©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Aspartate 698 within a Novel Cation Binding Motif in Integrin Is Required for Cell Adhesion (*)

(Received for publication, November 10, 1994; and in revised form, May 24, 1995)

Lan Ma Patricia J. Conrad Deborah L. Webb Marie-Luise Blue (§)

From the Institute for Bone and Joint Disorders and Cancer, Bayer Research Center, West Haven, Connecticut 06516

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The interactions of alpha(4)beta(1) integrin with vascular cell adhesion molecule (VCAM) and fibronectin play important roles in many physiological and pathological processes. To understand the mechanism of alpha(4)beta(1) integrin-mediated cell adhesion, we made mutant alpha(4) constructs. Three aspartic acid (Asp) residues in alpha(4), Asp-489, Asp-698, and Asp-811, were replaced with glutamic acids (Glu). The wild-type and mutant alpha(4) constructs were transfected into K562 cells, and stable transfectants with similar levels of alpha(4) surface expression were established. The Asp Glu substitutions did not affect alpha(4)beta(1) association or heterodimer formation as demonstrated by immunoprecipitation analysis. However, the glutamate substitutions at Asp-489 and Asp-698 severely impaired cell adhesion to VCAM and fibronectin, whereas the substitution at Asp-811 had no detectable effect on cell adhesion. In contrast to these results, isolated alpha(4)beta(1), containing the D489E or D698E substitution, was able to bind to VCAM, suggesting that these two residues are not critical for ligand recognition. In searching for a mechanism to explain inhibition of adhesion by Asp-489 and Asp-698 mutations, we found that the sequences flanking Asp-698 resemble the DxxxxxD-S-Sx divalent cation/ligand binding motif in beta integrins and the I-domains of alpha integrins. This suggests that Asp-698 in the alpha(4) integrin, which does not possess an I-domain, may also be involved in cation binding and may be part of a sequence functionally similar to that found in the I-domains of other alpha integrins.


INTRODUCTION

Integrins are structurally related cell surface receptors involved in cell-cell adhesion and cell attachment to the extracellular matrix. They are noncovalently associated heterodimers of alpha and beta subunits (1) . Many different alpha subunits are able to associate with the same beta subunit to form alpha/beta dimers with distinct ligand specificity. The ligand specificity of integrins are, therefore, believed to be determined largely by the structures of the alpha subunits.

The alpha(4)beta(1) integrin (VLA-4, CD49d/CD29) is expressed on lymphocytes, monocytes, bone marrow progenitors, and on some tumor cell lines(2, 3, 4, 5) . The ligands for the alpha(4)beta(1) integrin are vascular cell adhesion molecule (VCAM) (^1)and fibronectin. The interaction of alpha(4)beta(1) with its ligands is important in T cell activation, cell adhesion, inflammation, myogenesis, and tumor metastasis(5, 6, 7, 8, 9) . Furthermore, the alpha(4) integrin is critically involved in disease pathogenesis and progression in many animal models such as experimental autoimmune encephalomyelitis, autoimmune diabetes, and late phase antigen-induced asthma(10, 11, 12) . Elucidation of the mechanisms of alpha(4)beta(1)-ligand interaction is important in understanding these physiological or pathological processes and may have great therapeutic potential.

Divalent cations are required for integrins to bind to their ligands (1) . alpha(4) has a large extracellular domain and short cytoplasmic and transmembrane domains. The N-terminal half of the extracellular domain contains 7 homologous segments, which are conserved among all the integrin alpha subunits (Fig. 1). The 5th, 6th, and 7th homologous sequence segments from the N terminus have been identified as putative divalent cation binding sites(1, 13) . Masumoto and Hemler (13) demonstrated the importance of these divalent cation binding sites for alpha(4) function. Mutations at each of the three divalent cation binding sites (N283E, D346E, and D408E) of alpha(4) resulted in significant loss in the ability of alpha(4) to bind VCAM and fibronectin, whereas mutations at Cys-278, Cys-717, Cys-767, and Cys-828, downstream from the cation binding sites, did not affect interaction of alpha(4) with VCAM (13) .


Figure 1: Aspartic acid substitutions in alpha(4). Boxes indicate highly conserved regions in alpha integrins. M indicates a putative divalent cation binding site.



