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Originally published In Press as doi:10.1074/jbc.M001228200 on June 2, 2000

J. Biol. Chem., Vol. 275, Issue 33, 25652-25664, August 18, 2000
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The Role of alpha  and beta  Chains in Ligand Recognition by beta 7 Integrins*

Jonathan M. G. HigginsDagger §, Manuela CernadasDagger , Kemin Tan||, Atsushi Irie**, Jia-huai Wang||, Yoshikazu Takada**, and Michael B. BrennerDagger

From the Dagger  Lymphocyte Biology Section, Division of Rheumatology, Immunology, and Allergy, and the  Pulmonary and Critical Care Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School and the || Laboratory of Immunobiology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115 and the ** Department of Vascular Biology, Scripps Research Institute, La Jolla, California 92037

Received for publication, February 14, 2000, and in revised form, May 8, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Integrins alpha Ebeta 7 and alpha 4beta 7 are involved in localization of leukocytes at mucosal sites. Although both alpha Ebeta 7 and alpha 4beta 7 utilize the beta 7 chain, they have distinct binding specificities for E-cadherin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1), respectively. We found that mutation of the metal ion-dependent adhesion site (MIDAS) in the alpha E A-domain (D190A) abolished E-cadherin binding, as did mutation F298A on the A-domain surface near the MIDAS cleft. A docking model of the A-domain with E-cadherin domain 1 indicates that coordination of the alpha E MIDAS metal ion by E-cadherin Glu31 and a novel projection of Phe298 into a hydrophobic pocket on E-cadherin provide the basis for the interaction. The location of the binding site on the alpha E A-domain resembles that on other integrins, but its structure appears distinctive and particularly adapted to recognize the tip of E-cadherin, a unique integrin ligand. Additionally, mutation of the beta 7 MIDAS motif (D140A) abolished alpha Ebeta 7 binding to E-cadherin and alpha 4beta 7-mediated adhesion to MAdCAM-1, and alpha 4 chain mutations that abrogated binding of alpha 4beta 1 to vascular cell adhesion molecule-1 and fibronectin similarly reduced alpha 4beta 7 interaction with MAdCAM-1. Thus, although specificity can be determined by the integrin alpha  or beta  chain, common structural features of both subunits are required for recognition of dissimilar ligands.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Integrins are heterodimeric glycoproteins consisting of noncovalently associated alpha  and beta  subunits that play diverse roles in cell-cell and cell-matrix interactions. Integrins of the beta 1, beta 2, and beta 7 subfamilies are critical for leukocyte homing (1, 2). For example, binding of integrin alpha 4beta 7 to mucosal addressin cell adhesion molecule-1 (MAdCAM-1)1 on intestinal endothelial cells is required for the homing of naive lymphocytes to Peyer's patches (2). Integrins are also thought to play a role in microenvironmental homing or retention of lymphocytes at particular sites within a tissue (2). For instance, integrin alpha Ebeta 7 binds to epithelial cadherin (E-cadherin) and is involved in retention of lymphocytes within the epithelium (3-6).

Many integrin ligands are members of the immunoglobulin superfamily (such as VCAM-1 and MAdCAM-1) or possess domains that resemble the immunoglobulin fold (e.g. E-cadherin and the type III repeats of fibronectin). A unifying feature of these ligands is an integrin-binding surface containing an exposed acidic amino acid. For example, alpha 4beta 1 and alpha 4beta 7 bind to VCAM-1 and MAdCAM-1, respectively, on the face of domain 1 formed by the C, F, and G beta -strands, centered on an aspartate residue in the loop connecting the C and D strands (7-9). Domain 1 of E-cadherin contains a glutamate residue at the tip of the BC-loop essential for alpha Ebeta 7 binding (10, 11), and the tenth type III repeat of fibronectin has an acidic residue within an RGD sequence on the FG-loop required for alpha 5beta 1 binding (12). Other integrin ligands such as collagen I and iC3b that are not known to adopt immunoglobulin-like folds also possess vital acidic residues for alpha 2beta 1 and alpha Mbeta 2 binding, respectively (13, 14).

Approximately one-third of known alpha  chains (alpha 1, alpha 2, alpha 10, alpha 11, alpha D, alpha E, alpha L, alpha M, and alpha X) contain an inserted domain of ~200 amino acids related to the A-domains of von Willebrand factor. For those integrins tested, the A-domain is critically involved in ligand binding. For instance, isolated recombinant alpha 2, alpha L, alpha M, and alpha D A-domains share ligand binding properties with their parent integrins (15-18). The crystal structures of a number of integrin A-domains (alpha 1, alpha 2, alpha L, and alpha M) reveal a common "Rossmann fold" in which a six-stranded parallel beta -sheet is flanked by seven alpha -helices (19-22). Five conserved amino acids (DXSXS, T, and D) come together with two or three water molecules at the top of the domain to chelate a metal ion in what has been termed the metal ion-dependent adhesion site (MIDAS). Mutation of these residues abolishes metal binding to the alpha M A-domain (23) and severely curtails or eliminates ligand binding activity for all integrins tested (23-28). In one crystal form of the alpha M A-domain, a glutamate residue from a neighboring A-domain penetrates the MIDAS cleft and provides a sixth coordination site for the metal ion (19, 29). This "open" conformation of the A-domain is proposed to reflect the active ligand-bound conformation. Because most integrin ligands possess a critical exposed acidic residue, bona fide ligand recognition may use a similar mechanism (19). Crystal structures in which no ligand mimetic is bound in the MIDAS cleft have a "closed" conformation that likely reflects the inactive form of the A-domain (29, 30). Comparison of the unbound alpha 2 A-domain with the recently crystallized complex of this A-domain bound to a collagenous triple peptide confirms the model (31). This complex and mutational analyses of the alpha 2, alpha L, and alpha M integrins have revealed other residues on the upper surface of the A-domains surrounding the MIDAS cleft that contribute to the affinity and specificity of ligand binding (26, 29, 32-34).

This mechanism provides an elegant explanation of ligand binding by A-domain-containing integrins. However, most integrins do not possess an A-domain in the alpha  chain, yet they are also dependent upon acidic residues in their ligands and have a similar requirement for divalent cations. A potential explanation is the proposal that all integrin beta  chains have a region that adopts an A-domain-like fold (19, 35, 36). Indeed, all beta  chains possess a relatively conserved region of ~250 amino acids that contains a DXSXS sequence, which, together with conserved downstream oxygenated residues, could form a complete MIDAS site. In fact, mutation of such residues in beta 1, beta 2, beta 3, beta 5, and beta 6 integrins abolishes ligand binding activity (35, 37-45). However, it remains likely that non-A-domain-containing integrin alpha  chains also contribute directly to ligand binding. The seven repeats present in the N-terminal region of all integrin alpha  chains are proposed to fold into a "beta -propeller" of seven four-stranded beta -sheets (46), and mutations in the alpha 4 chain on the upper surface of the predicted second, third, and fourth blades of the propeller, for example, diminish the binding of alpha 4beta 1 to VCAM-1 (47-49). An alternative model proposes that this region of the alpha  chain forms "EF-hand-type" domains, but also postulates a role in ligand binding (50).

Here, we analyze the role of alpha  and beta  chains in the ligand binding activity of the beta 7 integrins alpha Ebeta 7 and alpha 4beta 7. This is important for a number of reasons. First, the ligand for alpha Ebeta 7, E-cadherin, is a unique integrin counter-receptor. Although a critical glutamate residue (Glu31) required for integrin binding has been identified, its structural context is unlike that of other ligands of integrins with alpha  chain A-domains (10, 11). Such integrins bind to counter-receptors that contain critical acidic residues presented on relatively flat surfaces (e.g. beta 2 integrin ligands ICAM-1 and -2) or on collagenous structures (9, 31). However, Glu31 in E-cadherin is on a highly exposed loop more akin to similar loops containing critical acidic residues in VCAM-1, MAdCAM-1, and fibronectin that bind to integrins lacking an alpha  chain A-domain (11). Also, alpha E has an "X"-domain of 55 amino acids immediately N-terminal to the A-domain that has 18 charged residues within a stretch of 21 amino acids and contains a post-translational cleavage site (see Fig. 1A) (51). For these reasons, there exists doubt as to the role of the A-domain in E-cadherin binding. Therefore, it is of great interest to determine if the alpha E A-domain is involved and what role the X-domain plays. Second, the beta 7 chain must be important in defining binding specificity since alpha 4beta 7 binds to MAdCAM-1, whereas alpha 4beta 1 does not (52-54); yet alpha Ebeta 7 and alpha 4beta 7 both utilize the beta 7 chain, but they have non-overlapping ligand binding specificities for E-cadherin and MAdCAM-1, respectively (54).2 Also, in a number of animal models of colitis and graft-versus-host disease, the administration of antibodies to either alpha 4beta 7 or alpha Ebeta 7 ameliorates the development of intestinal inflammation (55-58), and alpha Ebeta 7 potentially has a role in rejection of epithelial tissue within allografts (59). Therefore, there is considerable interest in the structural basis for ligand recognition by these integrins because of their important role in leukocyte trafficking and to aid the design of therapeutic small molecule inhibitors.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- DNA-manipulating enzymes were from New England Biolabs Inc. (Beverly, MA); oligonucleotides were from Integrated DNA Technologies Inc. (Coralville, IA); and other chemicals from Sigma. Purified human E-cadherin-Fc fusion protein was described previously (5); purified human MAdCAM-1-Ig (60) was kindly provided by Dr. Michael Briskin (Millennium Pharmaceuticals Inc., Cambridge, MA); and purified human IgG1 was from Calbiochem. MAdCAM-1-transfected L1-2 cells were provided by Dr. Eugene Butcher (Stanford University).

