©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
A Region of the Integrin VLA4 Subunit Involved in Homotypic Cell Aggregation and in Fibronectin but Not Vascular Cell Adhesion Molecule-1 Binding (*)

(Received for publication, September 18, 1995; and in revised form, November 20, 1995)

Marisa Muñoz (1) Juan Serrador (2) Francisco Sánchez-Madrid (2) Joaquín Teixidó (1)(§)

From the  (1)Centro de Investigaciones Biológicas, Departamento de Inmunología, Velázquez 144, 28006 Madrid, Spain and (2)Hospital de la Princesa, Servicio de Inmunología, Diego de León 62, 28006 Madrid, Spain

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The VLA-4 (alpha4beta1) integrin is involved in the adhesion of cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In order to study alpha4 structure-function relationships, we have expressed mutated alpha4 subunit by transfection into VLA-4-negative K562 cells. Substitutions at alpha4 residues Arg-Asp, which show the highest surface probability indexes inside the N-terminal alpha fragment, resulted in a reduction in the reactivity of all anti-alpha4 epitope A monoclonal antibodies (mAbs) tested, compared with the reactivity with anti-alpha4 epitopes B1, B2, and C mAb, both by transfectant flow cytometry, and by immunoprecipitation and SDS-polyacrylamide gel electrophoresis analysis of transfectant surface-iodinated proteins. In contrast, substitutions at nearby residues, Gln, Pro, and Ile did not affect the reactivity of any anti-alpha4 mAb representing the known alpha4 epitopes. Homotypic cell aggregation triggered by anti-alpha4 epitope A mAb was prevented in the transfectants expressing mutated alpha4 Arg-AspAsp residues, while cell aggregation was fully achieved with either anti-alpha4 epitope B2 or anti-beta1 mAb. Mutations at alpha4 residues Gln, Pro, and Ile did not affect the homotypic cell aggregation of the transfectants expressing these mutations. In addition, the adhesion of mutant Arg-Asp alpha4 transfectants to the connecting segment-1-containing fibronectin-40 (FN-40) fragment of fibronectin was diminished compared to wild type alpha4 transfectants, as well as to other mutant alpha4 transfectants. This adhesion to FN-40 was restored when the activating anti-beta1 TS2/16 mAb was present in the adhesion assays. In contrast, adhesion to VCAM-1 was not affected by mutations at Arg-Asp, nor at Gln, Pro, and Ile alpha4 residues. Altogether, these results indicate that alpha4 residues Arg and Asp are included in a region involved in homotypic cell aggregation, as well as in adhesion to FN-40, but not to VCAM-1.


INTRODUCTION

The alpha4 integrins play an important role in leukocyte extravasation during inflammation, lymphocyte traffic to lymphoid organs during a normal immune response, and hematopoietic progenitor cell adhesion to bone marrow stroma (reviewed in (1, 2, 3, 4) ). The alpha4 subunit can associate with the beta1 or the beta7 chains conforming the heterodimers alpha4beta1 (VLA-4) and alpha4beta7, respectively. VLA-4 interacts with the sequence EILDVPST within the alternatively spliced connecting segment-1 (CS-1) (^1)region of fibronectin(5, 6, 7) , and to domains first and fourth of vascular cell adhesion molecule-1 (VCAM-1)(8, 9, 10) . Interaction with other sequences on fibronectin(11, 12, 13) , as well as with thrombospondin(14) , requires VLA-4 to be activated. VLA-4 also interacts with the bacterial coat protein invasin in an activation-independent manner(15) . alpha4beta7 known ligands include the fibronectin CS-1 region, VCAM-1, and mucosal addressin cell adhesion molecule (MadCAM)(16, 17, 18, 19) .