A novel bivalent cation binding site has been identified in the integrin beta subunits(14, 15) . The aspartic acid in this cation binding sequence, conserved among beta subunits, is required for ligand binding(15, 16) . A similar cation binding motif is also found in the I-domain containing alpha subunits(15, 18) . Mutation at the aspartic acid residue in this cation binding site abolishes cell-ligand adhesion and cation binding(15, 17, 19) . Therefore, in addition to the essential role of the classical integrin cation binding sites in cell adhesion, beta subunits and the I-domains in alpha subunits also appear to be critically involved in the interaction of integrins with cations and ligands.

In an attempt to identify the ligand binding site in alpha(4), we made synthetic peptides derived from alpha(4) and examined their abilities to inhibit cell adhesion to VCAM. Our preliminary experiments showed that alpha(4) peptides, containing residues 482-491, 691-700, and 804-813, specifically inhibited the interaction of alpha(4)beta(1) with recombinant VCAM (20) , and the aspartic acid in these peptides appeared to be critical for inhibition. (^2)In this study, we have replaced the three aspartic acid residues, Asp-489, Asp-698, and Asp-811, with glutamic acid and examined the function of the mutated alpha(4) integrins in cell adhesion.


EXPERIMENTAL PROCEDURES

Materials

Restriction enzymes, T4 DNA ligase, and Klenow fragments were purchased from New England Biolabs, and the Sequenase 2.0 Sequencing Kit was obtained from United States Biochemical Corp. Taq DNA polymerase was obtained from Perkin-Elmer Cetus. Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma, and the 40-kDa fragments of human fibronectin (FN-40) were from Life Technologies, Inc. PBS (Bio-Whittaker) contains 10 mM phosphate (pH 7.0), 138 mM NaCl, 2.7 mM KCl, 0.5 mM MgCl(2), and 1 mM CaCl(2).

Antibodies

Antibodies used were as follows: mouse anti-human alpha(4) monoclonal antibodies C212.7(21) , HP2/1 (Amac, Inc., (4) ), B-5G10 (Upstate Biotechnology, (3) ), CIII376.2, a mouse monoclonal antibody raised against peptide LQEENRRDSWSYINSKSNDD in the cytoplasmic portion of alpha(4), mouse anti-human beta(1) monoclonal antibody K20 (Amac, Inc., (22) ), and goat anti-mouse IgG-fluorescein conjugate (Tago).

Mutagenesis

Site-directed mutagenesis was performed by overlap extension in the polymerase chain reaction using pBWT, a pBluescript SK plasmid containing human alpha(4)-cDNA inserted at XhoI/XbaI sites as a template as described (23) . The third base of the aspartic acid (Asp) codon for Asp-489, Asp-698, Asp-811, or Asp-698 and Asp-811 in alpha(4), was mutated from T to A using mutant oligonucleotide primers to generate the codon for glutamic acid (Glu). The mutant alpha(4) cDNA carrying the Asp Glu substitutions were cloned into pBluescript and sequenced to confirm the presence of the intended mutation and absence of any Taq polymerase errors. The four mutant alpha(4)-cDNAs were constructed in pBluescript by exchanging the restriction fragment containing the mutation for the corresponding wild-type sequence. The wild-type and mutant alpha(4) cDNAs were subcloned into pcDNA3 (Invitrogen) and designated as pcWT (wild-type), pcD489E, pcD698E, pcD811E (carrying Asp Glu substitution at Asp-489, Asp-698, and Asp-811, respectively), and pcLEV (carrying Asp Glu triple substitutions at Asp-489, Asp-698, and Asp-811).

Transfection

K562 cells were maintained in RPMI 1640 media with 5% fetal calf serum. Cells (5 10^6) were transfected with 20 µg of linearized alpha(4) pcDNA3 constructs by electroporation at 270 V/0.4 cm and 960 microfarads in 0.4 ml of PBS using a Gene Pulser (Bio-Rad). After 48 h, transfected cells were placed in medium containing 0.4 mg/ml Geneticin (Life Technologies, Inc.). Geneticin-resistant cells were subjected to several rounds of fluorescence-activated cell sorting (FACS) using mAb C212.7 and a FACStar Plus cytometer (Becton Dickinson) to enrich and to clone alpha(4) expressing stable transfectants.

