Evidence That the Integrin beta 3 and beta 5 Subunits Contain a Metal Ion-dependent Adhesion Site-like Motif but Lack an I Domain*

(Received for publication, December 19, 1996, and in revised form, March 5, 1997)

Emme C. K. Lin Dagger , Boris I. Ratnikov , Pamela M. Tsai , E. Rosalie Gonzalez , Shawn McDonald , Anthony J. Pelletier § and Jeffrey W. Smith

From the Program on Cell Adhesion and the Extracellular Matrix, The Burnham Institute, La Jolla Cancer Research Center, La Jolla and the § Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Addendum
REFERENCES


ABSTRACT

The amino-terminal domain of each integrin beta  subunit is hypothesized to contain an ion binding site that is key to cell adhesion. A new hypothesis regarding the structure of this site is suggested by the crystallization of the I domains of the integrin alpha L and alpha M subunits (Lee, J.-O., Rieu, P., Arnaout, M. A., and Liddington, R. (1995) Cell 80, 631-638; Qu, A., and Leahy, D. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10277-10281). In those proteins, an essential metal ion is bound by a metal ion-dependent adhesion site (MIDAS). The MIDAS is presented at the apex of a larger protein module called an I domain. The metal ligands in the MIDAS can be separated into three distantly spaced clusters of oxygenated residues. These three coordination sites also appear to exist in the integrin beta 3 and beta 5 subunits. Here, we examined the putative metal binding site within beta 3 and beta 5 using site-directed mutagenesis and ligand binding studies. We also investigated the fold of the domain containing the putative metal binding site using the PHD structural algorithm. The results of the study point to the similarity between the integrin beta  subunits and the MIDAS motif at two of three key coordination points. Importantly though, the study failed to identify a residue in either beta  subunit that corresponds to the second metal coordination group in the MIDAS. Moreover, structural algorithms indicate that the fold of the beta  subunits is considerably different than the I domains. Thus, the integrin beta  subunits appear to present a MIDAS-like motif in the context of a protein module that is structurally distinct from known I domains.


INTRODUCTION

Integrins are alpha beta heterodimers that mediate cell adhesion (1, 2). Integrins participate in development and tissue remodeling and are linked to several diseases. The integrins bind to many adhesive and extracellular matrix proteins. The focal points of this study are the alpha vbeta 3 and alpha vbeta 5 integrins, both of which recognize the Arg-Gly-Asp (RGD)1 tripeptide motif. The alpha vbeta 3 integrin binds to at least nine adhesive proteins and has two important biological functions. First, alpha vbeta 3 mediates the adhesion of osteoclasts to the bone surface (3), an event often considered to be the first step in bone resorption (4). Second, the alpha vbeta 3 integrin is expressed on the surface of angiogenic endothelial cells, where it is required for cell survival and further vessel development (5-7). It has been suggested that inhibitors of the alpha vbeta 3 integrin could be applied as antagonists of osteoporosis and tumor angiogenesis. The biological function of the alpha vbeta 5 integrin is less clear. This integrin can mediate cell adhesion to vitronectin. The alpha vbeta 5 integrin is also required for the internalization of adenovirus (8, 9), and it may be associated with angiogenesis (7).

All integrins require divalent cations to bind their ligands. An important clue to the structural basis for ion binding was revealed by the crystal structures of the I domains from the integrin alpha L and alpha M subunits (10, 11). Each I domain spans approximately 200 residues and is homologous to an "inserted" domain in a number of other proteins including von Willebrand factor (12). In alpha L and alpha M, the I domain is necessary and sufficient for ligand contact. These I domains contain a metal binding site called a MIDAS (metal ion-dependent adhesion site). This ion binding site consists of five liganding residues that can be separated into three groups. Each group of coordinating residues is located at separate positions within the primary amino acid sequence (10, 11). The first coordination group consist of the DXSXS sequence, where D is aspartate, X is any amino acid, and S is serine. The aspartate and both serines coordinate with metal ion. The second group, or coordination point, is a single threonine located 69 amino acids from the DXSXS. The third group is comprised of a single aspartate 102 residues from the DXSXS.

Interestingly, the DXSXS sequence is also present in the integrin beta  subunits (13), suggesting that they may also contain the MIDAS (10). If correct, this would provide a common structural basis for the regulation of all integrins by divalent metal ions. It would also imply that all integrins are regulated in a similar manner by metal ion. Despite this hypothesized similarity, integrins behave differently with respect to metal ions. For example, we recently demonstrated that the type of divalent ion present in the culture media regulates the way that the alpha vbeta 3 and alpha vbeta 5 integrins are organized on the cell surface (14). In fact, the same ion can direct the two integrins to completely different locations on the cell. This distinction suggests that the metal binding site within the beta  subunits is likely to have subtle but important structural differences that have an impact on receptor function.

To examine the hypothesized ion binding site within beta 3 and beta 5, the putative metal coordinating residues within each subunit were mutated to alanine. Results presented here are the first to show that Asp-119 and Asp-217 within beta 3 are important for the binding of soluble ligand to alpha vbeta 3. The homologous aspartic acids within beta 5 are also key to soluble ligand binding. Interestingly though, mutations at these aspartic acids do not completely abrogate cell adhesion. Their mutation decreases cell adhesion and reduces the apparent affinity of the integrin for metal ion. This finding indicates that each aspartic acid is likely to be part of a metal ion binding site that controls ligand contact. The similarity in spacing and function of these aspartates to the metal ligands in alpha L and alpha M indicate a similarity between the integrin beta  subunits and the MIDAS motif found in the I domains. However, evidence is presented here which argues that the fold of the beta  subunits is distinct from that of the I domains. The PHD structural algorithm predicts that the beta 3 and beta 5 subunits have little structural similarity with the I domains.