In addition to most types of leukocytes, VLA-4 is also expressed on various nonhematopoietic tumor cells such as melanomas (20) and during muscle differentiation at the stage of myotubes(21) . The alpha4 subunit is expressed on nonlymphoid tissues in developing mouse embryo(22, 23) , and it has been reported that the absence of a functioning alpha4 gene results in defects in placental and cardiac development, leading to embryonic lethality(24) . alpha4beta7 integrins are expressed on most lymph node T and B cells(25) , on subsets of CD4 memory T cells(18) , and on lymphocytes present in rheumatoid synovium(26) . The alpha4 subunit can be expressed at the cell surface as an uncleaved 150-kDa form, or as proteolytically cleaved fragments of 80 and 70 kDa, designated previously as alpha and alpha, respectively(27) .

Most data accumulated so far on the in vivo role of the alpha4 integrins have come from studies using anti-alpha4 mAb in animal models. Lung antigen challenge(28, 29, 30) , experimental allergic encephalomyelitis(31, 32) , ulcerative colitis(33) , contact hypersensitivity(34, 35) , and diabetes(36, 37, 38) , are among the processes where alpha4 integrins play a significant role. The inhibitory effect of the anti-alpha4 mAb in these processes comes from the blockade of VLA-4/ligand interactions, resulting in an inhibition of leukocyte recruitment.

Functional epitope mapping of the alpha4 subunit with a wide panel of anti-alpha4 mAb revealed the existence of three topographically distinct epitopes(39) . Epitope A anti-alpha4 mAb were able to induce homotypic aggregation, blocked partially adhesion to the CS-1-containing FN-40 fibronectin fragment and did not inhibit adhesion to VCAM-1. Epitope B mAb were subdivided into B1 and B2, both blocking adhesion to FN-40 and VCAM-1, with the difference that B2 mAb also triggered homotypic cell aggregation, while B1 mAb did not(39) . The only effect so far described for epitope C mAb is the blocking of homotypic aggregation (39) .

A precise localization of the alpha4 residues involved in VLA-4-mediated functions will contribute to the understanding of the interactions between VLA-4 and its ligands and could help in the designing of compounds aimed at blocking this interaction during unwanted inflammatory processes. In the present work we have analyzed the effect of amino acid substitutions at selected positions in the amino-terminal end of the alpha4 integrin subunit, in the adhesion of K562 cells expressing transfected mutant alpha4 to fibronectin and VCAM-1, as well as in homotypic cell aggregation.


MATERIALS AND METHODS

Antibodies, Cells, and VLA-4 Ligand Proteins

Monoclonal antibodies used in this study included P3X63(40) , TP1/36 (anti-CD43) (41) , TS2/16, Lia 1/2 and Lia 1/5 (anti VLA-beta1 subunit)(42, 43) , P1D6 (anti-alpha5)(44) , and the following anti-alpha4: HP1/3, HP1/1, HP1/7, HP2/1, HP2/4(45) , and B-5G10(46) . The human cell line K562 was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics (complete medium). alpha4 transfectant cells were maintained in the same medium containing 1.2 mg/ml G418 (Life Technologies, Inc.). The FN-40 (heparin II-binding domain) fragment from plasma fibronectin was prepared as described previously(7) . For the generation of recombinant soluble VCAM-1 (sVCAM-1), domains 1-4 of VCAM-1 were fused to the Fc portion of IgG1 in the pCD8IgG1 vector (47) and transfected into COS-7 cells. Supernatants from transfectants were precipitated with ammonium sulfate, and VCAM-1-4D-Fc was isolated by protein A-Sepharose (Pharmacia Biotech Inc., Sweden).