Flow Cytometry

Cells were incubated with primary mouse mAb in PBS containing 10% human serum (Life Technologies, Inc.) on ice for 20 min. After washing three times in PBS, the cells were treated with goat anti-mouse IgG coupled to fluorescein (Tago) in PBS containing 2% fetal calf serum. The cells were washed twice in PBS, resuspended in the same buffer, and analyzed using a FACScan cytometer (Becton Dickinson).

Immunoprecipitation and Western Blotting

Cells were washed with cold PBS, and samples were adjusted to equal cell numbers and cell densities of 10^7/ml. Cells were then incubated with PBS containing 0.2 mg/ml sulfosuccinimidobiotin (Pierce) and 14 mM glucose at room temperature. After 30 min, glycine was added to a final concentration of 4 mM, and the cell suspension was incubated on ice for 15 min. Cells were washed three times in PBS and then lysed on ice in buffer containing 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM MgCl(2), 1 mM CaCl(2), 1% Nonidet P-40. Cell lysates were precleared by centrifugation followed by incubation with GammaBind G Sepharose (Pharmacia Biotech) and recentrifugation. Aliquots of the lysates were incubated with specific integrin antibodies overnight at 4 °C, subsequently absorbed onto GammaBind G Sepharose, followed by washing in buffer containing 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM MgCl(2), 1 mM CaCl(2), 0.1% Nonidet P-40, and 0.05% Tween 20. Absorbed complexes were removed from the beads by heating in reduced SDS-PAGE sample buffer at 65 °C for 30 min, and the supernatants were analyzed on 4-12% polyacrylamide gradient gels (Novex). Proteins resolved on gels were transferred onto nitrocellulose membranes (Schleicher & Schuell), and the membranes were first incubated in 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5% nonfat dry milk for 1 h and then incubated with streptavidin-horseradish peroxidase conjugate (Boehringer Mannheim) in PBS containing 0.1% Tween 20. Biotinylated proteins were detected using the ECL kit (Amersham Corp.) according to manufacturer-suggested protocol.

Adhesion Assays

Assays for cell adherence to ligand proteins were carried out essentially as described(20) . Briefly, cells were labeled by incubating with 6-carboxyfluorescein diacetate and were added, with or without PMA or antibodies, to 96-well microtiter plates coated with recombinant VCAM, consisting of domains 1 to 3 of VCAM (VCAM1-3)(20), or coated with FN-40. Nonspecific sites on the plates were blocked with 1% bovine serum albumin. After a 25-min incubation at room temperature, unbound cells were removed by washing and low speed centrifugation with the plates inverted. Cells remaining bound to the plates were quantitated using a Cyto Fluor 2300 fluorimeter (Millipore). Nonspecific background cell binding was below 2% under these conditions.

Integrin-Ligand Binding Assays

Two approaches were used to assess the ability of alpha(4) integrin to bind to VCAM. In assay A, purified recombinant human VCAM1-3 was coupled to Sepharose 4B. 5 10^6 cells were surface-labeled with biotin and lysed in 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM MgCl(2), 1 mM MnCl(2), and 50 mM beta-octyl glucoside (Buffer A). Insoluble material was removed by centrifugation, and the supernatants were incubated with Sepharose 4B in Buffer A containing 5 mg/ml myoglobin. After centrifugation, the supernatants were then incubated with VCAM-Sepharose at 4 °C for 3 h. The VCAM-Sepharose was washed four times in Buffer A. One-half of the VCAM-Sepharose was incubated in Buffer A containing 20 mM EDTA overnight, and eluted alpha(4) was immunoprecipitated with mAb CIII376.2 and GammaBind G Sepharose. The biotinylated proteins bound to Sepharose were analyzed on 4-12% SDS-PAGE gels and detected with streptavidin peroxidase conjugate, using ECL following Western blotting. In assay B, CIII376.2, a mAb against the C-terminal peptide of alpha(4), was coupled to Sepharose. 1 10^7 cells were lysed in Buffer A or 50 mM Tris, pH 7.5, 150 mM NaCl, 0.5 mM MgCl(2), 1 mM CaCl(2), and 50 mM beta-octyl glucoside (Buffer B). After a brief centrifugation, the supernatants were first precleared with Sepharose 4B and then were incubated with CIII376.2-Sepharose or Sepharose (control) overnight at 4 °C. After washing, aliquots of the Sepharose were incubated with various concentrations of biotinylated VCAM1-3 in Buffer A or Buffer B containing 5 mg/ml myoglobin at 4 °C for 2 h. The Sepharose was recovered by centrifugation and washed in the same buffer. Biotinylated VCAM bound to the Sepharose was analyzed as described above. An aliquot of the cells (1 10^6) was surface-labeled with biotin, and the cell lysate was immunoprecipitated with mAb CIII376.2 as a control for alpha(4) surface expression.