Monoclonal Antibodies-- The mAbs used (all murine anti-human unless otherwise stated) were as follows: HML-1 (anti-alpha Ebeta 7, IgG2a) (61); 2G5 (anti-alpha Ebeta 7, IgG2a; Immunotech, Marseilles, France); BerACT-8 (anti-alpha Ebeta 7, IgG1) (62); alpha E7-1 (anti-alpha Ebeta 7, IgG2a) and alpha E7-2 and alpha E7-3 (anti-alpha Ebeta 7, IgG1) (63); LF61 (anti-alpha Ebeta 7, IgG1; Caltag Laboratories, Burlingame, CA) (64); ABB1.3 (anti-alpha Ebeta 7, IgG1; Immune Source, Los Altos, CA); Fib21, Fib30, and Fib504 (all anti-mouse and human beta 7, rat IgG2a) (65); B5E2 and B5G10 (anti-alpha 4, IgG1) (66); HP1/2 (anti-alpha 4, IgG1) (67); ACT-1 (anti-alpha 4beta 7, IgG1) (68); IV.3 (anti-Fcgamma receptor II/CD32, IgG2b; Medarex Inc., Annandale, NJ); MOPC21 (IgG1; Sigma); UPC10 (IgG2a; Sigma); and Y13.238 (anti-p21Ras, rat IgG2a; American Type Culture Collection, Manassas, VA).

Construction of alpha 4, alpha E, and beta 7 Expression Vectors-- Human alpha 4 cDNA, kindly provided by Dr. Martin Hemler (Dana-Farber Cancer Institute) (69), was inserted into the expression vector pAPRM8 (70) cleaved with SalI and NotI. Mutant forms of human alpha 4 in the vector pBJ-1 were described previously (48, 49). Wild-type alpha E and beta 7 cDNAs (5) were cloned into the HindIII and EcoRI sites of pAPRM8, respectively. To generate point mutations and deletions (see Fig. 1A), PCR amplification with mutagenic primers was used. Note that for the alpha E and beta 7 chains, residue 1 is defined as the first amino acid in the mature protein determined by peptide sequencing (51, 71, 72). For alpha E(Delta Glu163-Glu180), the primers 5'-AAGACCCCAGCACCAACCAGACCT-3' and 5'-TTTTTTGATGGCAATCTCGGTGCCAGCCAGAGCCCGGCGCTGCCTGG-3' were used for PCR; for alpha E(D190A), primers 5'-GGGGGGGATTGCCATCATCCTGGCTGGCTCAGGAA-3' and 5'-CTGCGAGGGGCTGGCGGAGAG-3'; for alpha E(G193A), primers 5'-GGGGGGGATTGCCATCATCCTGGATGGCTCAGCAAGCATTGAT-3' and 5'-CCCGGATCCTCAAAGGGCGTCTCCAAC-3'; for alpha E(Y354W), primers 5'-AATTTTGCGGCCGCGATATCCTGGCTGAGGGGAAGCTGAGTG-3' and 5'-GGGGGGGCTCAGCAGCCCATCCAGCGCCATCCAGTTGGTCAC-3'; and for beta 7(D140A), primers 5'-TGTTTCCTGTTTGGCTCTTCTACC-3' and 5'-GGGGGACGCGTTCCAGGTCGTCCTTCATGGAGTAGCTCAGGGCCATAAGGTA-3'. For the remaining mutations, a two-step PCR procedure was carried out using primers 1 and 2 and primers 3 and 4 in the initial amplification and primers 1 and 4 in the second round. In all cases, primers 1 and 4 were 5'-AATTTTGCGGCCGCGATATCCTGGCTGAGGGGAAGCTGAGTG-3' and 5'-CCCGGATCCTCAAAGGGCGTCTCCAAC-3', respectively. Primers 2 and 3 were as follows, respectively: for alpha E(R159S/R160S), 5'-CTGCCTGGCTGTGTTCACATC-3' and 5'-GTGAACACAGCCAGGCAGTCCTCGGCTCTGGAG-3'; for alpha E(D199A), 5'-TGGGGGATCAATGCTTCCTGA-3' and 5'-GGAAGCATTGATCCCCCAGCCTTTCAGAGA-3'; for alpha E(R202A/D205A), 5'-TGGGGGATCAATGCTTCCTGA-3' and 5'-GGAAGCATTGATCCCCCAGACTTTCAGGCAGCCAAAGCCTTCATCTCC-3'; for alpha E(G230A/V231A), 5'-TCCATACTGCACCAAGGCAAA-3' and 5'-GCCTTGGTGCAGTATGGAGCAGCGATCCAGACT-3'; for alpha E(D240A), 5'-CCGAAGGTCAAACTCAGTCTG-3' and 5'-ACTGAGTTTGACCTTCGGGCCGCCAGCCAGGAT-3'; for alpha E(F298A), 5'-TATGCCACCATCGGTGAGCAC-3' and 5'-CTCACCGATGGTGGCATAGCCGAGGACCCCCTC-3'; for alpha E(E325A), 5'-TCCCACCCCAATGGCAAAGCG-3' and 5'-TTTGCCATTGGGGTGGGAGCAGAATTTAAG-3'; and for alpha E(E345A/T346A), 5'-ATCCGGGTCTGAGGCGATCAG-3' and 5'-ATCGCCTCAGACCCGGATGCGGCCCATGCTTTC-3'. The mutations alpha E(Delta Glu176) and alpha E(P311H) were introduced inadvertently during PCR. Mutated alpha E PCR products were cleaved with PmlI plus BsaBI (for R159S/R160S, Delta Glu176, and Delta Glu163-Glu180), with BsaBI plus Eco47III (for D190A, G193A, D199A, R202A/D205A, G230A/V231A, D240A, and F298A), or with BlpI (for E325A, P311H/E345A/T346A, and Y354W) and inserted into similarly cleaved pAPRM8/alpha E. The beta 7(D140A) PCR product cleaved with BlpI plus MluI was inserted into similarly cleaved pAPRM8/beta 7. All regions generated by PCR were verified by automated DNA sequencing.

Generation of Transfected K562 and CHO Cell Lines-- K562 cells were maintained as described (11) and express no endogenous alpha E or beta 7 chains detectable by flow cytometry or immunoprecipitation (data not shown). K562 cells were transfected by electroporation with 10 µg of pAPRM8/alpha E or pAPRM8/alpha 4, 10 µg of pAPRM8/beta 7, and 1 µg of pGKpuro vectors (73). After selection in 2.5 µg/ml puromycin for 2 weeks, single cells expressing alpha Ebeta 7 or alpha 4beta 7 as determined by staining with anti-alpha Ebeta 7 or anti-alpha 4 mAbs, respectively, were obtained using an EPICS Elite ESP flow cytometer equipped with the Autoclone module (Coulter Corp., Miami, FL). For alpha Ebeta 7 transfectants, mAb alpha E7-1 was used, except for the mutants alpha E(Delta Glu163-Glu180)beta 7 and alpha E(G230A/V231A)beta 7, which did not stain strongly with this mAb, for which BerACT-8 was used. For alpha 4beta 7 transfectants, mAb B5E2 was used. K562 cells stably transfected with 1 µg of pGKpuro vector alone (K562 control cells) were used as controls. CHO cells were transfected with pBJ-1/alpha 4 and pBJ-1/beta 7, selected, and maintained essentially as described previously (48, 49).