Site-directed Mutagenesis and Transfections

The alpha4 cDNA in Bluescript (48) was subjected to site-directed mutagenesis using the Bio-Rad Muta-Gene kit, as recommended. Oligonucleotides representing alpha4 base pairs 394-420 (for R89S/D90A), 430-456 (for Q101H/P102L), 453-477 (for I108M), 513-544 (for G130R), and 538-568 (for D138Q/L139V), were used as primers containing selected mismatches. Base substitutions at sites of mutations raised new endonuclease restriction sites, which facilitated the identification of the mutations introduced that were later confirmed by DNA sequencing using a T7 sequencing kit (Pharmacia). Mutated alpha4 inserts were then excised and ligated into the pFNeo expression vector (49) in SalI-XbaI sites as described elsewhere(8) . For transfection, 25 µg of pFNeo vector containing mutant or wild type alpha4 was electroporated into 10^7 K562 cells, and after 48 h, 1.2 mg/ml G418 was added to the medium. G418-resistant cells were analyzed by flow cytometry, using a Coulter Epics XL, and enriched for VLA-4-positive cells by immunomagnetic bead selection (Dynal Co. Norway), using the anti-alpha4 mAb B-5G10.

Cell Surface Iodination and Immunoprecipitation

K562 cells were surface-labeled with Na[I] (Amersham Corp., UK) and solubilized as previously reported(27) . For immunoprecipitation, the supernatants were precleared with protein A-Sepharose beads, followed by incubation at 4 °C with antibodies. The immunocomplexes were harvested by incubation with protein A-Sepharose beads, boiled, and analyzed by SDS-PAGE using 7% polyacrylamide gels and nonreducing conditions.

Cell Adhesion and Aggregation Assays

For cell adhesion, transfectants or K562 cells were labeled in complete medium with the fluorescent dye BCECF-AM (Molecular Probes, The Netherlands), and added in RPMI medium containing 0.4% bovine serum albumin to 96-well dishes (Costar) (5 times 10^4 cells/well) previously coated with FN-40 or sVCAM-1. After incubation for 20 min at 37 °C, unbound cells were removed by three washes with RPMI medium, and adhered cells were quantified using a fluorescence analyzer (CytoFluor 2300, Millipore Co.). Homotypic cell aggregation assays were performed essentially as previously described(50) . Briefly, 10^5 cells in complete medium were incubated with 1/20 final dilution of culture supernatant mAb, and the degree of cell aggregation was measured at 3, 7, and 20 h in a semiquantitative manner using the method described by Rothlein and Springer(51) .


RESULTS

Expression of Mutated alpha4 Subunits

Analyses of hydrophilicity profiles according to Kyte and Doolitle (52) and surface probability according to Emini (see Kyte and Doolitle(52) ), indicated that within the first 200 amino acids in the amino-terminal end of the integrin alpha4 subunit there are several regions ranging from 4 to 10 amino acids long showing high indexes of surface probability (Fig. 1A). Residues Arg-Asp are contained inside one of these regions and show the highest surface probability index of this amino-terminal end. Other amino acid stretches displaying high indexes of surface exposure include amino acids Gln, Pro, Asp, and Leu (Fig. 1A). Therefore, we performed site-directed mutagenesis at residues Arg-Asp, Gln-Pro, and Asp-Leu, according to the scheme shown on Fig. 1B. We also made single mutations at residues Ile and Gly, which exhibit low surface exposure indexes (Fig. 1, A and B). The mutated, as well as wild type, full-length alpha4 cDNAs in the expression vector pFNeo were transfected into VLA-4-negative K562 cells, and the resulting geneticin-resistant transfectants were designated as shown in Fig. 1B.


Figure 1: A, surface probability profile of the first amino-terminal 200 amino acids from the integrin alpha4 subunit. The index of surface probability according to Emini (see Kyte and Doolitle(52) ) for each alpha4 residue is plotted. The amino acids subjected to site-directed mutagenesis are indicated. B, schematic diagram of the amino-terminal end of alpha4 and site-directed mutagenesis of the alpha4 cDNA. Shown are the double and single mutations introduced on selected alpha4 residues and the designation of the alpha4 transfectants. C denotes the cysteines included in the first 200 amino-terminal amino acids.



