Asp-698 and Asp-811 of the Integrin alpha 4-Subunit Are Critical for the Formation of a Functional Heterodimer*

Yvonka Zeller, Jens Lohr, Marei Sammar, Eugene C. ButcherDagger , and Peter Altevogt§

From the Tumor Immunology Programme, 0710, German Cancer Research Center, D-69120 Heidelberg, Federal Republic of Germany, the Dagger  Laboratory of Immunology and Vascular Biology, Department of Pathology and Digestive Disease Center, Stanford University, Stanford, California 94305, and the Center of Molecular Biology in Medicine, Veterans Administration Medical Center, Palo Alto, California 94304

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The amino acid motif LDV is the principal binding site for alpha 4 integrins in fibronectin, and homologous motifs are recognized in vascular cell adhesion molecule-1 and MAdCAM-1. Three conserved LDV motifs (LDV-1 to 3) occur in the ectodomain of the human and mouse alpha 4-subunit, the functions of which are unknown. We demonstrate here that alpha 4-transfected fibroblasts with mutation in LDV-1 (D489N) behaved like alpha 4-wild type but that LDV-2 (D698N) and LDV-3 (D811N) mutants were impaired in binding and spreading on alpha 4-specific substrates. On the RGD-containing fibronectin fragment FN-120 there was an inverse behavior; now the alpha 4-wild type and the LDV-1 mutant could not adhere whereas the two other mutants could. The beta 1 chain was critical for the differential integrin response. Biochemical analysis demonstrated that the LDV-2 and -3 mutations reduced the strength of the alpha 4beta 1 association, favored the formation of alpha 5beta 1, and prevented the expression of alpha 4beta 7 on the cell surface. Our results indicate that LDV-2 and LDV-3 are critical for the formation of a functional heterodimer. The presence of similar amino acid motifs in ligands and the alpha 4-subunit suggest that metal coordination plays an important role in integrin-ligand binding as well as for heterodimer formation.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Integrins are a class of heterodimeric cell-surface molecules that are important mediators of cell function. In addition to providing the necessary mechanical stability for cell-cell or cell-matrix contact, integrins can transduce signals out of cells and into the cell from the microenvironment. The cytoplasmic portion of integrin chains can interact with signal- and cytoskeleton-associated proteins and thereby influence cellular properties like shape, adhesion, motility, growth, and differentiation (for review see Refs. 1 and 2).

An essential feature of integrins is their complex regulation of function which despite their biological importance as adhesive structures is not fully understood. Both subunits form a non-covalently linked heterodimer that appears to be important for transport to the cell surface as well as for ligand binding. It is believed that integrins contain multiple ligand contact sites, and several regions and residues have been identified in both alpha  and beta  chains. All of the identified contact sites carry amino acid sequences with oxygenated side chains that can potentially bind divalent cations. It has been estimated that integrin heterodimers can bind up to five divalent cations. The observation that all integrin ligands possess a critical aspartate or glutamate residue in their contact site to the integrin has led to the proposal that a metal ion provides a bridge between ligand and receptor. In addition to ligand binding, divalent cations are believed to contribute to the regulation of receptor conformation (for review see Refs. 3-5).

The alpha 4 integrins, alpha 4beta 1 and alpha 4beta 7, are adhesion molecules that play an important role in hematopoiesis (6, 7), lymphocyte migration (8, 9), mouse skeletal muscle formation (10), placental or cardiac development (11), and possibly tumor metastasis (12, 13). VLA-4 (alpha 4beta 1) mediates the adhesion of cells to fibronectin (14-16) or the cytokine inducible endothelial cell ligand VCAM-11 (17, 18). The heterodimer alpha 4beta 7 is a homing receptor that mediates the lymphocyte entry into the gut-associated lymphoid tissues (8, 19). The ligand for alpha 4beta 7 is the addressin MAdCAM-1 (20). Only alpha 4beta 7-positive cells can bind to MAdCAM-1, whereas both alpha 4beta 1- and alpha 4beta 7-positive cells can bind to fibronectin and VCAM-1 (21, 22). Molecular studies have identified amino acid sequences in each ligand that are recognized by alpha 4 integrins. A dominant binding site in fibronectin involves the LDV motif in the HepII/IIICS region, and the peptide surrounding and encompassing these residues has been termed CS-1 peptide (23, 24). A homologous sequence IDS, present in domains 1 and 4 of VCAM-1, is essential for the binding to alpha 4 integrins under static conditions (25-27) or flow (28), and an LDT sequence in the first domain of MAdCAM-1 is important for alpha 4beta 7 binding (29). Three conserved LDV motifs occur in the extracellular sequence of the alpha 4-subunit in mouse and man which have been termed LDV-1-3 (30).

We previously reported that the purified alpha 4-subunit as well as an LDV-containing peptide derived from the 80-kDa N-terminal portion could support the binding of lymphocytes via alpha 4beta 1 or alpha 4beta 7 (30) which could be important for alpha 4-mediated homotypic cell aggregation (22, 31, 32). In another study Ma et al. (33) have suggested that the LDV sites, in particular LDV sites 1 and 2, may serve as additional cation binding motifs that are required for cell adhesion. To understand further the functional role of the LDV motifs in the alpha 4-subunit, we have mutated the central aspartic acid to asparagine in each site in a consecutive fashion and have examined the effect on cell adhesion and spreading. Our results suggest a role for LDV-2 and -3 but not LDV-1 in the formation of a functional alpha beta heterodimer. It is therefore possible that both ligand binding and the dimerization of alpha - and beta -subunits follow the same principal rule of metal ion coordination.