Cell-surface Labeling and Immunoprecipitation-- K562 or CHO transfectants were suspended at 107 cells/ml in phosphate-buffered saline with 0.2 mg/ml EZ-Link sulfosuccinimidobiotin (Pierce) and incubated for 30 min at 25 °C. Alternatively, K562 cells were labeled using Na125I as described (5). After washing, cells were lysed in TBS with 0.5% Triton X-100, 1 mM MgCl2, 1 mM CaCl2, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 µg/ml antipain, 1 µg/ml chymostatin, and 1 mM phenylmethylsulfonyl fluoride for 1 h at 4 °C. Clarified lysates were precleared with 0.4% (v/v) normal rabbit serum, 25% mAb IV.3 hybridoma supernatant, and 1.5% (v/v) protein G-Sepharose (Amersham Pharmacia Biotech) for 16 h at 4 °C. Subsequently, aliquots were incubated with monoclonal antibodies for 1.5 h at 4 °C. Then, 10 µl of protein G-Sepharose resin was added, and the incubation at 4 °C was continued for a further 1.5 h. Immobilized complexes were washed six times with TBS and 0.1% Triton X-100, 1 mM MgCl2, and 1 mM CaCl2.

SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting-- SDS-polyacrylamide gel electrophoresis and visualization of radiolabeled proteins were carried out as described (5). Immunoblotting onto polyvinylidene difluoride membrane (Millipore Corp.) was carried out as described (74). Blots were blocked with 1% BSA, phosphate-buffered saline, and 0.04% sodium azide for 16 h at 25 °C; probed with 40 ng/ml horseradish peroxidase-conjugated streptavidin (Pierce) in 0.1% Tween 20 and TBS (pH 7.4) for 1 h at 25 °C; and after washing, visualized with Chemiluminescent Reagent Plus (NEN Life Science Products). Densitometry was conducted using Scion Image software (Scion Corp., Frederick, MD).

Adhesion Assays-- Adhesion assays were carried out as described previously (5). Briefly, 96-well microtiter plates were coated with goat anti-human IgG polyclonal antibody and blocked with 1% BSA in TBS. Wells were then coated with human IgG1 Fc-containing proteins in 50 µl of TBS and 1 mM CaCl2 and blocked with 1% BSA, 2% human serum, and 1 mM CaCl2 in Hepes-buffered saline. Alternatively, transfected CHO cells were grown to near confluence in 96-well tissue culture plates. Transfected L1-2 cells, CHO cells (released with 0.5 mM EDTA in phosphate-buffered saline), or K562 cells were labeled with calcein/AM (Molecular Probes, Inc., Eugene, OR), and adhesion assays were carried out at 37 °C in Hepes-buffered saline with 0.1% BSA and 1 mM each MnCl2, MgCl2, and CaCl2 for 15 min (K562 cells) or 1 mM each MgCl2 and CaCl2 (15 min for CHO cells and 60 min for L1-2 cells). To correct for variations in integrin expression level between clones, we established that the percent adhesion seen with a panel of K562 clones was linearly dependent on the mean fluorescence intensity (MFI) of wild-type alpha Ebeta 7 on the surface determined by flow cytometry with anti-beta 7 mAb Fib504 (correlation coefficient = 0.91). The adhesion of each mutant clone was determined as a percentage of that seen for a clone expressing wild-type alpha Ebeta 7 at a comparable level (±50% wild-type MFI). Then, % wild-type adhesion = 100 × (% adhesion of test cells/MFI on test cells)/(% adhesion of K562-alpha Ebeta 7 WT/MFI on K562-alpha Ebeta 7 WT).

Biotinylation of E-cadherin-Fc-- Purified E-cadherin-Fc or human IgG1 at an equal concentration was dialyzed into 10 mM sodium borate with 0.5 mM CaCl2 (pH 8.4). After incubation with a 33-fold molar excess of aminohexanoylbiotin-N-hydroxysuccinimide ester (Zymed Laboratories Inc., South San Francisco, CA) for 50 min at 25 °C, the biotinylated protein was dialyzed into TBS with 1 mM CaCl2 (pH 7.4).

Flow Cytometry of Monoclonal Antibody and E-cadherin-Fc Binding to Transfectants-- K562 or CHO cells were incubated in staining buffer (Hepes-buffered saline with 1% BSA, 2% human serum, and 0.04% sodium azide) at 2 × 106 cells/ml with primary antibodies at 10 µg/ml (for MOPC21, ABB1.3, ACT-1, HP1/2, LF61, UPC10, 2G5, Y13.238, Fib21, Fib30, and Fib504) or a 1:200 dilution of ascites (for alpha E7-1, alpha E7-2, alpha E7-3, BerACT-8, HML-1, B5E2, and B5G10) for 1 h at 4 °C. After washing three times with staining buffer, the cells were incubated with a 1:50 dilution in staining buffer of fluorescein isothiocyanate-conjugated goat (Fab')2 anti-murine or anti-rat IgG and IgM (BIOSOURCE International) as appropriate for 1 h at 4 °C. Data was collected for 10,000 cells that excluded propidium iodide for each antibody/K562 clone combination using a FACSort flow cytometer (Becton Dickinson). Analysis of E-cadherin-Fc binding to K562 transfectants was carried out similarly, except that K562 cells at 106 cells/ml were incubated in staining buffer with 1 mM each MnCl2, MgCl2, and CaCl2 containing 100 µg/ml biotinylated E-cadherin-Fc or biotinylated human IgG1. The secondary reagent was 20 µg/ml neutravidin-conjugated Alexa 488 (Molecular Probes, Inc.). To correct the mAb or Fc fusion binding data for variations in integrin expression level between clones, binding was calculated relative to that of an anti-beta 7 reference mAb (Fib504) according to the following equation: % wild-type binding = 100 × ((a - b)/(c - d))/((e - f)/(g - h)), where a = MFI of test reagent on test K562 clone, b = MFI of isotype-matched negative control on test K562 clone, c = MFI of Fib504 on test cell, d = MFI of negative control mAb Y13.238 on test cell, e = MFI of test reagent on reference K562-WT transfectant cell, f = MFI of isotype-matched negative control on K562-WT cell, g = MFI of Fib504 on K562-WT transfectant cell, and h = MFI of negative control mAb Y13.238 on K562-WT transfectant cell.

Modeling of the Human alpha E A-domain-- Models of the human alpha E A-domain were based upon the open (1IDO (19)) and closed (1JLM (30)) human alpha M A-domain crystal structures. The program O was used to substitute the residues of human alpha E into the human alpha M structures.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Model of the alpha E Integrin A-domain-- To facilitate selection of residues for mutagenesis and to aid interpretation of the results, a three-dimensional model of the alpha E chain A-domain was generated. A FASTA screen (75) of the Swiss Protein Database with the human alpha E A-domain protein sequence revealed that it was most closely related to the A-domain of human alpha M (CD11b), with 38% identity over 192 amino acids. The alignment of the alpha E A-domain sequence with that of alpha M revealed a single insertion of two amino acids in alpha E compared with alpha M (Fig. 1A). Substitution of human alpha E residues into the human alpha M crystal structure in the open conformation (1IDO (19)) produced a model of the alpha E A-domain (Fig. 1B). No major changes in the backbone structure are necessary to accommodate the alpha E protein sequence, and the conserved MIDAS residues Asp190, Ser192, Ser194, Thr261, and Asp294 form a potential metal ion coordination site in a cleft at the top of the domain, as in known integrin A-domain structures. A notable difference from the alpha M (and alpha L) structure in the model is the extended alpha 1-beta B loop at the base of the A-domain due to the insertion of two amino acids mentioned above. The length of this loop is thus more similar to that in the alpha 1 and alpha 2 A-domains, but is unique in that a disulfide bond that may rigidify the region is predicted between the alpha 1-beta B loop and the start of the beta B-strand (Fig. 1, A and B).


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  		Fig. 1.   Model of the alpha E A-domain. A, alignment of the X-domain and A-domain of human alpha E with the corresponding region of human alpha M. The first line contains the X-domain of alpha E. There is no equivalent region in alpha M. The following three alignment lines show the A-domains of alpha E and alpha M. The location of alpha -helices and beta -strands in the alpha M A-domain crystal structure (19) are indicated, and the MIDAS residues are underlined. Residues in boldface are those mutated in this study; the triangle indicates the Delta Glu176 deletion; and the Delta Glu163-Glu180 deletion is italicized. Mutations that influence binding of E-cadherin are labeled with asterisks. The post-translational cleavage site of alpha E is indicated with a vertical arrow, and a predicted disulfide bond between Cys218 and Cys221 is marked. B, ribbon diagram of the alpha E A-domain model based on the crystal structure of the alpha M A-domain in theopen conformation (19) as described under "Results."