Expression of alpha4 by the transfectants was analyzed by flow cytometry using anti-alpha4 mAb recognizing alpha4 epitopes A (HP1/1, HP1/3, HP1/7), B1 (HP2/1), B2 (HP2/4), and C (B-5G10)(39) . Wild type alpha4 transfectants, here called 4M7, as well as QP(101-102)HL and I108M mutants, expressed all alpha4 epitopes at comparable levels ( Fig. 2and Table 1). However, the RD(89-90)SA mutants consistently showed a weaker staining with anti-alpha4 epitope A antibodies, compared to the staining with antibodies recognizing epitopes B1, B2, and C on the same cells ( Fig. 2and Table 1). On average, the decrease in the mean fluorescence values obtained in the RD(89-90)SA transfectants with the anti-alpha4 epitope A mAb HP1/1 and HP1/7 were 52 and 45%, respectively, of those obtained with the HP2/4 (epitope B2), and 48 and 38% compared with those obtained with B-5G10 (epitope C). Flow cytometry analysis of G130R transfectants showed only weak alpha4 expression detectable with the epitope C B-5G10 antibody, while no staining was detected with any anti-alpha4 antibody in the DL(138-139)QV transfectants through all rounds of transfections (Table 1).


Figure 2: Flow cytometry analysis of K562 alpha4 transfectants. 4M7 (wild type alpha4 transfectants), and the alpha4 mutant RD(89-90)SA, QP(101-102)HL, and I108M cells were incubated at 4 °C for 40 min with saturating concentrations of control P3X63 mAb, anti-beta1 (TS2/16) mAb, or the anti-alpha4 mAbs HP1/1, HP1/3, HP1/7 (Epitope A), HP2/1 (Epitope B1), HP2/4 (Epitope B2), and B-5G10 (Epitope C). After washing, cells were incubated at 4 °C for 30 min with FITC-conjugated secondary antibody (Dako, Denmark) and finally analyzed using a Coulter Epics XL flow cytometer.





We next surface iodinated wild type and mutant alpha4 transfectants, and after solubilization the cell extracts were immunoprecipitated with anti-alpha4 antibodies representing epitopes A, B1, and C, as well as with anti-beta1 antibodies, followed by SDS-PAGE. The 4M7, QP(101-102)HL, and I108M cells showed a characteristic pattern of alpha4 structural forms in alpha4-K562 transfectants(27) . Thus, most of alpha4 is expressed at the cell surface as a cleaved alpha form, with little expression of the uncleaved alpha form (Fig. 3). No differences in terms of alpha4 structure and expression levels with the various anti-alpha4 antibodies were found amongst these cells (Fig. 3). In contrast, the anti-alpha4 epitope A HP1/1 and HP1/7 immunoprecipitates from the RD(89-90)SA transfectants showed a dramatic reduction in the amount of cell surface alpha4 subunit compared with that found in HP2/1 and B-5G10 immunoprecipitates, which were identical to their counterparts in the other three transfectants (Fig. 3). Also, anti-beta1 immunoprecipitates from RD(89-90)SA cells did not change with respect to the anti-beta1 immunoprecipitates from 4M7, QP(101-102)HL, and I108M cells (Fig. 3). Together with the flow cytometry analyses, these results indicate that alpha4 residues Arg-Asp are contained within the region corresponding to alpha4 epitope A.


Figure 3: Immunoprecipitation of wild type and mutated forms of the alpha4 subunit. 4M7, RD(89-90)SA, QP(101-102)HL, and I108M alpha4 transfectants were labeled with I and solubilized, and the cell extracts were immunoprecipitated with saturating concentrations of the anti-alpha4 mAbs HP1/1 or HP1/7 (epitope A), HP2/1 (epitope B1), B-5G10 (epitope C), anti-beta1 mAb TS2/16, or control mAb P3. Immunoprecipitates were analyzed under nonreducing conditions by SDS-PAGE and autoradiography. The migration of the different structural forms of the alpha4 subunit and beta1 subunit is indicated. The migration of the alpha5 integrin subunit which is expressed by parental K562 is indicated, as obtained by immunoprecipitation with the anti-alpha5 P1D6 mAb (not shown).