RESULTS

Construction and Expression of Wild-type and Mutant alpha(4)

Asp-489, Asp-698, and Asp-811 of alpha(4) (Fig. 1) were substituted with glutamic acid by site-directed mutagenesis. Stably transfected cell lines expressing wild-type alpha(4) (K-WT) and alpha(4) containing glutamic acid substitutions at the three aspartic acid residues Asp-489, Asp-698, and Asp-811 (K-LEV) were established in K562. Five clones from each transfectant were selected, and the clones showed comparable levels of alpha(4) surface expression by immunofluorescence staining (data not shown). Clones K-WT.1 and K-LEV.5 were characterized in the present study. Surface expression of alpha(4)beta(1) integrin was analyzed by flow cytometry using mAb C212.7. As shown in Fig. 2A, alpha(4) surface expression on cell line K562 was not detectable. Both K-WT and K-LEV showed similar levels of alpha(4) surface expression (Fig. 2, E and I). The data suggest that the mutations at the three aspartic acid residues did not have a significant effect on the biosynthesis and surface expression of alpha(4). As shown in Fig. 2D, K562 expresses abundant beta(1) subunits. When transfected with pcWT, the surface expression of beta(1) increased significantly (Fig. 2H). The pcLEV transfectant also exhibited elevated beta(1) expression (Fig. 2L).


Figure 2: Surface expression of alpha(4)beta(1) by K562, K-WT, and K-LEV. Flow cytometric analysis of cell lines K562 (A-D), K-WT (E-H), and K-LEV (I-L) were carried out using the mAbs C212.7 (A, E, and I), HP2/1 (B, F, and J), B-5G10 (C, G, and K), and K20 (D, H, and L), followed by incubation with fluorescein-labeled goat anti-mouse IgG. Samples treated with integrin antibodies are represented as shaded profiles superimposed on the open profiles representing control staining.



To examine whether the Asp Glu triple substitutions cause significant changes in alpha(4) secondary structure, the mutant alpha(4) transfectant was analyzed using different alpha(4) mAbs for immunofluorescent staining (Fig. 2). As shown in Fig. 2I, the epitope for alpha(4) mAb C212.7 was retained on alpha(4)LEV, indicating that the mutations do not disrupt the structure of this epitope. Two other alpha(4) mAbs recognizing different epitopes on alpha(4), HP2/1 and B-5G10, were also tested. Both antibodies recognized alpha(4)LEV (Fig. 2, J and K). It has been shown that mAb C212.7 and HP2/1 block alpha(4) binding to VCAM and fibronectin^2(17, 24, 25) . The epitopes that mAb HP2/1 and B-5G10 recognize belong to previously defined B1 and C epitope groups of alpha(4), respectively(24) . The FACS analysis (Fig. 2) suggests that the mutations at Asp-489, Asp-698, and Asp-811 do not affect the structures of these epitopes, and, therefore, the mutations probably do not cause any significant change in the global secondary structure of alpha(4).

Surface expression of alpha(4) on K-WT or K-LEV transfectants was also analyzed by immunoprecipitation (Fig. 3). Both the alpha(4)WT and alpha(4)LEV proteins show identical patterns: they are present on the cell surface in a cleaved form (80 kDa and 70 kDa) and are associated with the endogenous beta(1) subunits (120 kDa). The results indicate that the Asp Glu substitutions do not affect the in vivo cleavage of alpha(4), alpha(4)beta(1) heterodimer formation, or the stability of the complex.


Figure 3: Analysis of alpha(4)beta(1) surface expression on K-WT and K-LEV by immunoprecipitation. K562, Jurkat, K-WT, and K-LEV were surface-labeled with biotin. Cell extracts were incubated without (Control) or with various antibodies as indicated. The lysates were then precipitated with protein G-Sepharose. Proteins bound to G-Sepharose were analyzed by SDS-PAGE and Western blotting.