EXPERIMENTAL PROCEDURES

Sequence Alignment and Structural Predictions

Amino acid sequences of beta 3 (residues 107-292) and beta 5 (residues 109-296) were each aligned with the I domain sequences of alpha L (residues 125-310) and alpha M (residues 128-318) using the multiple sequence alignment program, ClustalW (15). Without further manipulation, the putative metal ligands were identified in each beta  subunit by comparison to the known metal coordination sites within the MIDAS motifs of alpha L and alpha M.

The structures of beta 3, beta 5, and the I domains of alpha L and alpha M were analyzed using the PHD algorithm (16, 17). The algorithm compares the environment of a single residue within a data base of known crystal and NMR structures and then assigns the probability that a residue is in a helix or a sheet. The algorithm also assigns a reliability index from 0 to 9 to assist in distinguishing residues which could be present in either structure. In this analysis, structural predictions for individual residues were only accepted when the reliability index was greater than five. The amino acid sequences encompassing residues 107-292 in beta 3, residues 109-296 in beta 5, residues 125-310 in alpha L, and residues 128-318 in alpha M were subjected to this analysis. Individual structural elements in a predicted protein were considered to be a match (compared with elements in the known crystal structure of alpha L or alpha M) only if at least 50% of the residues in the element were predicted to be in the correct position and of the correct structure (helix or sheet).

Mutagenesis

The beta 3 and beta 5 cDNAs were cloned using the polymerase chain reaction. The beta 3 or beta 5 cDNA in the Bluescript II SK(+) plasmid (Stratagene) was used as the parental plasmid for mutagenesis. Point mutations were introduced using the Chameleon Double-stranded, Site-directed Mutagenesis Kit (Stratagene). 5'- Phosphorylated oligonucleotide primers were produced by Integrated DNA Technologies. The oligonucleotide sequences used to produce each of the beta 3 mutants are as follows: D119A, TACTACTTGATGGCCCTGTTTAC; T182A, ATGATATGAAGGCCACCTCCTTGCC; T183A, ATGATATGAAGACCGCCTCCTTGCC; D217A, CACGGAACCGAGCTGCCCCAGAGGG; E220A, GAGATGCCCCAGCGGGTGGCTTTG. The oligonucleotide sequences used to introduce each of the beta 5 mutations are as follows: D121A, CTGTACTACCTGATGGCCCTCTCCCTGTCC; N186A, GTTGTTTCCAGCTTGCGTCCCCT; S190A, GCGTCCCCGCCTTTGGGTTCCGCC; D220A, CGGAACCGAGCTGCCCCTGAGGGG; E223A, CCTGCGGGGGGCTTTGATGCAGTA. The point mutations are indicated in bold lettering. All mutations were confirmed by dideoxy sequencing, and the cDNAs were subsequently subcloned into the mammalian expression vector pcDNA3 (Invitrogen).

Cell Lines and FACS Analysis

Human embryonic kidney 293 cells were obtained from ATCC and maintained in Dulbecco's modified Eagle's medium (BioWhittaker) supplemented with 10% fetal calf serum (Irvine Scientific). Mutated constructs of human beta 3 or beta 5 cDNA in plasmid pcDNA3 were transfected into 293 cells using DOTAP transfection reagent (Boehringer Mannheim). Following selection in 500 µg/ml geneticin (Life Technologies, Inc.) for approximately 3 weeks, the top 5% of the positive fluorescent population of cells was obtained by sterile FACS using either the anti-alpha vbeta 3 monoclonal antibody (mAb) LM609 or the anti-alpha vbeta 5 mAb P1F6. Cells expanded from the sorted population were continuously monitored for high expression of transfected integrin throughout the duration of the study. FACS analysis was performed using standard protocols. Cells were incubated with mouse primary antibody against alpha vbeta 3 or alpha vbeta 5 (5 µg/ml), washed, and then incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibody (Caltag). All cells used for binding studies exhibited stable integration of the beta  subunit cDNA into the genome.

Antibodies and Synthetic Peptides

The anti-alpha vbeta 3 mAb LM609 was purchased from Chemicon. Nonspecific mouse IgG was obtained from Calbiochem. Monoclonal antibody P1F6 (anti-alpha vbeta 5) was purchased from Becton Dickinson. Synthetic peptides with sequences GRGDSP and SPGDRG were obtained from Coast Scientific.