Absence of Homotypic Aggregation by RD(89-90)SA Transfectants in Response to Epitope A Anti-alpha4 Antibodies

To characterize the effect of the mutations in the alpha4 subunit on VLA-4-mediated functions, we first analyzed the homotypic aggregation in the various alpha4 transfectants in response to different anti-alpha4 mAb recognizing distinct alpha4 epitopes(39) . We found that 4M7, QP(101-102)HL, and I108M cells aggregated equally well in response to epitope A anti-alpha4 mAb HP1/3 and HP1/7, as well as to the HP2/4 (epitope B2) and to the anti-beta1 mAb Lia 1/2 and Lia 1/5 ( Table 2and Fig. 4). However, the RD(89-90)SA transfectants showed a severely impaired capability to aggregate in response to HP1/3 and HP1/7 mAb, while displaying high cell aggregation in response to the HP2/4 and anti-beta1 mAb ( Table 2and Fig. 4). These effects were evident as early as 3 h after the addition of the antibodies, and were maintained for 20 h. The epitope C anti-alpha4 mAb B-5G10 inhibited the homotypic aggregation in all transfectants. On the other hand, the different transfectants, as well as the untransfected K562 cells aggregated similarly in response to the anti-CD43 mAb TP1/36 (Table 2). Consequently, the loss of cell aggregation induced by HP1/7 and HP1/3 mAb, allowed us the identification of residues Arg-Asp as part of the alpha4 epitope involved in homotypic cell aggregation.




Figure 4: Loss of homotypic cell aggregation in the RD(89-90)SA transfectants induced by the anti-alpha4 epitope A mAb HP1/7. 4M7, RD(89-90)SA, and QP(101-102)HL alpha4 transfectants (10^5 cells/well) were incubated at 37 °C with the anti-alpha4 epitope A mAb HP1/7 or with the the anti-alpha4 epitope B2 mAb HP2/4. Photographs were taken 3 h after the addition of antibodies.



RD(89-90)SA Transfectants Show a Reduced Adhesion to FN-40, but Not to VCAM-1

The cell adhesion capability to FN-40 and VCAM-1 of the different alpha4 transfectants was analyzed, either in the absence or in the presence of the anti-beta1 mAb TS2/16, which has been previously shown to enhance cell adhesion mediated by VLA beta1 integrins, including VLA-4, to a variety of ligands(53) . In the absence of TS2/16, 4M7, QP(101-102)HL, and I108M cells all adhered to FN-40 in a concentration-dependent manner, whereas the RD(89-90)SA transfectants consistently showed a diminished adhesion ability compared to wild type and to the other alpha4 mutant transfectants (Fig. 5). In average, the reduction of RD(89-90)SA cell adhesion to FN-40 compared to the 4M7 adhesion was about 40%. The adhesion to FN-40 of QP(101-102)HL was slightly higher than that of 4M7 cells and adhesion of I108 M was very similar to 4M7. All alpha4 transfectants analyzed in cell adhesion assays to FN-40 in the presence of TS2/16 showed an increase in adhesion to this ligand, and the RD(89-90)SA transfectant adhesion levels were restored to adhesion levels obtained with the alpha4 wild type 4M7, as well as with alpha4 mutant QP(101-102)HL and I108 M transfectants (Fig. 5). These results indicate that the potential of the alpha4beta1 heterodimer carrying substitutions at residues Arg and Asp in adopting a high affinity conformation is not affected by these mutations.


Figure 5: Adhesion of alpha4 transfectants to FN-40. 4M7, RD(89-90)SA, QP(101-102)HL, and I108M cells were labeled with BCECF-AM, incubated in the absence or in the presence of the anti-beta1 mAb TS2/16 (1/6 final dilution from culture supernatant) for 20 min at 37 °C, and added to 96-well microtiter plates coated with increasing concentrations of FN-40. After 20 min of incubation at 37 °C, plates were washed, and adhesion was quantified using a fluorescence analyzer. Each point represent the mean of triplicate determinations, with a standard deviation of <10%. K562 cells showed no adhesion at any concentration of FN-40 tested (not shown).