Impaired Adhesion of K-LEV to VCAM and Fibronectin

Adhesion of K-WT and K-LEV to immobilized recombinant human VCAM1-3 was compared. As illustrated in Fig. 4A, K-WT bound to VCAM strongly, whereas K-LEV was much less adherent to the same ligand. At ligand coating concentrations of 2.5 to 5 µg/ml, adhesion by K-LEV was approximately 4-6-fold lower than adhesion by K-WT. Similar results were obtained with adhesion to murine L-cell transfectants expressing the 7-domain form of human VCAM (data not shown)(20) . The control cell line K562, showed little, if any, binding to recombinant VCAM, indicating that the adhesion was dependent on transfection of alpha(4). As demonstrated in Fig. 4B, the adhesion of K-WT or K-LEV transfectants to VCAM could be blocked completely by mAb C212.7, which is directed against the alpha(4) subunit of VLA-4. The adhesion could also be blocked by an antibody against human VCAM (data not shown). This indicates that the adhesion of alpha(4) transfectants to immobilized VCAM is an alpha(4)- and VCAM-dependent specific interaction.


Figure 4: Adhesion of K-WT and K-LEV to VCAM. A, K-WT (bullet) and K-LEV () were tested for adhesion to microtiter plates coated with the indicated amounts of recombinant VCAM1-3. ♦, K562 cells. B, K-WT and K-LEV were tested for adhesion to microtiter plates coated with recombinant VCAM1-3 (2.5 µg/ml) in the presence of PMA (10 ng/ml) and the presence or absence of C212.7 Ab. Control wells were coated with bovine serum albumin only.



The avidity of integrins can be regulated through inside-out cell signaling. The avidity of VLA-4 increases upon activation with phorbol esters(26, 27) . Both K-LEV and K-WT could respond to PMA stimulation (Fig. 5A). However, PMA failed to fully restore the capability of K-LEV to bind VCAM. In addition, the alpha(4) antibody C212.7 triggered homotypic cell aggregation of K-LEV (data not shown), suggesting that the mutant VLA-4 integrin is also functional in outside-in cell signaling.


Figure 5: Effect of PMA stimulation on adhesion of K-WT and K-LEV. A, adhesion of K-WT (WT) and K-LEV (LEV) in the absence and presence of PMA (10 ng/ml) to recombinant VCAM1-3 at coating concentrations of 2.5 and 5 µg/ml. B, K-WT, K-LEV, and K562 were tested for adhesion to microtiter plates coated with the indicated amounts of FN-40 in the presence of PMA.



The ability of K-LEV to adhere to fibronectin was also tested. FN-40 is a chymotryptic fragment of human fibronectin(28) . It contains the sequence essential for alpha(4)beta(1) recognition (29) . Adhesion of K-WT to FN-40 required PMA stimulation. K-LEV failed to adhere to FN-40 under these conditions (Fig. 5B).

Impaired Adhesion of K-D489E and K-D698E to VCAM and Fibronectin

Mutations at all three aspartic acid residues of the alpha(4) subunit resulted in decreased cell adhesion to fibronectin and VCAM. To determine the contribution of each substitution, mutant alpha(4) constructs, pcD489E, pcD698E, and pcD811E, each of which contains a glutamic acid substitution at only one of the aspartic acid residues, were transfected into K562. The pcWT construct was used as a control. Stable clonal transfectants (K-WT, K-D489E, K-D698E, K-D811E) with similar alpha(4) surface expression were obtained following repeated rounds of cell sorting. K562, K-WT, K-D489E, K-D698E, and K-D811E were analyzed for alpha(4) surface expression (Fig. 6). As expected, the mutant and wild-type transfectants expressed alpha(4) at comparable levels, and the alpha(4) antibodies C212.7, HP2/1, and B-5G10 recognized all three mutant alpha(4) subunits (Fig. 6).


Figure 6: Analysis of surface expression of alpha(4)WT, alpha(4)D489E, alpha(4)D698E, and alpha(4)D811E. FACS analysis of cell lines K562 (A, F, and K), K-WT (B, G, and L), K-D489E (C, H, and M), K-D698E (D, I, and N), and K-D811E (E, J, and O) was carried out using the mAb C212.7 (A-E), HP2/1 (F-J), and B-5G10 (K-O), followed by incubation with fluorescein-labeled goat anti-mouse IgG. Fluorescence histograms of anti-alpha(4) mAb-treated samples are represented as shaded profiles superimposed on the open profiles of control samples.