    EXPERIMENTAL PROCEDURES
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Introduction
Procedures
Results
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References

Cell Culture-- Balb/3T3 fibroblasts were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 10 mM HEPES. CHO cells transfected with human full-length VCAM-1 or a 1-3 domain deletion mutant of VCAM-1 (Delta 1-3) were obtained from Dr. J. Clements (British Biotech, Oxford, UK) and maintained as described (34). All cells were kept at 37 °C, 5% CO2, and 100% humidity.

Antibodies-- mAb 5/3 against alpha 4 integrins has been described before (30). mAbs HMalpha 5-1 and HMbeta -1 are blocking hamster mAbs against mouse alpha 5 integrin and the beta 1-subunit, respectively (35). These mAbs were obtained from Dr. H. Yagita, Tokyo, Japan. mAbs Fib 30 and Fib 504 are blocking antibodies against the beta 7-subunit (36). mAb RMV-7 is a blocking antibody to mouse alpha v (37). mAb 9EG7 (38) was kindly provided by Dr. D. Vestweber (ZMBE Münster, Germany). mAbs were used in a purified form or as hybridoma supernatants.

Peptides-- The RGDS peptide was purchased from Sigma (Taufkirchen, Germany). The CS-1 peptide CEILDVPST was synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry and purified by preparative high pressure liquid chromatography. It was characterized further by analytical high pressure liquid chromatography and mass spectroscopy.

Cytofluorography-- The cell-surface staining of cells with saturating amounts of mAbs, either hybridoma supernatants or purified antibodies, and phycoerythrin-conjugated goat antibodies to rat Ig (SERVA, Heidelberg, FRG) or hamster Ig (Dianova, Hamburg, FRG) has been described elsewhere (39). For the detection of cytoplasmic staining cells were first incubated on ice with 0.5% formaldehyde in PBS. After washing in PBS cells were permeabilized with 0.05% saponin (Sigma) for 10 min at room temperature. The cells were washed again and stained as above.

Stained cells were analyzed with a FACScan fluorescence-activated cell analyzer (Becton Dickinson, Heidelberg, FRG). For enrichment of alpha 4-transfectants the cells were stained under sterile conditions with mAb 5/3 and sorted on a FACS Vantage using counter staining with propidium iodide to exclude dead cells.

For the activation of the 9EG7 epitope, the cells were pretreated with peptide at the indicated concentration for 15 min followed by staining with the mAb and phycoerythrin-conjugated antibodies to rat IgG in the presence of the activating peptides.

Site-directed Mutagenesis and DNA Transfection-- The mouse alpha 4-cDNA clone in Bluescript SK+ was obtained from Dr. B. Holzmann (University of Munich, Germany) and was used as template for site-directed mutagenesis with the Stratagene Chameleon kit (Stratagene, Heidelberg, Germany). In successive rounds of mutagenesis the Asp in positions Asp-489, Asp-698, and Asp-811 were changed to Asn as indicated in Fig. 1. Mutations were confirmed by DNA sequence analysis. Wild-type and mutated alpha 4-cDNAs were subsequently cloned in the pcDNA3 expression vector (Invitrogen) and transfected into Balb/3T3 fibroblasts using LipofectAMINE (Life Technologies, Inc., Eggenstein, Germany). Transfectants were selected for similar expression levels using FACS sorting (see above). The sorting of transfectants was repeated when the expression level of alpha 4 integrin dropped by more than 20 mean fluorescent units.

Cell Adhesion and Spreading-- Fibronectin or its fragments FN-120 or FN-40 (Life Technologies, Inc.) were coated to LABTEK glass chamber slides (Nunc, Wiesbaden, Germany) at a concentration of 10 µg/ml or as otherwise indicated for 16 h at 4 °C. Wells were blocked with 1% bovine serum albumin in PBS for 1 h at room temperature, washed with HBSS containing 10 mM HEPES, 2 mM Ca2+, and 2 mM Mg2+ (binding buffer) and used for the assay. For adhesion, cells (5-10 × 106/ml) were suspended in the same buffer, and 0.2-ml aliquots were added to the coated slides. The binding assay was performed for 10 min at 37 °C without shaking, and the slides were fixed in 2% glutaraldehyde/PBS after briefly dipping into PBS. For antibody or peptide blocking studies, cells were preincubated with purified antibody (10 µg/ml final concentration, or as otherwise indicated) or peptides (500 µg/ml or otherwise indicated) for 10 min at room temperature and then transferred to the chamber slides. For Mn2+ activation, the Ca2+ and Mg2+ ions in the buffer were substituted with the indicated concentrations of Mn2+. Cell binding was measured by counting six independent 10 × fields by video microscopy using IMAGE 1.55 software. To analyze cell spreading, the plated cells were incubated at 37 °C at a microscope stage, and pictures were taken for 2 h every 10 min and stored on an optical disc. Pictures were analyzed, and the percentage of spread versus non-spread cells in each frame was determined. Clearly visible pseudopodia formation served as criterium for spread cells.