Generation of K562 Cells Expressing Mutant alpha Ebeta 7 Integrins-- To localize sites on the human alpha Ebeta 7 integrin that are involved in binding to E-cadherin, we generated a series of site-directed mutants of both alpha  (Fig. 1A) and beta  chains. Mutations were made in the DXSXS MIDAS motifs of both alpha E (D190A) and beta 7 (D140A) chains and in a number of residues predicted by the model to be solvent-exposed at the top of the alpha E A-domain surrounding the MIDAS site (G193A, D199A, G230A/V231A, F298A, and E325A). Further mutations were made in prominent surface-exposed residues in other regions of the alpha E A-domain based upon the model (R202A/D205A, D240A, P311H/E345A/T346A, and Y354W). The double-basic post-translational cleavage site within the X-domain was removed by mutation (R159S/R160S); a deletion of a single residue within the highly charged region of the X-domain was generated (Delta Glu176); and a final mutant lacking the entire charged region of the X-domain was produced (Delta Glu163-Glu180).

After transfection of K562 cells with these mutant constructs together with the appropriate wild-type partner chain cDNA, we obtained cells expressing cell-surface alpha Ebeta 7 as determined by flow cytometry. K562 cell clones expressing the mutant forms of alpha Ebeta 7 were obtained by autocloning (see "Experimental Procedures"), and a number of clones expressing each mutant at similar levels as determined by surface staining with anti-beta 7 integrin mAb Fib504 were selected for further analysis.

To confirm that the mutations introduced into alpha Ebeta 7 did not prevent heterodimer formation, cells expressing each mutant were surface-labeled with biotin and immunoprecipitated with anti-beta 7 mAb Fib504 (Fig. 2A). The ratio of co-immunoprecipitated alpha E chain to beta 7 chain as determined by densitometry was within 2-fold of that determined for wild-type alpha Ebeta 7 in all cases, with the exception of alpha E(R159S/R160S)beta 7, confirming that these mutations do not have a major impact on heterodimer formation. The mutation R159S/R160S appeared to reduce the amount of alpha E chain co-immunoprecipitated with the anti-beta 7 antibody. However, this mutation removes two arginine residues from the alpha E chain that are possible targets of biotinylation and might affect the efficiency of labeling. Therefore, immunoprecipitation was performed after 125I surface labeling of K562-alpha E(R159S/R160S)beta 7 cells, and this revealed that the normal proportion of alpha E to beta 7 chain was present. Moreover, the R159S/R160S mutation, as expected, prevented the majority of the alpha E chain from being cleaved, resulting in the larger size of the alpha E chain (~175 kDa) compared with the wild type (150 kDa) under reducing conditions (Fig. 2B). Under nonreducing conditions, both the wild type and the R159S/R160S mutant have an alpha E chain of ~180 kDa (data not shown). A low level of residual cleavage of the mutant chain was seen that may be due to inefficient cleavage of the chain at a single arginine residue located two residues N-terminal of the bona fide dibasic site (see Fig. 1A). Immunoprecipitation from K562-alpha E(Delta Glu163-Glu180)beta 7 cells demonstrated the presence of a smaller alpha E chain, as expected, resulting from loss of the charged region (data not shown). However, this mutant, which displayed evidence of proteolytic degradation, was expressed at a low level, and less alpha E chain was precipitated with an anti-beta 7 chain mAb than for the other mutants. Another mutant with a deletion of the entire X-domain was not expressed at the cell surface and therefore was excluded from the analysis. This suggests that large deletions within the X-domain have some impact on the ability of the alpha E chain to associate with beta 7 and to be transported to the cell surface.


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  		Fig. 2.   Immunoprecipitation of alpha Ebeta 7 mutant heterodimers. K562 cells transfected with wild-type or mutant alpha Ebeta 7 were surface-labeled with biotin (A) or 125I (B) as described under "Experimental Procedures." Lysates were immunoprecipitated with anti-beta 7 mAb Fib504 and subjected to SDS-polyacrylamide gel electrophoresis on a 7.5% gel under reducing conditions as described under "Experimental Procedures." Delta E176, Delta Glu176.

Mapping of anti-alpha Ebeta 7 Monoclonal Antibody Epitopes Defines a Likely Binding Site for E-cadherin-- To probe the overall conformation of the mutant alpha Ebeta 7 integrins on the cell surface and to map antibody-binding epitopes, the reactivity of each mutant with a panel of anti-alpha Ebeta 7 mAbs was determined by flow cytometric analysis and compared with that of anti-beta 7 mAb Fib504 as a reference (see "Experimental Procedures"). mAbs Fib21, Fib30, and Fib504, which recognize three distinct epitopes on the beta 7 chain (65), all bound equally well to all the mutant alpha Ebeta 7 integrins (Fig. 3A). Thus, none of the mutations, including that of the beta 7 chain MIDAS motif (D140A), had any detectable influence on the conformation of the beta 7 chain. This analysis also supported the use of Fib504 as a reference mAb for subsequent analysis of anti-alpha E mAbs. All the mutant alpha Ebeta 7 integrins were recognized as well as the wild type by multiple mAbs that recognize different epitopes of alpha Ebeta 7 and that do not recognize denatured protein in immunoblotting (Refs. 63 and 71 and this study), suggesting that the mutant alpha Ebeta 7 integrins maintain their basic structure. Indeed, no significant difference was seen in the recognition by mAbs LF61 and ABB1.3 of any of the mutants (Fig. 3B).


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  		Fig. 3.   Epitope mapping of anti-alpha Ebeta 7 monoclonal antibodies. The binding of anti-beta 7 (A) or anti-alpha Ebeta 7 (B) mAbs to K562 cells expressing mutant forms of alpha Ebeta 7 was determined by flow cytometry. Binding is expressed as the percentage of binding obtained with anti-beta 7 mAb Fib504 and is corrected for variations in the expression level on different clones as described under "Experimental Procedures." None of the antibodies bound to K562 control cells. Each bar represents the mean ± S.D. determined from two to four different K562 clones analyzed in two to five separate experiments. Delta E176, Delta Glu176. *, significant difference versus alpha Ebeta 7 WT by Dunnett's multiple comparison test (p < 0.01).

The mutagenesis of alpha E mapped five of the eight anti-alpha Ebeta 7 mAbs tested to the alpha E A-domain (Fig. 3B). mAb alpha E7-1 bound similarly to all the A-domain mutants except one, alpha E(G230A/V231A)beta 7, to which its binding was reduced by >80%. Thus, the epitope of this mAb is highly likely to encompass Gly230 and/or Val231. Similarly, mAb alpha E7-2 binding was abolished only to alpha E(E325A)beta 7; mAb alpha E7-3 binding was abolished only to alpha E(F298A)beta 7; and mAb HML-1 and 2G5 binding was reduced by ~80 and 60%, respectively, only to alpha E(G193A)beta 7. Gly193, Gly230, Val231, Phe298, and Glu325 form a cluster of exposed residues surrounding the MIDAS-chelated metal ion at the top of the A-domain model (see Fig. 8A). Since alpha E7-1, alpha E7-2, alpha E7-3, HML-1, and 2G5 all block alpha Ebeta 7-mediated adhesion to E-cadherin, whereas LF61 and ABB1.3 do not (3, 5, 63),2 these results provide a preliminary mapping of the E-cadherin-binding site on alpha Ebeta 7 to the top of the A-domain surrounding the MIDAS site.

Interestingly, removal of the cleavage site within the unique alpha E X-domain (alpha E(R159S/R160S)beta 7) reduced the binding of mAb BerACT-8 by 70%, whereas all the other mAbs tested bound normally (Fig. 3B). Immunoprecipitation of alpha E(R159S/R160S)beta 7 with BerACT-8 suggests that the residual binding of this mAb is due to recognition of the small proportion of alpha E(R159S/R160S)beta 7 with a cleaved alpha E chain (data not shown). These findings suggest that the loss of BerACT-8 binding to the R159S/R160S mutant is not directly due to the loss of the two arginine residues, but rather that BerACT-8 recognizes an epitope that is fully exposed only when the X-domain is cleaved. However, the precise location of the BerACT-8 epitope was not determined in this study.