Binding of Fab-9 to alpha vbeta 3 Expressed on 293 Cells

Fab-9 is an RGD-containing, synthetically engineered antibody that has been optimized through phage display to bind to beta 3-integrins (18, 19). Fab-9 has at least a thousand-fold lower affinity for alpha vbeta 5 (18) and does not bind specifically to 293 cells transfected with beta 5. 293 cells expressing the beta 3 mutants were harvested from tissue culture flasks with 0.2 mM EDTA in phosphate-buffered saline (EDTA/PBS) and washed with cold Binding Buffer (Hanks' balanced salt solution lacking MgCl2, CaCl2, and MnCl2 (Life Technologies, Inc.), 50 mM HEPES, pH 7.4, 3 mg/ml bovine serum albumin) supplemented with 0.5 mM MgCl2 and 0.05 mM MnCl2. These cation conditions were optimized for Fab-9 binding. Fab-9 was labeled with Na125I (Amersham Corp.) using IODO-GEN (Pierce) to approximately 40,000 cpm/ng. Cells (3 × 106/ml) were incubated with increasing concentrations of 125I-Fab-9 at 14 °C for 70 min. Nonspecific binding was measured by including 20 mM EDTA in the incubations (18), although prior study in this lab has shown that competition with an excess of unlabeled Fab-9 yields nearly identical values. Following incubation, free 125I-Fab-9 was separated from cell-bound ligand by centrifugation through 20% sucrose, 50 mM Tris-buffered saline, pH 7.4, at 14,000 rpm for 3 min in disposable microcentrifuge tubes (Fisher). The bottoms of the tubes were cut off and counted in a gamma counter. All data represent the average of triplicate measurements. All assays were repeated at least twice yielding identical results. The affinity of alpha vbeta 3 for Fab-9 was calculated by Scatchard analysis (20).

To measure the apparent affinity of integrin for Mg2+, Fab-9 binding was measured as a function of Mg2+ concentration. The apparent affinity for cation was taken as the concentration of cation that supported half-maximal ligand binding.

Binding of Vitronectin to 293 Cells Expressing alpha vbeta 5

Ligand binding to cells expressing alpha vbeta 5 and mutant forms of beta 5 was measured with 125I-vitronectin (Vn) using a tracer format (21). For that purpose, vitronectin was purified from human serum as described previously (22) and labeled to high specific activity (150,000 cpm/ng) with Na125I. Cells expressing the mutants of beta 5 were harvested with EDTA/PBS and washed with Binding Buffer containing 0.5 mM MgCl2 and 0.02 mM CaCl2. Cells (107/ml) were incubated with 0.5 nM 125I-Vn and increasing concentrations of unlabeled Vn in cation-supplemented Binding Buffer at 14 °C for 70 min. Specific binding was determined by subtracting the EDTA-sensitive binding from total binding. The affinity of alpha vbeta 5 for vitronectin was derived using Scatchard analysis (20).

To measure the apparent affinity of integrin for divalent cation, the binding of 125I-Vn was measured across a range of metal ion concentration. The apparent affinity for cation was determined as the concentration of ion at which half-maximal ligand binding was observed.

Cell Adhesion Assays

Cell adhesion assays were performed as described previously (23). Briefly, Fab-9 (50 nM) or vitronectin (6 nM) were coated on 96-well plates by an overnight incubation at 4 °C. The plates were then blocked with 1% bovine serum albumin. Cells (1 × 105 cells/well) were harvested with EDTA/PBS and resuspended in Binding Buffer containing the appropriate cations as described for soluble ligand binding. Cells were allowed to adhere at 37 °C for 45-60 min. Non-adherent cells were removed by gentle washing, and adherent cells were detected by a colorimetric assay for lysosomal acid phosphatase (24). Color absorbance was detected at A405 nm.

Measuring the Binding of Antibody AP5

The binding of mAb AP5 to alpha vbeta 3 on 293 cells was measured as described previously (25). Briefly, cells were incubated with 50 µg/ml FITC-conjugated AP5 in the presence of varying concentrations of Ca2+ and analyzed by FACS. Mean fluorescence intensity was determined per 10,000 cells.


RESULTS

Sequence and Structural Comparison between the I Domain of alpha M and the Amino-terminal Domain of beta 3 and beta 5

As a first step toward characterizing the putative metal binding site in beta 3 and beta 5, their sequences were compared with the I domain of alpha M. The sequence of beta 3 (residues 107-292) was aligned with the I domain of alpha M using the multiple sequence alignment program ClustalW (15). The sequence of beta 5 was incorporated into this alignment using the extensive identity between beta 3 and beta 5. The DXSXS motif and an aspartic acid residue representing the third coordination group in the MIDAS align well in all three proteins. The beta  subunits diverge from the MIDAS motif at the middle metal coordinating position (Thr-209 in alpha M). The beta  subunits contain a small disulfide-bonded loop in this region, a structure absent in the I domains. Within this disulfide-bonded loop, beta 3 contains two threonine residues (Thr-182 and Thr-183) that could be homologues of the coordinating threonine in alpha M. Within the same disulfide-bonded loop of beta 5, Asn-186 and Ser-190 could potentially ligand with metal ion.

To examine further the homology between the I domain and the integrin beta 3 and beta 5 subunits, structural predictions were made using the PHD algorithm under strict conditions (16, 17). As shown in Fig. 1, the algorithm correctly predicted 11 of 13 structural elements in alpha M and 10 of 13 elements in the I domain of alpha L (not shown). These findings validate the fidelity of the algorithm. The algorithm predicted that beta 3 contains only 3 of the 13 structural elements in the I domains and that beta 5 may contain up to 2 of the I domain elements. Consequently, the overall fold of this domain in the beta  subunits is likely to be significantly different than that of the known I domains.