The mutant alpha(4) transfectants K-D489E, K-D698E, and K-D811E were compared with K-WT in adhesion assays for their abilities to bind VCAM and fibronectin. Transfectant/VCAM adhesion is shown in Fig. 7A. The percentage of K-D811E cells adhering to VCAM is similar to that of K-WT. However, the binding of K-D489E and K-D698E transfectants to VCAM was significantly lower than that of K-WT. Their adhesion to VCAM (at 3.5 µg/ml) was only 10-20% of that of K-WT. Even at a higher concentration of VCAM (7 µg/ml), K-D489E and K-D698E adhered poorly. These results suggest that aspartate 811 in alpha(4) is not essential for VCAM binding. In contrast, Asp-698 and Asp-489 are required for VLA-4-mediated cell adhesion.


Figure 7: Adhesion of K-WT (), K-D489E (▴), K-D698E (▪), and K-D811E (). A, transfectants were tested for adhesion to microtiter plates coated with the indicated amounts of recombinant VCAM1-3. B, transfectants were tested for adhesion to microtiter plates coated with the indicated amounts of FN-40 in the presence of PMA.



It is believed that the ligand binding sites on alpha(4) for VCAM and fibronectin are identical or overlapping(24, 25, 30) . To examine the effect of the Asp Glu substitutions on fibronectin binding, the abilities of K-D489E, K-D698E, and K-D811E to adhere to FN-40 were tested. As shown in Fig. 7B, K-WT adhered to FN-40 upon PMA activation. Adhesion of K-D811E was comparable to that of K-WT. However, K-D489E and K-D698E failed to adhere to FN-40 under the same conditions.

Binding of Wild-type and Mutant alpha(4)to VCAM

The direct binding of ligand to wild-type and mutant alpha(4) isolated from K-WT, K-LEV, K-D489E, and K-D698E was assessed using either surface-labeled cell extracts and VCAM-Sepharose or biotinylated VCAM1-3 and affinity-purified alpha(4)beta(1) (Fig. 8). As indicated in Fig. 8A, alpha(4)beta(1) complexes from both K-WT and K-LEV cells bound to VCAM-Sepharose in a cation-dependent manner. The amount of mutant alpha(4) bound to VCAM-Sepharose was comparable to that of wild-type. In other experiments, wild-type or mutant alpha(4)beta(1) was immobilized on CIII376.2-Sepharose and incubated with various concentrations of biotinylated VCAM1-3. At a VCAM concentration of 0.06 µg/ml, VCAM binding to either wild-type or mutant alpha(4)beta(1) was not detectable (data not shown). As shown in Fig. 8B, at VCAM concentrations of 0.3 and above, the mutant alpha(4)beta(1) from K-LEV, K-D489E, and K-D698E was able to bind VCAM even at low concentrations of cations, and the amounts of VCAM bound to the mutant forms of alpha(4)beta(1) were similar to that bound to the alpha(4)beta(1) from K-WT. These results suggest that substitutions at Asp-489 and Asp-698 are not critical for VCAM recognition.


Figure 8: Binding of VCAM to wild-type and mutant alpha(4). A, extracts of biotin-surface-labeled K-WT and K-LEV cells were precipitated with VCAM-Sepharose. Proteins bound to VCAM-Sepharose (P1) were eluted from VCAM-Sepharose in Buffer A containing 20 mM EDTA and immunoprecipitated with mAb CIII376.2 (P2). Both P1 and P2 were analyzed on 4-12% SDS-polyacrylamide gels. Arrows indicate the 80-kDa and 70-kDa forms of alpha(4). B, alpha(4) in K-WT, K-LEV, K-D489E, and K-D698E cells was immunoprecipitated with CIII376.2-Sepharose 4B (VLA-4) or Sepharose (control). 0.3 µg/ml biotinylated VCAM1-3 was incubated with VLA-4 immobilized on CIII376.2-Sepharose. Biotinylated VCAM bound was analyzed on 4-20% Tricine SDS-polyacrylamide gels.