Mutation of the MIDAS Motif of the alpha E or beta 7 Chain Abolishes Adhesion to E-cadherin-- The functional activity of the alpha Ebeta 7 mutants was tested by measuring the adhesion of the K562 transfectants to E-cadherin-Fc fusion protein immobilized on microtiter plate wells as described previously (5, 11). Thirty to forty percent of K562-alpha Ebeta 7 WT cells adhered to E-cadherin-Fc at saturating concentrations, whereas K562 control cells did not adhere (data not shown). Strikingly, mutation of the DXSXS sequence to AXSXS within the MIDAS motif of either the alpha E (D190A) or beta 7 (D140A) chain completely abolished detectable adhesion of transfected K562 cells to E-cadherin-Fc-coated wells (Fig. 4A). Thus, the coordination of metal ions by the MIDAS residues of the alpha E A-domain and the proposed A-like domain of beta 7 are likely to be critical for alpha Ebeta 7-mediated adhesion to E-cadherin.


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  		Fig. 4.   Analysis of the E-cadherin binding capacity of mutant alpha Ebeta 7 integrins. A, adhesion of K562-alpha Ebeta 7 mutants to E-cadherin-Fc. In all cases, flow cytometry to determine integrin expression level was conducted within 30 h of the adhesion assays; E-cadherin-Fc was coated at saturating concentrations between 20 and 100 ng/well; and the background adhesion to the same concentration of human IgG1 was subtracted. Each bar represents the mean ± S.E. pooled from four to nine determinations in triplicate from two to four independent experiments involving two to four different K562 clones. The results were combined as described under "Experimental Procedures." The lack of a bar indicates that no adhesion above that to IgG1 could be detected. *, p < 0.01 versus the wild type by Dunnett's multiple comparison test. B, E-cadherin-Fc directly binds to K562-alpha Ebeta 7 WT cells, but not to K562 control cells. The binding of 50 µg/ml biotinylated human E-cadherin-Fc or human IgG1 to transfected K562 cells was determined by flow cytometry in the presence of 1 mM each MnCl2, MgCl2, and CaCl2, using a neutravidin-conjugated Alexa 488 secondary reagent. C, the direct binding of 100 µg/ml E-cadherin-Fc to K562-alpha Ebeta 7 mutants was analyzed as described for B. Binding is expressed as the percentage of binding obtained with anti-beta 7 mAb Fib504 as described under "Experimental Procedures." Each bar represents the mean ± S.D. determined from two to four different K562 clones analyzed in two to three separate experiments. Asterisks indicate that no binding above that to IgG1 could be detected (p < 0.01 versus the wild type by Dunnett's multiple comparison test); double asterisks indicate a significant increase in binding over the wild type (p < 0.01). Delta E176, Delta Glu176

Phe298 at the Top of the alpha E A-domain Is Critical for Adhesion to E-cadherin-- K562 cells expressing alpha Ebeta 7 containing mutations G193A, D199A, R202A/D205A, G230A/V231A, D240A, P311H/E345A/T346A, E325A, and Y354W in the alpha E A-domain adhered to E-cadherin-Fc similarly to K562-alpha Ebeta 7 WT transfectants (Fig. 4A). Assays conducted at lower coating concentrations of E-cadherin-Fc or without manganese also did not reveal any differences from the wild type (data not shown). In contrast, K562-alpha E(F298A)beta 7 cells did not adhere to E-cadherin-Fc at any concentration tested (p < 0.01 versus the wild type by Dunnett's multiple comparison test). Phe298 is predicted to be prominently exposed at the top of the alpha E A-domain close to the MIDAS metal ion coordination site and thus is likely to be involved directly in binding to E-cadherin. The lack of structural data for the potential A-like domain of integrin beta  chains precluded a reliable similar analysis of the beta 7 chain.

Neither removal of the cleavage site from the X-domain of the alpha E chain (R159S/R160S) nor deletion of a single residue within the charged region (Delta Glu176) altered adhesion to E-cadherin-Fc (Fig. 4A). The results suggest that the fine structure of the charged region of the X-domain is not crucial for binding to E-cadherin and that post-translational cleavage in this region is not a prerequisite for adhesion to E-cadherin. Furthermore, an antiserum to the charged region of the X-domain that recognizes alpha Ebeta 7 on the cell surface by flow cytometry does not inhibit the adhesion of K562-alpha Ebeta 7 WT transfectants to E-cadherin-Fc (data not shown), indicating that this region does not participate directly in the binding of alpha Ebeta 7 to E-cadherin.

Direct Binding of Soluble E-cadherin-Fc to K562-alpha Ebeta 7 Transfectants Confirms and Extends Definition of the E-cadherin-binding Site-- Adhesion assays are not simply a measure of direct receptor to ligand binding because stable adherence of cells to immobilized ligands may require post-receptor events including modulation of the cytoskeleton. It is possible that mutation of alpha Ebeta 7 might affect signaling through the integrin and such downstream events rather than alter the direct binding to E-cadherin. To rule out this possibility and to provide a quantitative assay for direct binding of alpha Ebeta 7 to E-cadherin, we made use of the ability of soluble E-cadherin-Fc to bind directly to alpha Ebeta 7 (5). The binding of biotinylated E-cadherin-Fc to the surface of K562-alpha Ebeta 7 transfectants could be detected by flow cytometry using a neutravidin-conjugated Alexa 488 secondary reagent (Fig. 4B). No such binding was seen to K562 control cells. Saturation of binding was achieved between 50 and 100 µg/ml E-cadherin-Fc (data not shown). The binding of 100 µg/ml biotinylated E-cadherin-Fc to K562 cells transfected with each mutant form of alpha Ebeta 7 was assessed. In each case, the background binding of biotinylated human IgG1 was subtracted, and the results are expressed as the percentage of the binding seen to wild-type alpha Ebeta 7 after correcting for the level of surface expression determined using anti-beta 7 chain mAb Fib504.

The results (Fig. 4C) confirm those obtained in adhesion assays. Mutation of the MIDAS motif of either the alpha E or beta 7 chain completely abrogated the ability of E-cadherin-Fc to bind alpha Ebeta 7 (p < 0.01 versus the wild type by Dunnett's multiple comparison test). Also, the critical importance of Phe298 for binding to E-cadherin was reinforced. No direct binding of E-cadherin-Fc to alpha Ebeta 7 could be detected if this amino acid was mutated to alanine (p < 0.01). In addition, the direct binding assay highlighted the previously undetected role of Glu325 in binding to E-cadherin. Mutation of this amino acid to alanine unexpectedly increased the binding of E-cadherin-Fc to alpha Ebeta 7 2-fold over the wild type (p < 0.01). This residue is exposed on the surface of the alpha E A-domain near the MIDAS site, and it forms a crucial part of the epitope of the adhesion-blocking mAb alpha E7-2 (Fig. 3B). As in the adhesion assays, mutation of other residues in the A-domain, mutation of the X-domain cleavage site (R159S/R160S), and removal of a single amino acid within the charged region (Delta Glu176) did not alter E-cadherin-Fc binding.

Mutation of the MIDAS Motif of the beta 7 Chain Abolishes alpha 4beta 7-mediated Adhesion to MAdCAM-1-- A role for the MIDAS motif within the potential A-like domain of the beta 7 chain has been proposed for alpha 4beta 7-mediated adhesion to MAdCAM-1 based on beta 1/beta 7 chimera analysis and mapping of the adhesion-blocking antibody ACT-1 epitope to this region of the beta 7 chain (60). Transfection of both human alpha 4 and beta 7 chains into K562 cells conferred the ability to adhere to MAdCAM-1-Ig fusion protein, but not to human IgG1, as described by others (60). Adhesion to MAdCAM-1-Ig could be blocked by anti-human alpha 4beta 7 mAb ACT-1, and K562 control cells were unable to adhere to MAdCAM-1-Ig (data not shown). After transfection of K562 cells with alpha 4 and the beta 7 chain containing the D140A mutation within the MIDAS motif, we obtained clones that expressed the mutant alpha 4beta 7(D140A) as assessed by flow cytometry. A comparison of the staining obtained with mAbs to at least two different epitopes on the alpha 4 chain (B5G10, B5E2, and HP1/2) (76), three different epitopes on the beta 7 chain (Fib21, Fib30, and Fib504) (65), and an antibody that recognizes cell-surface beta 7 chain only in the context of alpha 4beta 7 (ACT-1) (60, 77) showed that the binding of these mAbs was unaffected and confirmed that the D140A mutation did not grossly affect the structure of alpha 4beta 7 (Fig. 5A). In addition, immunoprecipitation of wild-type alpha 4beta 7 and alpha 4beta 7(D140A) with either mAb ACT-1 or Fib504 demonstrated that heterodimerization was not affected (Fig. 5B) (data not shown). Despite this, K562-alpha 4beta 7(D140A) cells were unable to adhere to MAdCAM-1-Ig, even when the mutant integrin was expressed at higher levels than wild-type alpha 4beta 7 (Fig. 5C). Thus, an intact MIDAS motif within the A-like domain of the beta 7 chain is required for the adhesion of alpha 4beta 7 to MAdCAM-1 as well as for the adhesion of alpha Ebeta 7 to E-cadherin.