Fig. 1. Sequence alignment and structural prediction of the alpha M I domain with beta 3 and beta 5. The I domains of alpha L and alpha M and the corresponding regions in beta 3 (residues 107-292) and in beta 5 residues (109-295) were aligned using the program ClustalW (15). The positioning of the MIDAS residues in alpha L and alpha M are identical, so only alpha M is shown. The amino acid residues that coordinate metal ion in alpha M and the predicted coordinating residues in beta 3 and beta 5 are boxed. These same regions were subjected to structural prediction using the PHD algorithm (16, 17). The predicted structures were compared with the actual structure of alpha M as determined by crystallographic data (10). beta  strands are represented as hatched rectangles; alpha  helixes are shown as shaded rectangles, and turns/random coils are left open. Secondary structural elements according to the alpha M crystal structure are labeled as beta  sheet strands A-F and alpha  helixes 1-7 (10). Crystal structure is abbreviated as X and predicted structure as P. The position and length of each element in the figure is shown to scale.
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The Metal Ion Preference of alpha vbeta 3 and alpha vbeta 5 Is Similar to the I Domain of alpha M

As a second step in assessing the similarity between the metal binding site in the beta  subunits and the MIDAS, the ion preference of alpha vbeta 3 and alpha vbeta 5 was compared with that of alpha M (26). The I domain of alpha M will bind ligand in a series of metals, although Ca2+ and Ba2+ are largely ineffective in supporting binding. Therefore, we tested the same panel of metals for the ability to support ligand binding to alpha vbeta 3 and alpha vbeta 5. This analysis was done by measuring the binding of ligand to each alpha v-integrin as a function of the type of divalent metal ion present in the binding buffer. All metals were tested at a concentration of 1 mM to be consistent with the prior study of the I domain of alpha M (26). As shown in Fig. 2, most metal ions tested support the binding of vitronectin to alpha vbeta 5. However, Ca2+ and Ba2+ supported only minimal binding to alpha vbeta 5. A similar metal preference was observed for the binding of ligand to alpha vbeta 3 (not shown). The only significant difference between the two alpha v-integrins was the inability of Cd2+ to support ligand binding to alpha vbeta 3 (not shown). These data show that ligand binding to alpha vbeta 3, alpha vbeta 5, and the I domain is supported by similar metals.


Fig. 2. Integrin alpha vbeta 5 demonstrates a cation preference similar to an I domain. A panel of metal ions was tested for the ability to support 125I-vitronectin binding to alpha vbeta 5 expressed on kidney 293 cells. Each ion was included at a concentration of 1 mM. Binding is expressed as a percentage of maximum specific binding which was achieved in the presence of Co2+. The data represent the average of two experiments that yielded nearly identical results.
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Expression of the Mutant Forms of beta 3 and beta 5 in 293 Cells

To probe the structure of the ion binding site within each beta  subunit, we mutated putative metal liganding residues to alanine. In beta 3 these are Asp-119, which represents the DXSXS sequence; Thr-182 and Thr-183, which are hypothesized to make up the second coordination group; and Asp-217, which is thought to comprise the third coordinating group. Within beta 5 the putative coordinating residues are Asp-121, Asn-186, Ser-190, and Asp-220. Because of their proximity to the last putative coordination residue, we also mutated Glu-220 within beta 3 and Glu-223 within beta 5. Each cDNA construct was used to transfect 293 cells. Following antibiotic selection and FACS sorting, transfected cells were found to express nearly equivalent levels of each of the mutated integrins on the cell surface (Fig. 3). One mutant, beta 3 E220A, was not expressed on the cell surface, even though repeated attempts were made to transfect this mutant. To confirm proper heterodimer formation, the mutant integrins were immunoprecipitated from lysates of 125I-labeled cells using antibodies against alpha vbeta 3 (LM609) and alpha vbeta 5 (P1F6). Each antibody immunoprecipitated alpha v and the relevant beta subunit in an approximate 1:1 stoichiometry, confirming that the mutated subunits complex with alpha v (data not shown).


Fig. 3. beta 3 and beta 5 mutants are expressed on the cell surface with alpha v. Human kidney 293 cells were transfected with beta 5 (top row) or beta 3 (bottom row) cDNA containing point mutations at putative metal ion-coordinating residues (indicated in figure). Cells were maintained in antibiotic selection and FACS-sorted to obtain a population that expressed high levels of integrin. Here, FACS was used to analyze the expression level of the alpha vbeta 3 or alpha vbeta 5 heterodimer on each sorted cell population. Cells were incubated with a nonspecific mouse IgG (clear peak) or an antibody (shaded peak) that binds to the alpha vbeta 3 (mAb LM609) or alpha vbeta 5 (mAb P1F6) complex. The binding of primary antibody was detected with secondary antibody conjugated to FITC. Kidney 293 cells mock-transfected with the pcDNA3 expression vector did not exhibit any shift in fluorescence (not shown).
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Assessing the Ligand Binding Function of Mutated Forms of beta 3

The ligand binding function of each mutated form of alpha vbeta 3 was measured using the model ligand Fab-9 which has been characterized previously (18, 19, 27). This ligand was chosen because the binding affinity of soluble vitronectin for alpha vbeta 3 on 293 cells was too low to yield reproducible binding data. In these binding studies, the metal concentration was set to an optimal level. Wild-type alpha vbeta 3 bound to 125I-Fab-9 with an affinity of 9 ± 3 nM (n = 10). Within the detectable range of binding, the mutation of beta 3 residues D119A and D217A abolished binding of soluble Fab-9 to the cell surface (Fig. 4). Surprisingly, cells expressing the T182A and T183A mutations bound to 125I-Fab-9 with an affinity identical to that of wild-type alpha vbeta 3. To determine whether the mutations T182A and T183A had a more subtle effect on cation-dependent ligand binding, we measured their apparent affinities for Mg2+. The apparent affinities of T182A or T183A for Mg2+, as reported by Fab-9 binding, were identical to wild-type alpha vbeta 3 (Table I). Thus, unlike the corresponding threonines within alpha L and alpha M, neither of the candidate threonines within beta 3 appear to be crucial metal ligands.