When we analyzed cell adhesion to sVCAM-1, no significant differences among the 4M7, RD(89-90)SA, QP(101-102)HL, and I108M transfectants were observed, both in the absence and in the presence of TS2/16 (Fig. 6). In addition, the increment of cell adhesion to sVCAM-1 in the presence of TS2/16 was lower than that observed in the case of FN-40, and was observed mainly at lower sVCAM-1 concentrations (Fig. 6).


Figure 6: Adhesion of alpha4 transfectants to sVCAM-1. 4M7, RD(89-90)SA, QP(101-102)HL, and I108M cells were labeled with BCECF-AM and processed for adhesion to increasing concentrations of sVCAM-1 as in Fig. 5. Adhesion was carried out at 37 °C for 20 min and quantified in a fluorescence analyzer. Each point represent the mean of triplicate determinations, with a standard deviation of <10%. K562 cells showed no adhesion at any concentration of sVCAM-1 tested (not shown).




DISCUSSION

In the present study we show that residues Arg and Asp form part of the integrin alpha4 subunit epitope A. Substitutions at these residues resulted in a diminished capability of three anti-alpha4 epitope A mAb (HP1/1, HP1/3, and HP1/7) to recognize the alpha4 subunit expressed by RD(89-90)SA transfectants, which correlated with a decrease in cell adhesion to the FN-40 fragment of fibronectin and lack of homotypic cell aggregation of these transfectants. Anti-alpha4 mAb specific to epitopes B1, B2, and C recognized the alpha4 molecule in the RD(89-90)SA transfectants in a similar fashion to wild type alpha4 transfectants, and complete homotypic cell aggregation in the RD(89-90)SA transfectants was obtained using either anti-alpha4 epitope B2 or anti-beta1 mAb, indicating that the mutations did not affect the overall conformation of alpha4. These results indicate that the alpha4 residues Arg and Asp are involved in the interaction of VLA-4 with FN-40, as well as in homotypic cell aggregation triggered by epitope A anti-alpha4 mAb. It is unlikely that substitutions at Arg and Asp modified nearby cysteine disulfide bonds, since these residues do not appear to be in the middle of such a bond, according to the alpha4 disulfide bond pairing disposition shown earlier (54) , based in a comparison with the alphaIIb disulfide bond pairing previously assigned(55) . Substitutions at residues Arg and Asp did not affect the adhesion of RD(89-90)SA transfectants to VCAM-1, indicating that these residues do not appear to be essential in the interaction between VLA-4 and VCAM-1.

Interestingly, the Arg alpha4 residue posses the highest surface index probability of the alpha proteolytic N-terminal fragment. The alpha4 proteolytic cleavage site located between residues Lys-Arg and Ser(27) , also shows a very high surface index probability, probably reflecting a surface exposure to proteases. This suggests that the Arg and Asp residues indeed may be on the surface of the alpha4 subunit, which could account for their functional relevance.

The diminished adhesion of RD(89-90)SA cells to FN-40 in the absence of TS2/16 was in average 40% with respect to wild type alpha4 transfectants. This result is in accordance with a previous report showing that adhesion to FN-40 could be partially inhibited (up to 60%) by anti-alpha4 epitope A mAb(39) . The same studies indicated that alpha4 epitopes B1 and B2 were involved in the interaction of VLA-4 with FN-40, which could be completely blocked by a panel of anti-alpha4 mAb. Using alpha4 murine/human chimeric constructs expressed in mammalian cells, it has recently been shown that epitope A mAb mapped to the most alpha4 N-terminal 100 amino acids in one case(56) , whereas another report found that residues 1-38 contained the epitope A(57) . Moreover, they reported that B1 and B2 epitopes mapped to alpha4 residues 152-203(56) , while the other obtained different sites for the B1 (alpha4 195-268) and B2 (alpha4 108-182) epitopes(57) . In spite of these differences which could be solved by performing point mutations, it is clear that a region C-terminal to residue 108 contains the main site of alpha4 involved in the interaction with the CS-1 domain of fibronectin, and that, as demonstrated in this report, additional binding sites are found in a region which includes residues Arg and Asp. Our data confirm that these residues do not form part of the B1 and B2 epitopes. The possibility of the presence of several sites in alpha4 capable to interact with fibronectin could result in a potential strengthening of the VLA-4-mediated adhesion. It is conceivable that alpha4 residues Arg and Asp might form part of an alpha4 domain interacting with another site contained inside the FN-40 fragment of fibronectin, such as the H1 site (58) , and that perhaps these interactions might be differentially regulated.