Binding of cells to human VCAM-1-transfected CHO cell monolayers was done using Eu3+-labeled cells (40). Briefly, cells were loaded with EuCl3 for 1 h at 4 °C, washed with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, and resuspended in Hanks' balanced salt solution plus 2 mM Ca2+ and Mg2+. 100 µl of the cell suspension (5 × 105 cells/ml) were allowed to bind to the monolayer of VCAM-1-CHO cells in a 96-well plate for 30 min at 37 °C. Following the binding assay bound and non-bound cells were separated on the basis of buoyant density using Percoll as described (44). For detection of the bound Eu3+-labeled cells, the plates were inverted to remove the Percoll and fixative, then washed once in PBS, and refilled with 50 µl of PBS, and 50 µl of Europium enhancement solution (LKB Wallac, Turku, Finland) was added. The fluorescence was measured in a time-resolved fluorometer (Arcus 1230, LKB-Wallac, Turku, Finland) after 30 min.

Isolation of RNA and Reverse Transcriptase-PCR Analysis-- The isolation of total RNA from cells has been described in detail elsewhere (41). Total RNA (6 µg) was transcribed into cDNA using Moloney murine leukemia virus reverse transcriptase (Promega, Heidelberg, Germany) and oligo(dT)20 for priming. The RNA/DNA hybrid was treated with RNase H and used as template for PCR using an annealing temperature of 60 °C and 35 cycles of 80 s. The following primers for the beta 7 integrin subunit were used: forward, ATAGGTTTTGGCTCCTTCGTG; reverse, AGTGGAGAGTGCTCAAGAGTCACAGT. PCR products were separated on a 1% agarose gel containing 0.5 µg/ml ethidium bromide. The mouse beta 7 cDNA clone was obtained from G. Krissansen, University of Auckland, New Zealand.

Biochemical Analysis-- Lactoperoxidase-catalyzed iodination of intact cells was carried out as described (39). Following the labeling reaction, the cells were washed three times in PBS and lysed at 4 °C for 15 min in Tris-buffered saline containing either 0.3% CHAPS (Sigma), 1% IGEPAL CA-630 (Sigma) or 1% IGEPAL CA-630 in the presence of 2 mM Ca2+ and Mg2+ ions. Lysates were prepared by centrifugation in an Eppendorf centrifuge at 4 °C for 15 min and precleared with 30 µl of packed rat Ig coupled to Sepharose. Immunoprecipitations were carried out using mAb 5/3 coupled to Sepharose or mAbs HMbeta -1 preadsorbed to Protein G-Sepharose for 1 h at 4 °C. The precipitates were washed 4 times in the respective lysis buffers and eluted from the Sepharose by boiling for 2 min in non-reducing SDS sample buffer. SDS-polyacrylamide gel electrophoresis was performed on 7,5% slab gels. Gels were dried and exposed to x-ray-sensitive films (Kodak Biomax-MS) using the Biomax MS intensifying screen.

    RESULTS
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Procedures
Results
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References

Expression of Wild-type and Mutant alpha 4-Subunits-- The LDV motifs in the mouse alpha 4-subunit were mutated in positions Asp-489, Asp-698, and Asp-811 by changing Asp to Asn as outlined in Fig. 1. The wild-type, single, double, and triple mutated alpha 4-subunits (referred to as mut 1, mut 12, and mut 123) were transfected into alpha 4-negative Balb/3T3 fibroblasts and selected for equal cell-surface expression by FACS sorting. As shown in Fig. 2 the level of expression as detected by fluorescent staining with an alpha 4-specific mAb was comparable for all transfected cells. As revealed by staining with the respective mAbs, the expression levels of VLA-5, alpha v-integrins, as well as the beta 1 integrin subunit were not changed significantly following transfection. There was, however, induction of beta 7 surface expression in alpha 4-wt and mut 1 cells and little or undetectable expression in mut 12 and mut 123 cells, respectively. Induction of new beta  chains upon transfection was also observed in another study (42).


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Fig. 1.   Schematic representation of alpha 4-subunit mutations. Structures of mutant alpha 4-subunits used in this study are shown. Aspartic acid to asparagine point mutations were introduced into the mouse alpha 4-cDNA consecutively rendering the LDV motifs to LNV. Known divalent cation binding sites in the alpha 4-subunit are also indicated.


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Fig. 2.   Analysis of alpha 4 and beta 1 expression on transfected fibroblasts. Indirect immunofluorescence staining of the cells used in the present study is shown. Cells were stained by indirect immunofluorescence using mAb 5/3 (alpha 4 integrin subunit), HMbeta -1 (beta 1 integrin-subunit), HMalpha 5-1 (alpha 5 integrin subunit), Fib 30 (beta 7 integrin subunit), and RMV-7 (alpha v integrin subunit) followed by phycoerythrin-conjugated goat anti-rat IgG or anti-hamster IgG, respectively. For negative control the first antibody was omitted, and the peak fluorescence is indicated with an arrow.

alpha 4-Transfectants Are Altered in Adhesion-- We investigated the adhesion of the different cells to immobilized fibronectin. As shown in Fig. 3A all cells could readily bind. The binding of non-transfected 3T3 cells is due to the expression of other integrins than alpha 4 that can support adhesion to fibronectin (alpha 5beta 1, alpha v integrins see below). The binding site for alpha 4 integrins is located on the FN-40 chymotryptic cleavage fragment of fibronectin. As shown in Fig. 3B the binding of alpha 4-wt and mut 1 cells to FN-40 was of similar magnitude and blocked in the presence of the mAb to the alpha 4-subunit. A partial blocking was obtained with mAbs against the beta 1- or the beta 7-subunit suggesting that the binding was mediated by alpha 4beta 1 and alpha 4beta 7 integrins. The mut 123 cells, and to a lesser extend mut 12 cells, showed a significant reduction in binding ability. Untransfected 3T3 cells did not bind.