Fig. 4. Mutation of acidic residues in beta 3 eliminates binding of soluble ligand. Human 293 cells expressing mutant alpha vbeta 3 were incubated in solution with 125I-Fab-9 at 14 °C. Specific binding (black-square) was calculated as the difference between total (square ) and nonspecific binding (open circle ), which was measured in the presence of EDTA. In nearly identical experiments we found the level of nonspecific binding to be the same when excess Fab-9 was used as a competitor. Each point is the average of triplicate data points. Scatchard plots (bound (B) versus bound/free (B/F)) and calculated affinities (Kd) are shown in the insets. The correlation coefficients of each Scatchard plot are as follows: wild-type (WT), r2 = 0.97; T182A, r2 = 0.92; and T183A, r2 = 0.85. No specific binding was detected for D119A and D217A. Each figure is representative of at least two experiments in which identical results were obtained. Ligand binding to cells expressing mutant alpha vbeta 3 was always done in parallel with cells expressing WT alpha vbeta 3.
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Table I. The apparent affinities of beta 3 mutants T182A and T183A for magnesium (Mg2+)

Human 293 cells expressing wild-type (WT) alpha vbeta 3 or mutants T182A and T183A were allowed to bind 125I-Fab-9 as a function of increasing Mg2+ concentrations. The apparent affinity of integrin for Mg2+ was determined as the concentration of Mg2+ at which half-maximal ligand binding occurred. Human 293 cells expressing wild-type (WT) alpha vbeta 3 or mutants T182A and T183A were allowed to bind 125I-Fab-9 as a function of increasing Mg2+ concentrations. The apparent affinity of integrin for Mg2+ was determined as the concentration of Mg2+ at which half-maximal ligand binding occurred.

Cell line Apparent affinity for Mg2+ n

nM
WT 0.5 4
T182A 0.5, 0.7 2
T183A 0.5, 0.7 2

Cell adhesion is a multimeric interaction between clustered integrins and a non-diffusable matrix. Therefore, it can often proceed even when the affinity between integrin and ligand is very low. Consequently, we measured the effect of mutations within the beta 3 subunits on cell adhesion to immobilized Fab-9. Surprisingly, cells expressing beta 3 mutated at Asp-119 and Asp-217 adhered to Fab-9 (Fig. 5), even though they failed to bind soluble ligand. The mutated forms of beta 3 supported adhesion to a level that usually reached approximately 40% that of wild-type beta 3. More importantly, both mutations also exhibited a shift in the apparent affinity for metal ion. This was measured by determining the level of metal ion that supported half-maximal adhesion. The study was done with Mn2+ because it has the highest affinity for the integrin. The D119A mutation exhibited an apparent affinity for ion that was approximately 6-10-fold lower than that of wild-type beta 3. The mutation at Asp-217 was even more deleterious, exhibiting an apparent affinity for metal that was 15-20-fold lower than wild-type beta 3. These are the first data to demonstrate that mutations at putative metal-coordinating residues within an integrin beta  subunit shift the ion response curve. This can be interpreted to indicate that Asp-119 and Asp-217 contribute to metal binding affinity.


Fig. 5. Cells expressing alpha vbeta 3 mutants adhere to Fab-9. Human kidney 293 cells expressing mutant alpha vbeta 3 were allowed to adhere to immobilized Fab-9 at 37 °C as a function of Mn2+ concentration. Adhesion was inhibited by inclusion of mAb LM609 or GRGDSP peptide (not shown). Each data point represents the mean of triplicate wells and is expressed as a percentage of maximal adhesion for each cell line. This experiment was repeated five times yielding similar results.
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Assessing the Ligand Binding Function of Mutant Forms of the beta 5 Subunit

The binding of vitronectin to wild-type alpha vbeta 5 on 293 cells was initially characterized in conditions containing 500 µM Mg2+ and 20 µM Ca2+. Under these cation concentrations, the binding of vitronectin was specific and saturable with a Kd of 9 nM. Vitronectin binding to cells expressing wild-type alpha vbeta 5 could be completely inhibited with function-blocking mAb P1F6 or GRGDSP peptide (data not shown). Each mutant of alpha vbeta 5 was evaluated for its ability to bind soluble vitronectin. The binding of vitronectin was assayed as a function of the concentrations of Mg2+ or Mn2+ (Fig. 6). The titration of Mn2+ was carried out to only 5 mM because artifactual binding of vitronectin was detected above this concentration. In this experiment, the data are expressed as a percentage of maximal binding to wild-type alpha vbeta 5 which was always measured in parallel. Cells expressing alanine mutations at Asp-121 and Asp-220 failed to bind to soluble Vn in either Mg2+ or Mn2+. In the radioligand binding assay that was employed, we were only able to detect vitronectin binding to integrin when the Kd was below 500 nM. Since wild-type alpha vbeta 5 has a Kd of 9 nM for soluble vitronectin, we conclude that mutations at Asp-121 and Asp-220 cause at least a 55-fold reduction in the affinity of the integrin for vitronectin. These data are consistent with the role of each aspartate in metal coordination and with nearly identical data obtained for beta 3 (see above). The mutation of Asn-186 and Ser-190 to alanine had no effect on ligand binding. Thus, we were unable to identify a residue in beta 5 that corresponds to the metal coordinating threonine (Group 2) in alpha L and alpha M. It is also interesting to note that the mutation of Glu-223 to alanine eliminated the ability of alpha vbeta 5 to bind soluble vitronectin. Although this residue is not homologous to any of the metal ligands in the MIDAS, our data indicate that it has a role in ligand binding function. It may, in fact, substitute for the missing second metal ligand (see "Discussion").