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  		Fig. 5.   A MIDAS mutation in the beta 7 chain abolishes adhesion of K562-alpha 4beta 7 cells to MAdCAM-1-Ig. A, the beta 7 mutation D140A does not alter the binding of anti-alpha 4beta 7 (ACT-1), anti-alpha 4 (B5G10, B5E2, and HP1/2), and anti-beta 7 (Fib21, Fib30, and Fib504) mAbs to K562-alpha 4beta 7 transfectants. Antibody binding was determined as described under "Experimental Procedures." None of the antibodies bound to K562 control cells. Each bar represents the mean ± S.D. determined from four different K562 clones analyzed in three separate experiments. B, the beta 7 mutation D140A does not alter alpha 4beta 7 heterodimer formation. K562 cells transfected with wild-type alpha 4beta 7 or alpha 4beta 7(D140A) or K562 control cells were surface-labeled with biotin. Proteins immunoprecipitated with anti-alpha 4beta 7 mAb ACT-1 were detected as described in the legend to Fig. 2. C, the mutation D140A in the beta 7 chain MIDAS motif of alpha 4beta 7 abolishes detectable adhesion to MAdCAM-1-Ig. The adhesion of K562 transfectants to human MAdCAM-1-Ig or IgG1 was determined as described under "Experimental Procedures." The expression level of alpha 4beta 7(D140A) on the cell surface by flow cytometry was higher than that of the wild type. The data are the mean ± S.D. (n = 3) and are representative of three separate experiments with two different clones.

Mutations of the alpha 4 Chain That Reduce alpha 4beta 1-mediated Adhesion Also Reduce alpha 4beta 7-mediated Adhesion to MAdCAM-1-Ig-- A number of residues in the second, third, and fourth repeats of the alpha 4 chain have been implicated in the binding of alpha 4beta 1 to VCAM-1 and fibronectin (47-49). To determine if the same residues are involved in the binding of alpha 4beta 7 to MAdCAM-1, we transfected CHO cells with previously described mutant alpha 4 chains together with the wild-type beta 7 chain. CHO cells were used in this case because K562 cells were found to express endogenous human alpha 4 chain at significant levels upon transfection with beta 7 (data not shown). We generated CHO cells expressing alpha 4beta 7 with single amino acid substitutions in the alpha 4 chain (Y187A, G190A, and Y202A) (48) or in which predicted loops in the second and third repeats of the alpha 4 chain were replaced with corresponding regions of the alpha 5 chain (R2, residues 112-131; and R3a, residues 151-164) (49). All the mutants were expressed at levels greater than that of wild-type alpha 4beta 7 as determined by flow cytometry with anti-alpha 4beta 7 mAb ACT-1; anti-beta 7 mAbs Fib21, Fib30, and Fib504; and anti-alpha 4 mAbs B5G10 and B5E2 (data not shown). Immunoprecipitation with anti-beta 7 mAb Fib504 from surface-biotinylated CHO transfectants also confirmed the presence of alpha 4beta 7 heterodimers for all the mutants (Fig. 6A). As previously found for alpha 4beta 1 (48, 49), none of the mutations altered the binding of mAb B5G10 to alpha 4beta 7. The binding of anti-alpha 4beta 7 mAb HP1/2 to the R2 mutant was reduced by ~85%, consistent with the similarly reduced binding of mAb HP2/1 (which recognizes the same epitope (76)) to alpha 4beta 1 (49).


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  		Fig. 6.   Mutations of the alpha 4 chain that reduce alpha 4beta 1-mediated adhesion also reduce alpha 4beta 7-mediated adhesion to MAdCAM-1-Ig. A, the mutations of alpha 4 do not prevent alpha 4beta 7 heterodimer formation. Transfected CHO cells were surface-labeled with biotin, and proteins were immunoprecipitated with anti-beta 7 mAb Fib504 as described in the legend to Fig. 2. To correct for different levels of integrin expression (see below) and to ease comparison between clones, varying ECL exposures of the different mutants, immunoprecipitated in the same experiment, are shown. Note that changes in the number and location of lysine and arginine residues, the major targets for biotinylation, may alter the efficiency of alpha 4 chain labeling for the R2 and R3a mutants. B, the adhesion of CHO transfectants to human MAdCAM-1-Ig at 0.1 µg/well was determined as described under "Experimental Procedures." In all cases, the expression level of the mutant integrin on the cell surface by flow cytometry was higher than that of the wild type (MFI with anti-alpha 4beta 7 mAb ACT-1: 43 for the wild type, 285 for Y187A, 62 for G190A, 72 for Y202A, 253 for R2, 377 for R3a, and 3 for the control). The data are expressed as the mean percentage ± S.D. (n = 3) of adhesion obtained compared with CHO-alpha 4beta 7 WT cells and are representative at least two separate experiments involving each clone. The mean percentage adhesion of all CHO transfectants to human IgG1 was ~1%, and that of CHO-alpha 4beta 7 WT cells to MAdCAM-1-Ig was 39%. C, CHO cells expressing wild-type or mutant alpha 4 integrins were cultured to near confluency in 96-well tissue culture plates, and fluorescently labeled L1-2 cells expressing MAdCAM-1 (4 × 105 cells/well) were added and incubated for 1 h at 37 °C. Data are shown as percentage ± S.D. (n = 3) of added cells that were adherent. Parent L1-2 cells showed only background level binding. *, significant decrease in adhesion versus wild-type alpha 4beta 7 by Dunnett's multiple comparison test (p < 0.01).

Transfection with both wild-type alpha 4 and beta 7 chains conferred upon CHO cells the ability to adhere to MAdCAM-1-Ig fusion protein and to MAdCAM-1-transfected L1-2 cells, but not to human IgG1 or to untransfected L1-2 cells (Fig. 6, B and C) (data not shown). Approximately 40% of CHO-alpha 4beta 7 cells adhered to MAdCAM-1-Ig, and this could be blocked by anti-alpha 4beta 7 mAb ACT-1. Untransfected CHO cells were unable to adhere to MAdCAM-1-Ig (Fig. 6B). A 2-fold greater percentage of CHO-alpha 4(Y202A)beta 7 cells adhered to MAdCAM-1-Ig compared with CHO-alpha 4beta 7 WT cells, presumably due to the higher level of alpha 4(Y202A)beta 7 expression (Fig. 6B). However, CHO cells expressing alpha 4(G190A)beta 7 did not adhere to MAdCAM-1-Ig even though they also had higher surface expression than wild-type alpha 4beta 7 transfectants. In addition,CHO-alpha 4(Y187A)beta 7 cells adhered less well than CHO-alpha 4beta 7 WT cells to MAdCAM-1-Ig despite the higher cell surface expression of the mutant integrin. CHO cells expressing alpha 4(R2)beta 7 were unable to adhere to MAdCAM-1-Ig, but the alpha 4(R3a)beta 7-expressing cells adhered well. Similar results were obtained when the adhesion of MAdCAM-1-expressing L1-2 cells to the CHO transfectants was assessed (Fig. 6C), although in this system, the defect in CHO-alpha 4(Y187A)beta 7 cell adhesion was more pronounced. In summary, the G190A and R2 mutations abolished alpha 4beta 7-mediated adhesion to MAdCAM-1, whereas the Y202A and R3a mutations did not substantially reduce adhesion, and Y187A had an intermediate effect.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Despite the distinctive nature of E-cadherin as the only integrin ligand known that is a cadherin and the presence of unique structural features within the alpha E chain that could contribute to ligand binding, the E-cadherin-binding site includes the A-domain of the alpha E chain, and its location appears similar to that defined for other integrin-ligand interactions. Mutation of the conserved MIDAS motif within the alpha E A-domain (D190A) abolishes E-cadherin binding. This is consistent with the requirement for magnesium or manganese ions (5) and an exposed acidic residue within E-cadherin for alpha Ebeta 7 binding (10, 11) and strongly suggests a direct role for a metal ion coordinated by the MIDAS motif in the interaction. This result also shows that, contrary to previous concepts (9), A-domain-containing integrins do not require the critical acidic residue of the ligand to be on a flat surface since Glu31 of E-cadherin is on the highly exposed BC-loop at the top of domain 1 in a context most similar to that of the RGD sequence in fibronectin, rather than on a fairly level surface at the end of the C strand as in ICAM-1 and -2.