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Fig. 3.   Adhesion of cells to fibronectin and its fragment FN-40. Adhesion of 3T3 fibroblasts and alpha 4-transfectants to fibronectin (A) and to the FN-40 fragment (B) coated at 10 µg/ml is shown. Blocking of cell adhesion is shown for alpha 4-wt cells using the indicated mAbs at 10 µg/ml. The mAb HMalpha 5-1 was used as control.

To analyze whether a differential binding could be detected on other alpha 4-ligands, we tested the ability of the cells to bind to VCAM-1 stably expressed in CHO cells. As shown in Fig. 4A, the alpha 4-transfectants were indistinguishable in their adhesion to the full-length 7-domain VCAM-1 that contains two binding sites in domains 1 and 4, respectively. In contrast, a 1-3 domain deletion mutant of VCAM-1 (VCAM Delta 1-3) having only one binding site showed a clearly reduced binding of mut 12 and mut 123 cells (Fig. 4B). As expected, the 3T3 cells gave only background binding to VCAM-1-CHO.


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Fig. 4.   Adhesion of cells to VCAM-1. Adhesion of 3T3 fibroblasts and alpha 4-transfectants to VCAM-1-transfected CHO cells is shown. A, CHO cells carrying a full-length 7-domain VCAM-1; B, CHO cells carrying a 1-3 domain deleted form of VCAM-1 (VCAM Delta 1-3).

Additional analysis of mut 123 cells were carried out on FN-40 by increasing the coating density of immobilized protein. Fig. 5A shows that although the adhesion of mut 123 cells was enhanced under these conditions, the level of alpha 4-wt cells was not reached. The adhesion of mut 123 cells was also improved in the presence of Mn2+ ions that can activate alpha 4 integrins. As shown in Fig. 5B, mut 123 cells required a concentration of approximately 250 µM Mn2+ ions to reach a similar level of adhesion to FN-40 as non-activated alpha 4-wt cells.


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Fig. 5.   Effect of FN-40 coating concentration and Mn2+ on cell adhesion. Comparison of adhesion between alpha 4-wt and mut 123 cells to the FN-40 fragment at increasing coating concentration (A) and in the presence of increasing concentration of Mn2+ ions (B) is shown.

Altered Cell Spreading on FN-40-- We studied the spreading of alpha 4-transfectants on FN-40, and the results are depicted in Fig. 6. After 40 min nearly all alpha 4-wt and mut 1 cells had spread on the FN-40 substrate, and this spreading was prevented in the presence of the alpha 4-specific mAb 5/3. The mut 12 cells, although partially able to adhere, were unable to spread on FN-40, and the spreading of mut 123 cells was similarly decreased when compared with alpha 4-wt cells. As expected, 3T3 fibroblasts could not spread on FN-40.


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Fig. 6.   Spreading of 3T3 and alpha 4-transfectants on FN-40. Spreading of 3T3 fibroblasts and alpha 4-transfectants on the FN-40 fragment coated at 10 µg/ml is shown. The spreading of cells was monitored by video microscopy, and pictures were taken at 20-min intervals. Data shown were taken at 40 min after plating of the cells, and the percentage of cells spread was evaluated. Blocking of cell spreading is shown for alpha 4-wt cells using mAb 5/3 at 10 µg/ml. The mAb HMalpha 5-1 was used as control.

The spreading of mut 12 and mut 123 cells was dependent on the duration time of the assay. As shown in Fig. 7A the defects of mut 12 and mut 123 cells in spreading were most prominent between 20 and 100 min of the assay. At later time points the percentage of spread cells became similar to alpha 4-wt or mut 1 cells. These observations suggested that the mutations in Asp-698 and Asp-811 impaired early events in alpha 4-mediated spreading which, however, disappeared with time.


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Fig. 7.   Kinetic of cell spreading on FN-40 and effects of Mn2+ ions. A, kinetic of cell spreading on FN-40 for 3T3 fibroblasts and alpha 4-transfectants. The spreading of cells was monitored by video microscopy, and pictures were taken at 20-min intervals. B, effect of Mn2+ concentration on cell spreading of 3T3 fibroblasts and alpha 4-transfectants.

We investigated the effect of Mn2+ ions on the spreading of mut 12 and mut 123 cells on FN-40. Fig. 7B reveals at a concentration of approximately 100 µM Mn2+ ions both mutants had acquired roughly 80% of the spreading rate of alpha 4-wt cells on FN-40. A further increase was seen at higher Mn2+ ion concentration; however, the mutant cells remained less efficient than alpha 4-wt cells. This could not be attributed to a lower alpha 4 expression level in mutant cells since the phenomenon was also seen when alpha 4 expression as detected by fluorescent staining was higher than on alpha 4-wt cells (not shown). Thus, the mutant alpha 4 integrin receptors could be activated by Mn2+ ions yet, due to an intrinsic defect, were less avid than the wild-type alpha 4 integrin.