Fig. 6. Mutation of acidic residues in beta 5 abolishes soluble vitronectin binding. Human 293 cells expressing the mutant forms of alpha vbeta 5 were allowed to bind to soluble 125I-vitronectin in the presence of increasing concentrations of Mg2+ (black-square) or Mn2+ (open circle ). Each point represents an average of triplicate data points. Nonspecific binding was determined by inclusion of EDTA in the binding reaction and was subtracted from the total binding to obtain specific binding. Nonspecific binding was typically less than 5-20% of the total binding. Each panel of this figure is representative of at least two experiments in which identical results were obtained. Ligand binding to cells expressing mutant beta 5 integrin (indicated in figure) was always done in parallel with cells expressing wild-type beta 5.
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The ability of each mutant form of alpha vbeta 5 to mediate adhesion to immobilized vitronectin was also measured (Table II). The substitution of alanine at Asp-121 and Glu-223 of beta 5 resulted in complete abrogation of cell adhesion, whereas alanine substitutions at beta 5 residues Asn-186 and Ser-190 had no effect on the ability of the cells to adhere to vitronectin. In contrast, beta 5 containing D220A mediated cell adhesion, although the absolute level of adhesion at saturation was lower than wild-type alpha vbeta 5. The apparent affinity of this mutant form of alpha vbeta 5 for metal ion was 5-50-fold lower than that exhibited by wild-type alpha vbeta 5 (45-62 versus 1-10 µM). This observation is consistent with a role for Asp-220 in coordinating metal ion and is also consistent with the fact that the homologous residue in beta 3 (Asp-217) contributes to metal binding affinity.

Table II. The effect of point mutations in beta 5 on cell adhesion to vitronectin

Cell lines expressing point mutations in beta 5 were allowed to adhere to immobilized vitronectin in the presence of a range of Mg2+ or Mn2+. The apparent affinities for metal ion were determined as the metal concentration at which half-maximal adhesion occurred. The range of apparent affinities that are listed were compiled from at least three separate experiments. Adhesion of cells containing the beta 5 mutations was always compared with the adhesion of 293 cells expressing wild-type alpha vbeta 5. Cell lines expressing point mutations in beta 5 were allowed to adhere to immobilized vitronectin in the presence of a range of Mg2+ or Mn2+. The apparent affinities for metal ion were determined as the metal concentration at which half-maximal adhesion occurred. The range of apparent affinities that are listed were compiled from at least three separate experiments. Adhesion of cells containing the beta 5 mutations was always compared with the adhesion of 293 cells expressing wild-type alpha vbeta 5.

Cell line Apparent affinity
Mg2+ Mn2+

µM
Wild-type 80-180 1-10
D121A No adhesion No adhesion
N186A 125-350 2-3
S190A 125-300 4-15
D220A 1000-2500 45-62
E223A No adhesion No adhesion

Mutations at Asp-119 and Asp-217 of beta 3 Fail to Disrupt the Function of the Inhibitory Ca2+-binding Site

Integrins contain two classes of ion binding sites, one that promotes ligand binding, called a Ligand Competent site, and another that inhibits ligand binding, called an Inhibitory site (27-30). The monoclonal antibody AP5 binds to the amino-terminal domain of the beta 3 subunit and reports the occupation of the Inhibitory Ca2+-binding site (25). As an extension of the present study, we measured the effect of each point mutation within beta 3 on the sensitivity of the binding of the AP5 antibody to Ca2+. As shown in Fig. 7, the binding of AP5 to wild-type alpha vbeta 3 and to both beta 3 D119A and beta 3 D217A was blocked by Ca2+. Thus, Asp-119 and Asp-217 are not part of the Inhibitory ion binding site.