In contrast, the mutation R159S/R160S within the extra X-domain of the alpha E chain prevents the normal post-translational cleavage of the alpha E chain, but does not alter the binding of E-cadherin to alpha Ebeta 7. Deletion of a single amino acid within the charged region of the X-domain also has no detectable effect on E-cadherin binding. Since an anti-X-domain antiserum also fails to influence adhesion to E-cadherin, we speculate that the alpha E X-domain does not contribute directly to the E-cadherin-binding interface. The X-domain may be important in optimizing the orientation of the A-domain for ligand binding since it is located at the junction between the second blade of the predicted beta -propeller and the A-domain of alpha E. Such a role would be consistent with the presence of an X-domain in murine and rat alpha E chains, but its low sequence homology and poor length conservation compared with human alpha E (78, 79). Alternatively, the X-domain might be involved in interactions with yet unidentified alpha Ebeta 7 ligands.

Residues predicted from our alpha E A-domain model to reside in loops close to the MIDAS cleft also were implicated in E-cadherin binding and suggest a wider interaction surface that is involved in ligand recognition at the top of the A-domain. The binding of five blocking monoclonal antibodies to alpha Ebeta 7 mapped to a cluster of residues (Gly193, Gly230/Val231, Phe298, and Glu325) encircling the MIDAS on the surface of the A-domain (Fig. 7A). The mutation F298A abolished detectable binding of E-cadherin to alpha Ebeta 7 and is located in the beta D-alpha 5 loop. This hydrophobic residue is predicted to be prominently exposed on the top surface of the A-domain (Fig. 7B). The mutation E325A, in the beta E-alpha 6 loop also at the top of the A-domain (Fig. 7B), increased the direct binding of E-cadherin to alpha Ebeta 7 2-fold and thus also may be close to the cadherin-binding surface.


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  		Fig. 7.   Residues at the E-cadherin-binding site of the alpha E A-domain. Shown are space-filling models of the alpha E A-domain in the open conformation viewed from the top. The MIDAS residues predicted to coordinate magnesium are colored pink. In A, residues colored red are those that significantly reduce the binding of the mAbs shown in parentheses. Mutation of residues in blue had no detectable effect on mAb binding. Since the five mAb indicated all block E-cadherin binding to alpha Ebeta 7, this provides a preliminary mapping of the E-cadherin-binding site to the upper surface of the alpha E A-domain surrounding the MIDAS cleft. In B, mutation of residues colored red abolishes detectable binding, and the mutation E325A in green increases the binding of E-cadherin to alpha Ebeta 7, confirming the location of the E-cadherin-binding site on the alpha E A-domain. Mutation of residues in blue did not have a significant influence on E-cadherin binding.

Using this information, we docked a model of human E-cadherin (11) onto the alpha E A-domain. Importantly, we utilized a model of the alpha E A-domain based on the open form of the alpha M A-domain, as this is likely to be the active conformation (31). The critical Glu31 residue in the BC-loop of E-cadherin domain 1 was maintained at a distance suitable for interaction with the magnesium ion chelated by the A-domain MIDAS motif. The E-cadherin molecule was then rotated relative to the A-domain structure to optimize the interaction and to minimize steric interference. Strikingly, the best orientation of the two structures places Phe298 of the alpha E A-domain into a hydrophobic pocket formed between the BC- and FG-loops of E-cadherin domain 1 (Fig. 8A). This interaction and the coordination of the MIDAS metal ion by Glu31 in E-cadherin provide the most important contributions to the contact between the two molecules. This is consistent with the finding that the mutation E31A in E-cadherin and the F298A and MIDAS mutation D190A in alpha Ebeta 7 all abolish detectable binding between the two proteins. Our previous study identified further residues in E-cadherin that contribute to alpha Ebeta 7 binding (11). Interestingly, mutation of Asn27, Lys30, and Glu89 in the BC- and FG-loops of E-cadherin reduces adhesion of K562-alpha Ebeta 7 cells to E-cadherin-Fc. These amino acids are all found at the proposed interface between E-cadherin and the A-domain (Fig. 8B). Asn27 and Glu89 could potentially form a hydrogen bond and a salt bridge, respectively, with Arg331 in the A-domain, and Lys30 in E-cadherin could form another salt bridge with Asp196 in the A-domain. Arg331 was not mutated in this study, but the model predicts that it may be an important component of the E-cadherin-binding site. This proposal is consistent with the marked reduction (>90%) that mutation of Glu89 causes in K562-alpha Ebeta 7 adhesion to E-cadherin (11). We also examined the interaction of E-cadherin with an alpha E A-domain model in the closed conformation that is likely to represent the inactive form (30, 31). E-cadherin residues Asn27, Lys30, and Glu89 all interact with a region of the A-domain that undergoes a marked conformational change upon transition between the two forms (19, 29-31) (Fig. 8B). Indeed, in the active form of the A-domain, Asp196 and Arg331 both move into more favorable positions for interaction with E-cadherin. Glu325 is also in this region of the A-domain, but is not predicted to make direct contacts with E-cadherin. This raises the possibility that the E325A mutation may increase binding to E-cadherin because it favors adoption of the active conformation.


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  		Fig. 8.   Docking model of the A-domain of alpha E with E-cadherin. A, detail of the contact site between the tip of E-cadherin domain 1 (blue) and the A-domain model in the open conformation (pink) showing the most important contributions to the contact. Phe298 of alpha E projects into a hydrophobic pocket in E-cadherin formed by Gly32, Val34, Val88, and the aliphatic portions of Lys28 and Ser82. Glu31 of E-cadherin coordinates the MIDAS magnesium ion of alpha E (in magenta). The inset shows the orientation of E-cadherin domain 1 relative to the alpha E A-domain. B, another view of the contact site showing interactions predicted to be favorable when the A-domain is in the open or "active" conformation. Structural elements that occupy different conformations in the open and closed forms of the alpha E A-domain model are shown in magenta. E-cadherin residues Asn27, Lys30, and Glu89, which were suggested by previous mutagenesis (11) to contact alpha Ebeta 7, are indicated. The human alpha E A-domain models are described under "Results," and the model of human E-cadherin was described previously (11).

The potential projection of Phe298 into a distinct pocket in E-cadherin domain 1 suggests a novel mode of integrin-ligand interaction that contributes to the specificity of alpha E for E-cadherin. The equivalent of Phe298 in alpha L, Thr243, is involved in binding to ICAM-1 and -2, but forms part of a relatively flat binding surface (20, 32, 33), and His258 of alpha 2 is involved in collagen I binding, but forms a hydrogen bond with the main chain of collagen (31, 34). Also, the alpha 1 and alpha 2 integrin A-domains contain an extra four-amino acid alpha -C helix in the beta E-alpha 6 loop that markedly alters the topology of the A-domain surface and regulates access to the collagen-binding site (21, 22, 31, 34, 80). Although hydrophobic residues are involved in all integrin A-domain interactions examined, the penetration of a prominent aromatic residue into a hydrophobic pocket of the ligand is not predicted for alpha L binding to ICAM-1 or -2 (20, 33, 81) and is not involved in alpha 2 A-domain binding to collagen (31). Interestingly, the amino acids implicated in binding of the alpha E A-domain to E-cadherin are similar to residues on the closely related A-domain of alpha M that are involved in binding iC3b (see Fig. 1A). The residue equivalent to Phe298 in the alpha M A-domain is also phenylalanine, and the mutation F246K abolishes alpha M binding to iC3b (29), whereas the F246A mutation reduces binding by 50% (26). Strikingly, the mutation D273K, the residue in the alpha M A-domain corresponding to Glu325, also leads to a doubling of ligand binding activity, in this case for iC3b (29). The results suggest that the alpha E and alpha M A-domains share common features for recognition, despite the dissimilarity of their ligands.

We found residues that could be mutated in the alpha E A-domain with no detectable influence on E-cadherin binding, but that are important for ligand binding by alpha M and alpha L A-domains. The mutations G193A, D199A, and G230A/V231A in alpha E did not cause a significant change in E-cadherin binding, whereas the mutations G143M, D149K, and E178A/E179A in alpha M all reduced iC3b binding by >90% (29), and M140Q, E146A, and T175A in alpha L all reduced ICAM-1 or -2 binding (32, 33). It is possible that in some instances, the more dramatic amino acid substitutions made in the studies of alpha Mbeta 2 and alpha Lbeta 2 are responsible for the detection of more important residues. For example, the M140Q mutation reduced ICAM-1 binding by 65% (32), but M140A had no effect on ICAM-1 or -2 binding in another study (33). Alternatively, it may be that E-cadherin has a smaller footprint on the alpha E A-domain than iC3b on the alpha M A-domain or ICAM on the alpha Lbeta 2 A-domain. Indeed, the docking model of E-cadherin with the alpha E A-domain does suggest limited contact between the molecules. This is due in part to the position of the critical Glu31 residue of E-cadherin at the tip of the first domain (Fig. 8A, inset), in marked contrast to the flat surfaces of ICAM-1 and -2 that bind the alpha L A-domain. It also possible that other residues on the surface of the alpha E A-domain that we did not mutate in this study contribute to E-cadherin binding. However, these results implicate common ligand-binding loops on the surface of all A-domains that surround the MIDAS cleft, but highlight integrin-specific differences that are critical to defining ligand binding specificity.