Presence of the alpha 4-Subunit Affects Spreading of Cells on FN-120-- We next analyzed the behavior of the cells on FN-120, which contains the RGD-binding site in fibronectin. Fig. 8 shows that 3T3 fibroblasts were able to spread on FN-120. Unexpectedly, the alpha 4-wt cells and mut 1 cells could not or only poorly spread on FN-120. In sharp contrast, in mut 12 and mut 123 cells spreading was again observed.


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Fig. 8.   Spreading of 3T3 and alpha 4-transfectants on FN-120. Spreading of 3T3 fibroblasts and alpha 4-transfectants on the FN-120 fragment coated at 10 µg/ml. The spreading of cells was monitored by video microscopy, and pictures were taken at 20-min intervals. Data presented were taken 40 min after plating the cells.

As indicated in Fig. 9, similar findings were made when the cells were analyzed for adhesion to FN-120. The adhesion of 3T3, mut 12, and mut 123 cells could be blocked in the presence of an RGDS peptide (Fig. 9A), by mAbs against the beta 1 integrin chain or by a mAb against alpha 5 integrins, respectively (Fig. 9B). mAb RMV-7 against alpha v integrins had no or only marginal effects (data not shown). This observation suggested that on FN-120 the cells used mainly the alpha 5beta 1 integrin for adhesion and also indicated that mut 12 and mut 123 cells used similar integrin receptors for adhesion to FN-120 than 3T3 fibroblasts.


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Fig. 9.   Inhibition of cell adhesion on FN-120. A, blocking of cell adhesion on FN-120 with RGDS peptide at 500 µg/ml. B, blocking of cell adhesion using the indicated mAbs at a concentration of 10 µg/ml.

The differential behavior of the cells on FN-120 could not be explained by differences in cell-surface expression of the relevant cell surface receptors since the density of alpha 5 and beta 1 was comparable on all cells (see Fig. 2). To demonstrate further that the effects observed were caused by the alpha 4-subunit and not due to an intrinsic failure of the fibroblasts, we sorted revertant cells that had lost alpha 4 expression. These alpha 4-negative or low expressing cells were fully restored in their ability to spread on FN-120 (data not shown). We concluded, that the alpha 4-wt or the mut 1 subunit altered the function of the RGD-binding integrins in transfected cells. The removal of Asp-698 and Asp-811 by mutagenesis released these RGD-binding integrins from the block of function.

Modulation of the 9EG7 Epitope by Soluble Ligand-- To demonstrate a differential activity of either the alpha 4beta 1 or RGD-binding integrins also in response to soluble ligands, the expression of the 9EG7 epitope, which is a conformation-dependent epitope of the beta 1 chain (38), was investigated. The expression of this epitope can be enhanced by Mn2+ ions or soluble ligand (43). The binding site for mAb 9EG7 has been mapped to the cysteine-rich membrane proximal site of the beta 1 chain (43). As shown in Fig. 10A, the alpha 4-wt transfected cells and mut 1 cells showed staining with mAb 9EG7 which was of similar magnitude as 3T3 cells. In contrast, both mut 12 and mut 123 cells showed a consistently higher staining suggesting that the epitope was more accessible. These differences were not seen with mAb HMbeta -1 which binds to a site distinct from the 9EG7 epitope on the beta 1 chain.


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Fig. 10.   Modulation of the 9EG7 epitope by soluble ligand. A, comparison of expression levels for the beta 1 integrin-associated epitopes recognized by mAbs 9EG7 and HMbeta -1, respectively. B, modulation of 9EG7 epitope expression by CS-1 peptide in 3T3 cells, alpha 4-wt, and mut 123 cells. C, modulation of 9EG7 epitope expression by RGDS peptide in 3T3 cells, alpha 4-wt, and mut 123 cells.

The modulation of the 9EG7 epitope expression following addition of soluble ligand in the form of the CS-1 peptide for alpha 4 integrins or the RGDS peptide for RGD-binding integrins, respectively, are shown in Fig. 10, B and C. An increase in the concentration of CS-1 peptide led to a drastic up-regulation of 9EG7 epitope expression in alpha 4-wt cells. The mut 123 cells increased the expression of the 9EG7 epitope only marginally, whereas 3T3 fibroblasts showed no increase. Addition of RGDS peptide to the cells showed an inverse behavior (Fig. 10C). Now the 3T3 cells gave an appreciable dose-dependent response in the expression of the 9EG7 epitope. Also the mut 123 cells showed a dose-response curve comparable to 3T3 fibroblasts, although these cells started at a much higher expression level (see above). In contrast, the alpha 4-wt cells gave a poor response to the RGDS peptide consistent with the previous notion that RGD-binding integrins were impaired in these cells.

LDV-2 and -3 Mutations Affect the Stability of the alpha 4beta 1 Heterodimer-- The functional data had indicated that mutations in the alpha 4-subunit were able to regulate the integrin response in the transfected cells and that the common beta 1 chain might be of crucial importance. To address this question further biochemical analyses were carried out. Immunoprecipitation with the alpha 4-specific mAb showed the typical pattern of alpha 4 integrins on SDS-polyacrylamide gel electrophoresis consisting of the 150-kDa band representing the intact alpha 4 chain and the smaller bands of 80 and 70 kDa that are proteolytic cleavage fragments of the alpha 4 chain (see Fig. 11A). The mutations slightly affected the electrophoretic mobility of these fragments.