Fig. 7. Calcium inhibits the binding of AP5 to WT alpha vbeta 3 and to the D119A and D217A mutants. Human 293 cells expressing the noted integrin were incubated with 50 mg/ml FITC-labeled AP5. The concentrations of Ca2+ were varied from 4 to 500 mM. The mean fluorescence intensity of 10,000 cells is presented for each point. Binding of AP5 to mutants T182A and T183A was also inhibited by Ca2+ (not shown).
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DISCUSSION

The primary objectives of this study were to examine the possibility that the amino-terminal portion of the integrin beta  subunit contains a MIDAS-like metal binding site and to assess whether this motif in the integrin beta  subunit is positioned at the apex of an I domain structure. The simplest step in this analysis involved a comparison of the two structures. The I domains and the amino-terminal portion of the integrin beta  subunits have similar hydropathy profiles (10) and also exhibit some sequence homology, particularly at residues known to ligand with metal. Both observations suggest the potential for a common fold. Here, we examined this possibility in more detail using the PHD algorithm, which generates a predicted structure based on the propensity of individual residues within a given local environment to exist in a helix, a sheet, or a disordered loop. Importantly, the algorithm relies on known crystal and NMR structures to predict tertiary structure from the primary sequence. It is reported to have a success rate of approximately 70% (17). The PHD algorithm correctly predicted 10 of 13 structural elements within the I domains of alpha L and alpha M, attesting to its ability to identify the major elements within an I domain. In contrast, the algorithm predicted that only 2-3 of the 13 I domain elements are present in the corresponding positions of beta 3 and beta 5. Although the beta  subunits appear to have some sequence similarity with the I domains, an in-depth analysis using a sophisticated algorithm suggests that the three-dimensional structure of the integrin beta  subunits is likely to be significantly different from that of the I domains. Based on this analysis it does not appear that the integrin beta  subunits contain an I domain-like region. This does not exclude the possibility that a metal-binding MIDAS motif could be presented in the context of a different backbone structure.

Therefore, a series of biochemical studies were performed to further assess metal and ligand binding to the alpha vbeta 3 and alpha vbeta 5 integrins. As a first step, the ion specificity of the ligand binding event was tested. Indeed, both integrins have an ion preference that is remarkably similar to that reported for the I domain of alpha M (26). Although some minor differences exist between alpha vbeta 3 and alpha vbeta 5, transition state metal ions like Co2+ and Mn2+, as well as the cation Mg2+, support ligand binding. Divalent ions like Ca2+ and Ba2+ were far less effective. Although we know the regulation of ligand binding to alpha v-integrins to be complex and that it can involve regulation by two classes of ion binding sites (27, 28, 30, 32), this simple test shows that the ligand binding event for alpha v-integrins has an ion specificity that is more similar to that of an I domain (26) than to an EF-hand (33).

A more detailed analysis of metal binding involved the mutation of the putative metal coordinating residues within beta 3 and beta 5. This approach identified two distantly spaced aspartic acid residues that greatly influence receptor function. These are Asp-119 and Asp-217 in beta 3 and Asp-121 and Asp-220 in beta 5. By sequence alignment, each aspartate appears to be a homologue of metal ligands in the MIDAS motifs of alpha L and alpha M. Substitution of any of these aspartates with alanine reduces the affinity of the alpha v-integrins for soluble ligands by at least 50-fold. Interestingly, mutation of Asp-119 and Asp-217 within beta 3 and Asp-220 in beta 5 did not completely abrogate receptor function because integrins with these mutations could still mediate cell adhesion. Despite the inability of each mutated integrin to bind soluble ligand, the ability of mutants at beta 3 residues Asp-119 and Asp-217 and beta 5 Asp-220 to mediate cell adhesion proves that these aspartates are not absolutely essential for ligand contact. It is important to emphasize that cell adhesion to an immobilized substratum is the summation of multivalent receptor-ligand contacts brought about by integrin clustering. In addition the ligand is immobilized and cannot freely diffuse; therefore, cell adhesion can often be observed even when the affinity between ligand and integrin is too low to measure in soluble ligand binding assays. Thus, another interpretation of this result is that each aspartate contributes to ligand binding affinity. We believe this to be a reasonable inference especially since FACS analysis indicates that each mutant is expressed on the cell surface at a level equivalent to the wild-type integrin. However, because the mutant integrins fail to interact with soluble ligand, we are unable to provide a quantitative measure of the difference in ligand binding affinity.

It is also key to assess whether the mutations at putative metal coordination sites alter the affinity of the integrin for metal ion. Unfortunately, the inability to generate milligrams of recombinant integrin, and the relatively low affinity of the integrin for ion, makes a direct measure of this parameter nearly impossible. We were, however, able to assess metal binding affinity indirectly by measuring the apparent affinity of the integrin for metal ion as reported by ligand binding. This was accomplished by measuring cell adhesion across a range of metal ion. From this analysis it is evident that the mutation of Asp-119 and Asp-217 in beta 3, and Asp-220 in beta 5, reduces the apparent affinity of each integrin for metal ion. Mutation of each aspartic acid lowered the apparent affinity for either Mn2+ or Mg2+ by 10-20-fold. This is the first evidence we are aware of in which an aspartic acid within an integrin beta  subunit has been shown to influence metal ion affinity. The simplest interpretation of this finding is that each of these aspartic acid residues is part of a metal ion binding site. Without a direct measure of metal binding affinity to each mutant, and without a three-dimensional structure, these aspartates cannot be unequivocally assigned as metal ligands. Yet, because each of the aspartate residues in question aligns well with metal ligands in the MIDAS motif, this finding strongly implies a similarity in the way the two protein modules ligand with ion at the first and third coordination groups.