Asp-489 and Asp-698 Flanking Sequences

To understand the mechanism of inhibition of cell adhesion mediated by alpha(4) with D489E and D698E substitutions, we compared the flanking sequences at these sites with corresponding sequences in other alpha integrins. Comparison of the Asp-489 flanking sequences with corresponding sequences in other alpha chains showed that this aspartic acid is conserved among most integrin alpha chains. Therefore, it seems unlikely that this residue plays an important role in discriminating between different ligands. It may be involved in a common integrin structure required for cell adhesion. Analysis of the Asp-698 flanking sequences showed that the DISFLLDVSSLS sequence resembles the DxxxxxD-S-Sx cation binding motif found in beta subunits and the I-domain of other alpha integrins (Fig. 9).


Figure 9: Comparison of the new putative cation binding motif in alpha(4) to similar sequences in other human alpha and beta integrins. The amino acid sequences are deduced from the nucleotide sequence data obtained from the GenBank data base, with the following accession numbers: alpha(1) (X68742), alpha(2) (M28249), alpha(4) (X16983), alpha(9) (D25303), alpha(e) (L25851), alpha(l) (Y00796), alpha(m) (M18044), alpha(x) (Y00093), beta(1) (X07979), beta(2) (M15395), beta(3) (M35999), beta(4) (X51841), beta(5) (M35011), beta(6) (M35198), beta(7) (M62880), and beta(8) (M73780). Alignment was done using the PILEUP program of the University of Wisconsin Genetics Computer Group. The conserved amino acid residues are in shaded boxes. The residues, which have been shown to be required for integrin function, are shaded in black.




DISCUSSION

We have demonstrated that glutamic acid substitutions at Asp-489 and Asp-698 severely impair the ability of alpha(4)-transfected cells to adhere to VCAM and fibronectin. A third mutation at Asp-811 had no detectable effect on cell adhesion to VCAM or fibronectin. However, direct binding experiments with isolated alpha(4) and VCAM suggest that Asp-489 and Asp-698 are not critical for ligand recognition. We found that the Asp-698 residue in alpha(4) resides in a sequence resembling the cation/ligand binding DxxxxD-S-Sx motif found in beta integrins and the I-domain of alpha integrins, implicating Asp-698 in cation-ligand interactions. Therefore, Asp-489 and Asp-698 may regulate cell adhesion through a novel and/or cation-dependent mechanism.

To investigate the effect of D489E and D698E substitutions on gross conformation of alpha(4), transfected cells with mutated alpha(4)s were analyzed for disruption of anti-alpha(4) epitopes, loss of alpha(4)/beta(1) chain association or alpha(4) cleavage, and impairment of signal transduction. As shown in Fig. 2and Fig. 6, alpha(4)LEV, alpha(4)D489E, alpha(4)D698E, and alpha(4)D811E were all recognized by a panel of mAbs against various distinct epitopes of alpha(4). The glutamic acid substitutions at Asp-489, Asp-698, and Asp-811 of alpha(4) also did not affect the ability of alpha(4) to associate with the beta(1) subunit or the in vivo cleavage of alpha(4) (Fig. 3). Therefore, the decreased cell adhesion by K-D489E and K-D698E is unlikely to be caused by disruption of alpha(4) secondary structure.

The mutant alpha(4) molecules also maintained the flexibility required for undergoing certain conformational changes during signaling. Integrins mediate outside-in and inside-out cell signaling. VLA-4-mediated cell adhesion can be rapidly and dramatically augmented by phorbol esters(26) . PMA activation (inside-out signaling) does not change the level of VLA-4 expression, and it is not dependent on de novo protein synthesis(26, 27) . We have shown that the adhesion of mutant alpha(4) transfectants to ligand increases in response to PMA treatment (Fig. 5A). A number of anti-alpha(4) antibodies, when binding to alpha(4)beta(1) integrin on the cell surface, cause homotypic cell adhesion (outside-in signaling, (31) ). Anti-alpha(4) antibody also triggered homotypic aggregation of the mutant alpha(4) transfectants (data not shown). These observations are consistent with the view that the Asp Glu substitutions did not change the global secondary structure of alpha(4).