The finding that alpha4 residues Arg and Asp are included in the region promoting homotypic cell aggregation agrees with the alpha4 epitope mapping studies mentioned earlier(56) . Homotypic cell interaction mediated by VLA-4 might play a relevant role in processes such as extracellular matrix invasion by melanoma cells during metastasis, where homotypic interactions between these cells results in a reduction of invasion(20) . In this regard, it has been reported that the alpha4 integrin chain can directly interact with alpha4beta1 and alpha4beta7(59) , suggesting that cells could use the alpha4 integrins to interact in a homotypic manner. The results from the present work do not distinguish between a role of Arg and Asp alpha4 residues in the initial steps leading to cell aggregation, or as part of the alpha4 molecule directly interacting with other alpha4 integrins or with unknown ligands.

Residues Gln and Pro are also included in an alpha4 region with high surface probability index. However, substitutions at these residues did not affect binding of any anti-alpha4 mAb to the alpha4 subunit expressed by the QP(101-102)HL transfectants, nor altered the adhesive functions of these cells. Transfectants expressing the alpha4 subunit with mutations at residue Gly showed only minor reactivity with anti-alpha4 epitope C mAb, while no stable transfectants were obtained with mutations at residues Asp and Leu. Both Asp and Gly residues are highly conserved among integrin alpha subunits, which could suggest that alteration at this region of the alpha4 subunit might result in an improper folding of the molecule and/or an inability to associate with the beta1 subunit.

Previous studies have shown that mutations at the alpha4 putative divalent cation sites (included in the region between residues 281 and 414) resulted in a reduction of VLA-4 interaction with both CS-1/fibronectin and VCAM-1(60) . Our results show that a region included within the first N-terminal 100 amino acids of the integrin alpha4 subunit participates in certain VLA-4-mediated cell adhesion functions, namely cell aggregation and binding to fibronectin. The alpha4 residues involved in the interaction of VLA-4 with VCAM-1, as well as those residues forming part of the epitope B1 and B2 involved in adhesion to CS-1, remain to be identified. The results from the present study provide an insight into the integrin alpha4 regions involved in the interaction of VLA-4 with its ligands. This information could be useful in future designing of products aimed at interrupting these interactions in several inflammatory and autoimmune pathologies.


FOOTNOTES

*
This work was supported by a grant from SmithKline Beecham-CDTI (Spain). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Centro de Investigaciones Biológicas, Dept. de Inmunología, Velázquez 144, 28006 Madrid, Spain. Tel.: 34-1-5611800 (ext. 4392); Fax: 34-1-5627518.

(^1)
The abbreviations used are: CS-1, connecting segment-1; VCAM-1, vascular cell adhesion molecule-1; sVCAM-1, soluble VCAM-1; MadCAM, mucosal addressin cell adhesion molecule; BCECF-AM, 2`,7`-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester; mAb, monoclonal antibody; FN-40, fibronectin-40.


ACKNOWLEDGEMENTS

We thank José Luis Alonso and Reyes Tejedor for their help in the generation of sVCAM-1, Pedro Lastres and Mayte Vallejo for their assistance in flow cytometry and with the fluorescence analyzer, and Dr. M^a Luisa Botella for her help in DNA sequencing.


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