This study also identifies an important distinction between the metal ligands in the MIDAS and in the beta  subunits. In the MIDAS motif, the second coordination group is a single threonine that coordinates with bound metal. Based on the alignment presented in Fig. 1, we hypothesized that the analogous threonine in beta 3 was at residue Thr-182 or Thr-183. Interestingly, the beta 5 subunit lacks this threonine, and we originally hypothesized that this difference in sequence at a metal ligand was key to the way in which beta 3 and beta 5 differentially organize on the cell surface in response to metal ions (14). However, the data presented here indicate neither Thr-182 nor Thr-183 within beta 3 makes a significant contribution to metal-dependent ligand binding, nor do Asn-186 and Ser-190 within beta 5. Thus, the distinct organization patterns of alpha vbeta 3 and alpha vbeta 5 on the cell surface do not appear to be related to a difference in metal coordination in this region of the beta subunit. The inability to identify a metal ligand within the beta subunits that is analogous to the second coordination group in the MIDAS is also a clear distinction in the way the two ion binding sites are structured.

Each integrin beta  subunit contains the sequence DXPE. The aspartate in this motif corresponds to Asp-217 in beta 3 and Asp-220 in beta 5. Here, we present evidence that the glutamic acid in this motif is important for integrin function. Transfection of 293 cells with a cDNA in which beta 3 Glu-220 is mutated to alanine failed to yield a cell line in which the alpha vbeta 3 heterodimer was expressed. The simplest interpretation of this observation is that beta 3 Glu-220 is required for proper folding or for assembly of a heterodimer with alpha v. In contrast, the mutation of Glu-223 of beta 5 to alanine allows association with alpha v and expression on the cell surface but eliminates ligand binding function. Other reports in the literature also point to the region surrounding the DXPE motif as an important domain that may be part of the ligand binding cleft. Two independent studies showed that synthetic peptides encompassing beta 3 residues 211-222 and 217-231 could block ligand binding to the alpha IIbbeta 3 integrin (31, 34).

In conjunction with the present study, these data suggest a hypothesis regarding the RGD binding site. Collectively the two lines of data indicate that the DXPE sequence may come together with the DXSXS motif to form a metal binding site that is also part of the RGD binding cleft. In this respect, the integrin beta subunits appear to contain a site that is similar to the MIDAS motif, where metal-coordinating residues are distantly spaced in the primary sequence but come together in the tertiary structure of the protein to make contact with a metal ion. This similarity must be interpreted in the context of several key differences between the beta  subunits and the I domains. Structural algorithms indicate that the beta  subunits lack similarity to the I domains, so the beta  subunits are likely to contain a MIDAS within a different backbone. Within the putative metal binding domain, the beta  subunits contain two disulfide bonds, whereas the I domains do not. The two domains function differently as well. The alpha vbeta 3 and alpha vbeta 5 integrins bind the RGD motif and require the association of both subunits for this function.

A final objective of the present study was to classify the putative ion binding site within the beta  subunits. There are two classes of metal ion binding sites on alpha vbeta 3 and on alpha 5beta 1 (25, 27, 30). These two cation binding sites have opposing effects on ligand binding. One class of site(s), called Ligand Competent sites, must be occupied for ligand to bind (23, 27, 30). The second class of sites are called Inhibitory sites because, when occupied with Ca2+, these sites interfere with ligand binding. The Inhibitory sites are allosteric to the ligand binding site and act by increasing the ligand dissociation rate (27). The data presented here provide the first mutational evidence that the Ligand Competent and Inhibitory sites are separate. The elimination of the Inhibitory cation binding site by mutation would be expected to decrease the ligand dissociation rate, thereby increasing overall ligand affinity. Inactivation of the Ligand Competent cation binding site would prevent ligand binding. Mutations at beta 3 Asp-119 and Asp-217 clearly belong to the class of sites called Ligand Competent sites. Importantly, the Inhibitory cation binding site appears to remain functional in both mutated integrins because the binding of the reporter antibody AP5 remains sensitive to Ca2+. Thus, mutations at Asp-119 and Asp-217 within beta 3 disrupt the Ligand Competent site without affecting the activity of the Inhibitory cation binding site.


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grants CA 56483 and AR 42750 (to J. W. S.) and GM 53489 (to A. J. P.).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.
Dagger    Supported by a postdoctoral fellowship from the Cancer Research Institute.
   Established Investigator of the American Heart Association and Genentech. To whom correspondence should be addressed: Program on Cell Adhesion and the Extracellular Matrix, The Burnham Institute, La Jolla Cancer Research Center, 10901 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-646-3121; Fax: 619-646-3192.
1   The abbreviations used are: RGD, Arg-Gly-Asp; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; MIDAS, metal ion-dependent adhesion site; Vn, vitronectin; WT, wild-type.

Addendum

While this paper was in review, reports on the same topic were published (Puzon-McLaughlin, W., and Takada, Y. (1996) J. Biol. Chem. 271, 20438-20443; Tozer, E. C., Liddington, R. C., Sutcliffe, M. J., Smeeton, A. H., and Loftus, J. C. (1996) J. Biol. Chem. 271, 21978-21984; Goodman, T. G., and Bajt, M. L. (1996) J. Biol. Chem. 271, 23729-23736). In each report, similar mutations were made in other integrins, yielding similar data. It should be noted, however, that the interpretation of the data are somewhat different. Based on the data presented here, we are reluctant to classify the amino-terminal regions of beta 3 and beta 5 as "I domains." As discussed above, we believe there is sufficient reason to suspect that the beta 3 and beta 5 subunits bind to metal using a MIDAS-like motif but that the backbone of the domain containing the MIDAS is structurally distinct from the known conformation of the I domains.


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