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Fig. 11.   Biochemical analysis of alpha 4beta 1 stability. Cells were labeled with 125I and lysed in the presence of the following: 1) 0.3% CHAPS; 2) 1% IGEPAL CA-630; 3) 1% IGEPAL CA-630 plus 2 mM Ca2+ and Mg2+ ions. A, lysates were incubated with mAb 5/3 coupled to Sepharose. Note that in mutant cells the 70- and 80-kDa alpha 4-subunits migrate slightly differently from wild-type alpha 4-transfectants. B, lysates were incubated with mAb HMbeta -1 adsorbed to protein G-Sepharose. All samples were analyzed by SDS-polyacrylamide gel electrophoresis under nonreducing conditions. C, shorter exposure of B. The bracket shows the area of the gel. The position of the alpha 5-subunit is indicated and was confirmed by precipitation with mAb HMalpha 5-1 in parallel experiments.

We analyzed the stability of the alpha 4beta 1 heterodimer from each cell type in the presence of different detergents. Following cell-surface iodination, aliquots of each transfectant were solubilized in 0.3% CHAPS, 1% IGEPAL CA-630, or 1% IGEPAL CA-630 in the presence of 2 mM Ca2+ and Mg2+ ions and the lysates were subjected to precipitation using mAbs to the alpha 4- or beta 1-subunit. As shown in Fig. 11, A and B, the different solubilization conditions preserved the heterodimer in alpha 4-wt and mut 1 cells since the 80- and 70-kDa subunits were present in both alpha 4 and beta 1 precipitates in similar amounts. Mut 12 and mut 123 cells showed a different behavior. Here the 70- and 80-kDa subunits were equally present only in the alpha 4-specific precipitates, whereas in the anti-beta 1 immunoprecipitates the heterodimer was only detectable in the presence of Ca2+ and Mg2+ (compare mut 12 or mut 123 A versus B). IGEPAL CA-630 alone almost completely disrupted the heterodimer, whereas 0.3% CHAPS showed a partial dissociation of the complex. This differential sensitivity toward detergents indicated that in mut 12 and mut 123 cells the alpha 4beta 1 heterodimer was significantly weaker or present in much smaller amounts than in alpha 4-wt or mut 1 cells. There was also a clearly reduced presence of beta  chains in the alpha 4-specific precipitates of mut 12 and mut 123 cells (see Fig. 11A).

The additional analysis of the beta 1-associated proteins by shorter exposure of the gels presented in Fig. 11B revealed another important finding. As shown in Fig. 11C the alpha 5 integrin chain was found to be complexed to beta 1 only in mut 12 and mut 123 cells and was barely detectable in alpha 4-wt and mut 1 cells.

LDV-2 and -3 Mutations Prevent Surface Expression of alpha 4beta 7-- Finally we reinvestigated the failure of mut 12 and mut 123 cells to express beta 7 at the cell surface following transfection. We used mAb DATK32 that recognizes a conformational epitope of alpha 4beta 7 when it is associated (34). Fig. 12A shows that alpha 4-wt and mut 1 cells showed cell-surface staining with this mAb, whereas mut 12 and mut 123 cells were negative. This was not due to the lack of beta 7 message that was detectable by PCR analysis of reverse-transcribed cDNA in all transfected cell lines as well as in 3T3 cells (Fig. 12B). Indeed, cytoplasmic staining of permeabilized cells with a beta 7 mAb revealed the presence of beta 7 protein in all transfectants (not shown). We concluded that the mutations in the alpha 4-subunit most likely affected the formation of the alpha 4beta 7 heterodimer or its transport to the cell surface.


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Fig. 12.   Analysis of alpha 4beta 7 expression in transfected fibroblasts. A, staining of cells with the alpha 4beta 7-specific mAb DATK 32. B, analysis of beta 7 mRNA by reverse transcriptase-PCR. Lane 1, positive control amplification from a mouse beta 7 cDNA clone; lane 2, 3T3 fibroblasts; lane 3, alpha 4-wt; lane 4, mut 1; lane 5, mut 12; lane 6, mut 123; and lane 7, negative control amplification with primers only.

    DISCUSSION
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Abstract
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Procedures
Results
Discussion
References

The goal of the present study was to assess the functional role of the three LDV sites in the mouse alpha 4-subunit by site-directed mutagenesis. We find that Asp right-arrow Asn substitution in LDV-2 and -3 but not in LDV-1 affects adhesion and spreading of transfected fibroblasts on alpha 4-specific substrates. The alpha 4 mutant cells were differentially affected in adhesion and spreading on FN-40. Whereas the mut 12 cells were mainly compromised in spreading, the mut 123 cells were affected in both adhesion and spreading suggesting that the mutation of the LDV-3 site was giving an additional effect. In the presence of Mn2+ ions the defects of mut 12 and mut 123 cells could be largely restored, and the lack of spreading on FN-40 was overcome by prolonged assay times. This suggested that the receptor was not completely non-functional and that LDV-2 and -3 were probably the most important in events early after alpha 4beta 1 receptor engagement. Ma et al. (33) have performed similar mutagenesis studies using K562 cells transfected with human alpha 4-subunits in which the LDV sites were mutated to LEV. These authors demonstrated impaired adhesion of D489E and D698E mutants (equivalent to LDV-1 and -2) on FN-40 and a 1-3 domain form of VCAM-1. There was, however, no effect in D811E mutants (33). The reasons for the discrepancies between the previous study and our data are not entirely clear but could be due, at least in part, to the choice of amino acid exchanges. Whereas Asp right-arrow Glu mutations retain the negative charge in the amino acid side chain, our Asp right-arrow Asn mutations abolished it. Also the type of mutagenesis (single versus accumulating mutations) is different, and cell spreading was not analyzed by Ma et al. (33). Based on sequence comparison it was proposed that Asp-698 and possibly -489 (identical with LDV-2 and -1) may be putative metal-binding sites that might be required to stabilize a protein-protein interaction. The reason why these sites might be important for alpha 4 integrin function remained unclear.