We also found that mutation of residues in the alpha 4 chain of alpha 4beta 7 can abrogate binding to MAdCAM-1. The mutations G190A and Y187A substantially reduced adhesion of alpha 4beta 7-expressing cells to MAdCAM-1, whereas the mutation Y202A did not. In addition, the replacement of alpha 4 residues 112-131 with the corresponding residues of alpha 5 abolished MAdCAM-1 recognition, whereas a similar substitution of residues 151-164 did not. The residues that influence adhesion lie on the upper surface of the second and third blades of the predicted beta -propeller (46, 49), consistent with a role in ligand binding. Strikingly, this pattern of abrogation of alpha 4beta 7-mediated adhesion to MAdCAM-1 is identical to that previously reported for alpha 4beta 1-mediated adhesion to VCAM-1 and the CS-1 fragment of fibronectin (48, 49). Although it remains possible that there are residues on alpha 4 that interact with VCAM-1 and fibronectin but not with MAdCAM-1 or vice versa, we clearly implicate a similar region of the alpha 4 molecule in ligand binding for both alpha 4beta 1 and alpha 4beta 7.

Since alpha Ebeta 7 and alpha 4beta 7 have distinct, non-overlapping ligand binding specificities for E-cadherin and MAdCAM-1, respectively (54),2 clearly the alpha  chain must determine this difference in ligand binding specificity. Indeed, we have shown that mutations in both the alpha E and alpha 4 chains can abrogate the binding of these integrins to their ligands. Similarly, since alpha 4beta 7 binds to MAdCAM-1, whereas alpha 4beta 1 binds to VCAM-1 but not MAdCAM-1 (52-54), in this case, the beta  chain must be important in defining specificity. As expected, mutation of the MIDAS motif of the beta 7 chain (this study) or of the beta 1 chain (47) abolishes ligand binding in both cases. Despite this, however, we have demonstrated that the beta 7 chain MIDAS is critically involved in ligand binding for both alpha Ebeta 7 and alpha 4beta 7 and that the alpha 4 chain is critical in ligand binding for both alpha 4beta 7 and alpha 4beta 1. In each case, a similar binding site is implicated on the shared chain even when the integrin is engaged in interactions with different counter-receptors. These results indicate that changes in the combinations of alpha  and beta  chains that make up integrin heterodimers can yield different ligand binding specificities despite the utilization of similar binding surfaces on each chain.

Like the beta 1 chain, beta 7 can pair with alpha  chains either containing or lacking a classical A-domain. It is known that the MIDAS motif of the beta  chain is involved in ligand binding in the context of the A-domain-containing integrins alpha Lbeta 2 and alpha Mbeta 2 (43-45) and in ligand binding in the context of alpha  chains that lack A-domains such as alpha 4beta 1, alpha 5beta 1, alpha IIbbeta 3, alpha Vbeta 5, and alpha Vbeta 6 (35, 37-42). We have shown for the first time that the MIDAS motif of a single beta  chain, beta 7, is critical whether it is paired with an A-domain-containing (alpha E) or A-domain-lacking (alpha 4) alpha  chain. The results stress that in every case studied, the beta  chain has a critical role to play in ligand binding, whatever the nature of the alpha  chain. Because of this, it seems likely that the majority of integrin ligands will possess residues that are critical for interaction with alpha  and beta  chains. Such a mechanism has been proposed for the binding of the alpha 5beta 1 integrin to fibronectin, where the RGD sequence in the tenth type III repeat together with a "synergy region" in the ninth type III repeat contribute to integrin binding (82). Similarly, along with the critical CD-loop in domain 1 of VCAM-1 and MAdCAM-1, an "accessory site" in domain 2 has been implicated in the binding of the alpha 4beta 1 and alpha 4beta 7 integrins (9, 83-85). For these non-A-domain-containing integrins, it is interesting that although the primary binding site in the ligand includes a critical exposed acidic residue, the accessory site need not. Indeed, it has been suggested that the MIDAS site of the beta 1 chain coordinates the acidic residue of the RGD sequence, whereas the synergy site interacts with the alpha 5 chain (86). For A-domain-containing integrins containing MIDAS motifs in both the alpha  and beta  chains, it might be expected that two acidic residues within the ligand are vital for ligand binding. In general, however, only one such residue has been identified in these ligands. In some cases, it is possible that the necessary pairing of acidic residues might be achieved by dimerization of ligand. Indeed, both ICAM-1 and E-cadherin are thought to form dimers on the cell surface (87-89). It also remains possible that the beta  chain A-like domain may regulate ligand binding by influencing the conformation of the A-domain in the alpha  chain (30), but this is more difficult to reconcile with the similar role of the beta 7 chain MIDAS in integrins with and without an alpha  chain A-domain. Further structural data will be required to resolve these issues.

    ACKNOWLEDGEMENTS

We thank Drs. Michael Briskin, Chafen Lu, and Dominic Picarella (Millennium Pharmaceuticals Inc.) for providing important reagents and for valuable discussions; Dr. Eugene Butcher for providing MAdCAM-1-expressing L1-2 cells; and John Daley (Dana-Farber Cancer Institute) for help with autocloning.

    Note Added in Proof

While this manuscript was under review, Ruiz-Velasco et al. also reported that mutation of alpha 4 residue Tyr187 in alpha 4beta 7 reduces MadCAM-1 binding (90).

    FOOTNOTES

* This work was supported by grants from the Crohn's and Colitis Foundation of America (to J. M. G. H.) and the National Institutes of Health (to M. B. B., Y. T., and J.-h. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Brigham and Women's Hospital, Smith Bldg., Rm. 538D, One Jimmy Fund Way, Boston, MA 02115. Tel.: 617-525-1105; Fax: 617-525-1010; E-mail: jhiggins@rics.bwh.harvard.edu. The atomic coordinates of the human E-cadherin domain 1 and alpha E A-domain models are available upon request.

Published, JBC Papers in Press, June 2, 2000, DOI 10.1074/jbc.M001228200

2 J. M. G. Higgins and M. B. Brenner, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: MAdCAM-1, mucosal addressin cell adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1; MIDAS, metal ion-dependent adhesion site; ICAM, intercellular cell adhesion molecule; mAb, monoclonal antibody; PCR, polymerase chain reaction; CHO, Chinese hamster ovary; TBS, Tris-buffered saline; BSA, bovine serum albumin; MFI, mean fluorescence intensity; WT, wild-type.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Distribution and Evolution of von Willebrand/Integrin A Domains: Widely Dispersed Domains with Roles in Cell Adhesion and Elsewhere
Mol. Biol. Cell, October 1, 2002; 13(10): 3369 - 3387.
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N. Wright, A. Hidalgo, J. M. Rodriguez-Frade, S. F. Soriano, M. Mellado, M. Parmo-Cabanas, M. J. Briskin, and J. Teixido
The Chemokine Stromal Cell-Derived Factor-1{alpha} Modulates {alpha}4{beta}7 Integrin-Mediated Lymphocyte Adhesion to Mucosal Addressin Cell Adhesion Molecule-1 and Fibronectin
J. Immunol., May 15, 2002; 168(10): 5268 - 5277.
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U. G. Strauch, R. C. Mueller, X. Y. Li, M. Cernadas, J. M. G. Higgins, D. G. Binion, and C. M. Parker
Integrin {{alpha}}E(CD103){{beta}}7 Mediates Adhesion to Intestinal Microvascular Endothelial Cell Lines Via an E-Cadherin-Independent Interaction
J. Immunol., March 1, 2001; 166(5): 3506 - 3514.
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E. Corps, C. Carter, P. Karecla, T. Ahrens, P. Evans, and P. Kilshaw
Recognition of E-cadherin by Integrin alpha Ebeta 7. REQUIREMENT FOR CADHERIN DIMERIZATION AND IMPLICATIONS FOR CADHERIN AND INTEGRIN FUNCTION
J. Biol. Chem., August 10, 2001; 276(33): 30862 - 30870.
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