocyte adhesion to cytokine-stimulated EC. Affinity chromatography data demonstrates direct interaction of this peptide with both a4pl and a5pl integrins. The dual activity profile of RC*D(ThioP)C* is unique and could have important therapeutic application in treating inflammatory diseases.

Acknowledgments-We thank Drs. N. Orida, D. Thueson, Y. Iwa- sawa, and G. Yakatan for comments and critical review of this manuscript and acknowledge Dr. W. Scholz for flow cytometric analysis of cells. We also express our gratitude toward Tanabe Sei- yaku Co., Ltd, Osaka, Japan for continued support in this area.

REFERENCES

1. Springer, T. A. (1990) Nature 346,425-434 2. Shimizu, Y., Van Seventer, G. A., Horgan, K. J., and Shaw, S. (1990)

Immunol. Reu. 114.109-143 3. Hemler, M. E. (1990) Annu. Reu. Immunul. 8,365-400 4. Wayner, E. A,, and Carter, W. G. (1987) J. Cell Biol. 105,1873-1884 5. Kramer, R. H., and Marks, N. (1989) J. Biol. Chem. 264,4684-4688 6. Gehlsen, K. R., Dillner, L., Engvall, E., and Ruoslahti, E. (1988) Science

7. Wayner, E. A,, Garcia-Pardo, A,, Humphries, M. J., McDonald, J. A., and

8. Pierschbacher, M. D., and Ruoslahtl, E. (1984) Proc. Natl. Acad. Sei.

9. Pierschbacher, M. D., and Ruoslahti, E. (1984) Nature 309,30-33

2 4 , 1228-1229

Carter, W. G. (1989) J. Cell Biol. 109,1321-1330

U. S. A. 8 1 , 5985-5988

10. Ruoslahti, E., and Pierschhacher, M. D. (1987) Science 238,491-497 11. Pytela, R., Pierschbacher, M. D., Ginsberg, M. H., Plow, E. F., and Ruos-

12. Vogel, B. E., Tarone, G., Giancotti, F. G., Gailit, J., and Ruoslahti, E.

13. El?:!, M. J., Urry, L. A,, and Hemler, M. E. (1991) J. Cell Bid. 112,169-

lahti, E. (1986) Science 2 3 1 , 1559-1562

(1990) J. Biol. Chem. 265,5934-5937

14. Pierschbacher, M. D., and Ruoslahti, E. (1987) J. Biol. Chem. 262,17294-

15. Guan, J.-L., and Hynes, R. 0. (1990) Cell 60,53-61

101

17298

RCD Peptide Inhibits a4Pl and a5Pl Integrin Function 20359 16.

17. 18.

19.

20.

21. 22.

23. 24.

25.

26.

27.

28.

29. 30.

31. 32. 33.

34.

35.

36.

37.

38.

39.

Garcia-Pardo, A., Wayner, E. A., Carter, W. G., and Ferreira, 0. C. (1990)

Wayner, E. A., and Kovach, N. L. (1992) J. Cell Biol. 116,489-497 Osborn, L., Hession, C., Tizard, R., Vassallo, C., Luhowskyj, S., Chi-Rosso,

Bevilacqua, M. P., Pober, J. S., Mendrick, D. L., Contran, R. S., and G., and Lobb, R. (1989) Cell 59, 1203-1211

Masinovsky, B., Urdal, D., and Gallatin, M. (1990) J. Immunol. 145,2886- Gimbrone, M. A. (1987) Proc. Natl. Acad. SCL. U. S. A. 84,9238-9242

Harlan, J. M. (1985) Blood 3,513-525 2895

Haverstick. D. M.. Cowan. J. F.. Yamada. K. M.. and Santoro. S. A. (1985)

J. Immunol. 144,3361-3366

Blood 66,946-952 . . , ,

Gartner, T. K., and Bennett, J. S. (1985) J. Biol. Chem. 260,11891-11894 Plow. E. F.. Pierschbacher. M. D.. Ruoslahti. E.. Maraerie. G.. and

Pytela, R., Pierschbacher, M. D., and Ruoslahti, E. (1985) Proc. Natl. Acad. Ginsberg, 'M. H. (1985) Proc. Natl..Acad. Sci. i/. S: A. 85,8057-8061

Scr U. S. A. 82,5766-5770 Cardarelli, P. M.,-Yamagata, S., Taguchi, I., Gorscan, F., Chiang, S., and

Lobl, T. (1992) J. Biol. Chem. 267,23159-23164 Sarnanen, J., Ali, F., Romoff, T., Calvo, R., Sorenson, E., Vasko, J., Storer,

B., Berry, D., Bennett, D., Strohsacker, M., Powers, D., Stadel, J., and

Kirchhofer, D., Gailit, J., Ruoslahti, E., Grzesiak, J., and Pierschbacher, Nichols, A. (1991) J. Med. Chem. 34,3114-3125

Engvall, E., and Ruoslahti, E. (1977) Znt. J. Cancer 20, 1-5 M. D. (1990) J. Biol. Chem. 265, 18525-18530

Stewart, J. M.,, and Young, J. D. (1984) Solid Phase Peptide Synthesis,

von E. Kamber (1971) Helu. Chim. ACTA 94. 927-930 Pierce Chemlcal Company, Rockford, IL

Gimbrone, M. A., Jr. (1976) Prog. Hemostask.Thromb. 3, 1-28 Cardarelli, P. M., and Pierschbacher, M. P. (1986) Proc. Natl. Acad. Sci.