The ability of K-LEV, K-D489E, and K-D698E to adhere to VCAM or fibronectin was severely impaired as demonstrated in Fig. 4, 5, and 7. To determine whether impaired adhesion was due to the inability of mutant alpha(4) to recognize ligand, direct ligand binding studies with alpha(4) isolated from K-LEV, K-D489E, and K-D698E and purified VCAM were carried out (Fig. 8). The ability of alpha(4) Asp-489 or alpha(4) Asp-698 to bind VCAM argues that Asp-489 and Asp-698 are not critical for ligand recognition. Therefore, the D489E and D698E substitutions may affect adhesion and the function of the alpha(4)beta(1) integrin in vivo by another mechanism.

Both Asp-489 and Asp-698 are in an alpha-helix/turn/beta-sheet structure, whereas Asp-811 is in the middle of a beta-sheet according to the Chou-Fasman prediction(32) . The turn regions where Asp-489 and Asp-698 reside have fairly high surface probability and flexibility, whereas Asp-811 has low surface probability. This suggests that Asp-489 and Asp-698 may be exposed on alpha(4) and have the ability to interact with other proteins.

One mechanism of impairing integrin function is disruption of divalent cation binding sites. Divalent cations are required for integrins to bind to their ligands(1, 35, 36) . Masumoto and Hemler (13) demonstrated the importance of the three putative divalent cation binding sites for alpha(4) function. Mutations at each of the three divalent cation binding sites resulted in significant inhibition of alpha(4)-mediated cell adhesion to VCAM and fibronectin. It has been reported recently that certain aspartic acid residues in the I-domains of alpha(M) and alpha(2) are required for cell adhesion(18, 19) . In both cases, the essential aspartic acid residue resides in the DxxxxxD-S-Sx sequence (Fig. 9) as proposed by Bajt and Loftus(14) . Furthermore, Michishita et al. (19) have also demonstrated that the same aspartic acid residue in alpha(M) is required for cation binding. In beta(1) and beta(3) subunits, the aspartic acid residues in this motif have also been proven to be critical for ligand binding(37, 38) . A point mutation in the beta(3) subunit of the alphabeta(3) integrin has been identified in the Cam variant of Glanzmann's thrombasthenia(33) . The substitution of the aspartic acid residue within this motif in beta(3) to tyrosine resulted in a perturbed interaction with divalent cations and the inability of alphabeta(3) to recognize ligand. We have compared the Asp-698 flanking sequence with those of relevant sequences from different alpha and beta subunits (Fig. 9). The sequence around Asp-698 of alpha(4) resembles the divalent cation binding motif mentioned above and thus implicates Asp-698 in divalent cation binding. Our finding that Asp-698 is critical for the interaction of alpha(4)-transfected cells with VCAM and fibronectin supports this hypothesis. Therefore, Asp-698 may be part of a novel cation binding motif in alpha(4), which is required for alpha(4) function.

In summary, we have identified two aspartates, Asp-489 and Asp-698, in alpha(4), critical for alpha(4)beta(1)-mediated cell adhesion. We found that the sequences flanking Asp-698 resemble the cation binding motif DxxxxxD-S-Sx, recently identified in the ligand binding domain (I-domain) of alpha integrins. Since alpha(4) does not contain an I-domain, this raises the possibility that the region of alpha(4) containing the DxxxxxD-S-Sx motif is functionally similar to that of the I-domain. The DxxxxxD-S-Sx motif in the I-domain of alpha integrins appears not to be required for ligand recognition as has been shown in studies with alpha(2)(34) and alpha(M)(39) . Our present findings are consistent with these results. Asp-689 may affect cell adhesion by increasing ligand accessibility or maintaining a binding pocket with high ligand affinity through binding of divalent cation. Alternatively, given that cell adhesion is a complex process, one or both aspartate residues (Asp-489, Asp-698) may be critical for efficient cell adhesion through an interaction with an as yet unknown cofactor.


FOOTNOTES

*
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 and reprint requests should be addressed. Tel.: 203-937-2375; Fax: 203-937-6923.

^1
The abbreviations used are: VCAM, vascular cell adhesion molecule; FN-40, the 40-kDa chymotryptic fragment of human fibronectin; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; FACS, fluorescence-activated cell sorting; PBS, phosphate-buffered saline.

^2
M.-L. Blue and P. J. Conrad, unpublished observation.


ACKNOWLEDGEMENTS

We would like to thank Maura Macaro for flow cytometric analysis and cell sorting.


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