Our results provide suggestive evidence that the LDV-2 and -3 sites are important for a functional interaction with the beta chain. We postulate that in the presence of a transfected alpha 4-subunit the formation of a functional alpha 4beta 1 heterodimer is dominant. The formation of other heterodimers, i.e. alpha 5beta 1, is suppressed presumably due to the lack of available beta 1 chains, and these alpha -subunits reach the cell surface by other means. The mutations of LDV-2 and -3 impair the ability of alpha 4 to interact with beta 1 and favor again the formation of alpha 5beta 1 at the cell surface.

Evidence for this hypothesis came from several independent approaches. First we studied the binding of alpha 4-transfectants to FN-120. We found that in the presence of a transfected alpha 4-wt or mut 1 subunit the fibroblasts were unable to adhere and spread via alpha 5beta 1 on FN-120. In contrast, mut 12 and mut 123 cells could again spread and adhere and behaved similar to the parental 3T3 cells. We could exclude the possibility that the observed differences in the binding were the results of phenotypic changes in the transfectants since the expression levels of alpha 5, alpha v, alpha 4, and beta 1 were comparable. Furthermore, back selection of transfectants for alpha 4 loss or low expressing variants fully restored the ability to spread on FN-120, supporting the view that the suppression was due to the presence of the alpha 4-subunit and not an intrinsic failure of the cells. Importantly, the dependence of the phenomenon on amino acid substitutions in the extracellular part of the molecule argued against an involvement of cytoplasmic proteins that may be required for integrin function. It rather appeared that cis-type of interactions with other proteins at the membrane surface was the reason.

An important observation was that in the binding of cells both to FN-40 and to FN-120 the beta 1 chain was involved. In particular, the inverse behavior of the transfectants on both types of substrates was striking. This led us to consider that the beta 1 chain might be a decisive factor in regulating the integrin response in the mutants. That indeed the beta 1 chain was crucial was supported by studies on the 9EG7 epitope that has been characterized as a ligand-induced binding site of the beta 1 chain (43). Interestingly, the epitope for this mAb was located in the cysteine-rich site of the beta 1-subunit (43) which is juxtaposed to the site on the alpha 4-subunit where Asp-698 and Asp-811 are located. The level of staining for this epitope in the absence of any ligand was consistently higher on mut 12 and mut 123 cells than on alpha 4-wt or mut 1 cells. Thus, the LDV-2 and -3 mutations in the alpha 4 chain induced a change in the accessibility of this particular beta 1 chain epitope that is usually up-regulated in the presence of bound ligand or activating divalent cations but also by the addition of EDTA (41). To study the effect of ligand binding on the 9EG7 epitope, we exposed the alpha 4beta 1 heterodimer to CS-1 peptide in solution. A clear-cut up-regulation was seen in alpha 4-wt cells. In contrast, there was only little change in mut 123 cells indicating that the epitope was already fully exposed in the first place and could not be up-regulated much further. These data demonstrated that the phenotype of the alpha 4-subunit, either mutated or not, was mirrored in the 9EG7 epitope presumably indicating a conformational change of the beta 1-subunits imposed by the respective alpha 4 chain. Thus, the 9EG7 epitope acted as an indicator of a change in the alpha beta conformation.

More direct proof that the LDV-2 and -3 mutation affected the conformation of the alpha beta heterodimer came from biochemical studies. By using different detergents for the solubilization of the cells, we noticed a much decreased stability of the alpha 4beta 1 heterodimer in mut 12 and mut 123 cells. In the absence of divalent cations the heterodimer could not resist the detergent milieu as evidenced by the reduced presence of beta 1 chains in the alpha 4-specific precipitates and the failure to detect the alpha 4 80 and alpha 4 70-kDa fragments in anti-beta 1-specific precipitates of mut 12 and mut 123 cells. This could mean that the complex was only very poorly associated or even free alpha 4 chain was present at the surface of these cells. Due to the lack of heterodimer-specific mAbs in the mouse, at the present we cannot distinguish between these two possibilities. Precipitation analysis of beta 1-associated integrin subunits also revealed that the alpha 5 chain was detectable only in mut 12 and mut 123 cells and not present or only weakly present in alpha 4-wt and mut 1 cells. Thus, the precipitation analysis reflected the results from the functional analysis of mutant cells.

Further evidence for a role of LDV-2 and -3 in heterodimer formation came from studies on the expression of alpha 4beta 7 in mutant cells. At the cell surface a alpha 4beta 7 heterodimer was only seen in alpha 4-wt and mut 1 cells, whereas in mut 12 and mut 123 cells it was not detectable. Despite this the beta 7-subunit was available in all mutant cells as detected by reverse transcriptase-PCR and cytoplasmic staining. It is likely that the mutant alpha 4-subunits had a decreased ability to interact with the beta 7 chain thus preventing the assembly of the heterodimer or its transport to the cell surface.