Pierschbacher, M. D., Hayman, E. G., and Ruoslahti, E. (1983) Proc. Natl. U. S. A. 83,2647-2651

Acad. Sci. U. S. A. 80.1224-1227 Bevilacqua, M. P., Prober, J. S., Wheeler, M. E., Cotran, R. S., and

Elices, M. J., Osborn, L., Takada, Y., Crouse, C., Luhowskyl, S., Hemler,

Hernler, M. E., Huang, C., Takada, Y., Schwarz, L., Strominger, J. L., and

Teixido, J., Parker, C. M., Kassner, P. D., and Hemler, M. E. (1992) J.

Yamada, K. M., and Kennedy, D. W. (1984) J. Cell Biol. 99,29-36

Gimbrone, M. A., Jr. (1985) J. Clin. Inuest. 76, 2003-2011

M. E., and Lobb, R. R. (1990) Cell 60,577-584

Clabby, M. L. (1987) J. Biol. Chem. 262, 11478-11485

Biol. Chem. 267,1786-1791

40. Holzmann, B., and Weissman, I. L. (1989) EMBO J. 8, 1735-1741 41. Chan, B. M. C, Elices, M. J., Murphy, E., and Hemler, M. E. (1992) J. Biol.

42. Szekanecz, Z., Humphries, M. J., and Ager, A. (1992) J. Cell Sci. 101,885-

43. Ager, A,, and Humphries, M. J. (1990) Int. Immunol. 2, 921-928 44. Vonderheide, R. H., and Springer, T. A. (1992) J. Exp. Med. 175, 1433-

Chem. 267,8366-8370

894

1442 45. Pulido, R., Elices, M. J., Campanero, M. R., Osborn L. Schiffer, S. Garcia-

Pardo, A,, Lobb, R., Hemler, M. E., and Sanchkz-Madrid, F. ('1991) J.

46. Takada, Y., Elices, M. J., Crouse, C., and Hemler, M. E. (1989) EMBO J. Biol. Chem. 266,10241-10245

47. Clayberger, C., Krensky, A. M., McIntyre, B. W., Koller, T. S., Parham, F., Brodsky, F., Linn, D. J., and Evans, E. L. (1989) J . Immunol. 138,1510-

8, 1361-1368

1514 48. Bezyrczyk, J. L., and McIntyre, B. W. (1990) J. Imrnurzol. 144,777-784 49. Campanero, M. R., Pulido, R., Ursa, M. A., Rodriguez-Moya, M., de

Landkuri, M. O., and Sanchez-Madrid, F. (1990) J. CellBiol. 110,2157- 2165

50. Pytela, R., Pierschbacher, M. D., and Ruoslahti, E. (1985) Cell 40, 191- 198

52. D'Souza, S. E., Ginsberg, M. H., Matsueda, G. R., a n f Plow, E. F. (1991) 51. Garcia-Pardo, A., and Ferreira, 0. C. (1990) Imrnunolo y 69, 121-126

53. D'Souza, S. E., Ginsberg, M. H., and Plow, E. F. (1991) Trends Biochem. Nature 350,66-68

Sei l d 7AG-35n 54. Argraves, W. S., Suzuki, S. Arai H., Thompson K. Pierschbacher, M. D.,

55. Kishimoto, T. K., O'Connor, K., Lee, A., Roberts, T. M., and Springer, T.

56. Fitzgerald, L. A., Steiner, B., Rall, S. C. J., Lo, S. S., and Phillips, D. R.

57. Suzuki, S., and Naitoh, Y. (1990) EMBO J. 9, 757-763

59. Sheppard, D., Rozzo, C., Starr, L., Quaranta, V., Erle, D. J., and Pytela, R. 58. Ramaswamy, H., and Hemler, M. E. (1990) EMBO J. 9, 1561-1568

60. Erle, D. J., Ruegg, C., Sheppard, D., and Pytela, R. (1991) J. Biol. Chem.

61. Moyle, M., Napier, M. A., and McLean, J. W. (1991) J. Biol. Chem. 266,

62. Sasaki, M., Kato, S., Kohno, K., Martin, G. R., and Yamada, Y. (1987)

", I" -" and Ruoslahti, E. (1987)'J. Ceh Biol. 105, lIi3-il90

A. (1987) Cell 48,681-690

(1987) J. Biol. Chem. 262,3936-3939

(1990) J. Biol. Chem. 265, 11502-11507

266, 11009-11016

19650-19658

Proc. Natl. Acad. Sci. U. S. A. 84,935-939