The alpha 4 and other integrins can physically interact with transmembrane-4 superfamily proteins like CD81 (TAPA-1) and others in the cell membrane of different cell lines (44). The binding site for TAPA-1 in the alpha 4 chain is not entirely clear but has been mapped outside the alpha 4 cytoplasmic tail (44). Transmembrane-4 superfamily proteins can associate with several integrins, but a direct role in the regulation of alpha 4 integrin function has so far not convincingly been demonstrated. Although a possible interaction of these molecules with alpha 4 in our transfected fibroblasts has to be considered, we do not regard this as a reasonable mechanism to explain our results.

Sanchez-Aparicio et al. (45) have reported that stimulation of human cells with the beta 1-specific mAb TS2/16 could not only activate alpha 4beta 1 but also led to recognition of the RGDS sequence in fibronectin. Thus, the conformational change induced in alpha 4beta 1 by the mAb resulted in the ability to recognize the RGD sequence. Could the mutated alpha 4-subunit in our transfectants induce a similar change? The data presented in Fig. 9 argue against this possibility. It is evident that the adhesion of mut 12 and mut 123 cells to FN-120 was dependent on alpha 5beta 1 since it was blocked by the respective mAb but not by the mAb against the alpha 4 integrin.

Divalent cations regulate integrins in a complex way. All integrin-ligand interactions are divalent cation-dependent, and putative metal ion-binding sites have been identified in alpha - and beta -subunits (see Refs. 3-5). The alpha -subunit of all integrins contain 3-4 divalent cation binding modules with homology to the EF-hand Ca2+-binding motifs (46). In the I domain which is present in many but not all alpha -subunits a metal ion-dependent adhesion site is present that coordinates Mg2+ (47), and a metal ion-dependent adhesion site-like motif is also present in the most conserved region of beta  integrin subunits (47). All these regions are involved in ligand binding implying that the ligand-binding pocket is complex and involves both subunits. Since the ligand epitopes recognized by integrins are often short acidic peptide motifs with central oxygenated amino acids, it has been suggested that receptor-bound cation might act as an integrin-ligand bridge. Recently a model was proposed for the alpha  chain 7-fold repeats that represent about 40% of the extracellular portion of the alpha  chains (48). This model predicts a beta -propeller domain composed of seven blades made out of beta -sheets around a central axis (48). The I domain is inserted into the beta -propeller between the second and the third blade and is predicted to sit on the upper rim ot the beta -propeller domain (49). The beta -propeller model and the crystal structure of the alpha -subunit I domain from CD11a and CD11b (47, 49) have allowed for the first time to propose a dynamic quaternary structure model of integrin-ligand interaction sites (5).

Outside the region of ligand contact the structure and conformation of integrin subunits is less well defined. It is known that the alpha - and beta -subunits form a heterodimer also in the absence of ligand and that withdrawal of divalent cations by chelating agents destabilizes the alpha beta heterodimer (50-52). It is quite possible that the conformation of the alpha beta heterodimer also depends on multiple interactions that, at least to some part, uses metal ion bridging for stabilization. The metal ion-dependent adhesion site-like domain of several integrin beta -subunits was suggested to be important for the association with the alpha -subunit (53-55). Interestingly, the beta -propeller model predicts that the EF-hand Ca2+-binding motifs are located near one another on the lower surface of the beta -propeller domain and might be involved in interactions with the beta -subunit rather than in ligand binding (49). The LDV-2 and -3 sites are located on the stalk region of the alpha 4-subunit. Due to this localization they are probably not directly involved in ligand binding but may be necessary to establish a proper association with the beta  chain. The lack of this putative association affects the affinity of the receptor and leads to defects that seem to be important in the early phase of ligand binding. These defects in receptor function of mutant cells were restored, although not completely, in the presence of Mn2+ ions and by prolonged assay times. It is possible that the presence of Mn2+ enforced additional binding sites. Collectively, our data suggest that LDV-2 and -3 sites represent important contact sites between alpha 4 and its beta -subunits. The presence of similar amino acid motifs in ligands and the alpha 4-subunit suggests that metal coordination plays an important role in integrin-ligand binding as well as for heterodimer formation.

    ACKNOWLEDGEMENTS

We thank Volker Schirrmacher for support and stimulating discussions. We are grateful to Dr. Rüdiger Pipkorn for peptide synthesis and Drs. Dietmar Vestweber and Hideo Yagita for generous gifts of antibody.

    FOOTNOTES

* This work was supported by a grant from the Deutsche Forschungsgemeinschaft (to P. A.).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: Tumor Immunology Programme, 0710, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Federal Republic of Germany. Tel.:06221-423713; Fax: 06221-423702; E-mail: p.altevogt{at}dkfzheidelberg.de.

1 The abbreviations used are: VCAM-1, vascular cell adhesion molecule-1; mAb, monoclonal antibody; PBS, phosphate-buffered saline (lacking Ca2+ and Mg2+); FACS, fluorescence-activated cell sorter; wt, wild type; mut, mutant; CHO, Chinese hamster ovary; PCR, polymerase chain reaction; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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