Molecular Determinants of Arg-Gly-Asp Ligand Specificity for beta 3 Integrins*

(Received for publication, September 4, 1996, and in revised form, November 19, 1996)

Thomas J. Kunicki Dagger , Douglas S. Annis and Brunhilde Felding-Habermann

From the Roon Research Center for Arteriosclerosis and Thrombosis, Division of Experimental Hemostasis and Thrombosis, Department of Molecular and Experimental Medicine, and the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The Arg-Tyr-Asp (RYD) and Arg-Gly-Asp (RGD) sequences within the third complementarity-determining region of the heavy chain (H3) of murine recombinant Fab molecules OPG2 and AP7, respectively, are responsible for their specific binding to the platelet integrin alpha IIbbeta 3. In this study, we evaluated the influence of divalent cation composition and single amino acid substitutions at key positions within H3 on the selectivity of these Fab molecules for integrin alpha IIbbeta 3 versus the vitronectin receptor alpha Vbeta 3. The parent Fab molecule OPG2 (H3 sequence, HPFYRYDGGN) binds selectively to alpha IIbbeta 3 and not at all to any other RGD-cognitive integrin, particularly alpha Vbeta 3, under any divalent cation conditions. The binding of the AP7 Fab molecule (HPFYRGDGGN) to alpha IIbbeta 3 is not affected by the relative composition of calcium, magnesium or manganese. However, AP7 binding to alpha Vbeta 3, either expressed by M21 cells or as the purified integrin, is supported by manganese and inhibited by calcium. If the flanking asparagine 108 residue within the AP7 H3 loop is replaced by alanine (HPFYRGDGGA), the resulting Fab molecule AP7.4 binds selectively to alpha Vbeta 3 in a cation-dependent manner, but does not bind at all to alpha IIbbeta 3 under any conditions. AP7.4 binding to alpha Vbeta 3 is supported by manganese, completely inhibited by calcium, and largely unaffected by magnesium. This behavior mimics that of the adhesive protein, osteopontin, another ligand that binds preferentially to alpha Vbeta 3. Despite these differences in specificity for alpha IIbbeta 3 and alpha Vbeta 3, AP7 and AP7.4 remain selective for the beta 3 integrins and do not bind to cell lines that express the RGD-cognitive integrins alpha Vbeta 5 or alpha 5beta 1. These results confirm that subtle changes in the amino acid composition immediately flanking the RGD or RYD motifs can have a profound effect on beta 3 integrin specificity, most likely because they influence the juxtaposition of the arginine and aspartate side chains within the extended RGD loop sequence.


INTRODUCTION

The two members of the beta 3 integrin subgroup are alpha IIbbeta 3, which is required for platelet cohesion mediated by the binding of fibrinogen or von Willebrand factor (1-3); and alpha Vbeta 3, the ubiquitous vitronectin receptor that mediates a variety of cellular processes, including migration, tumor cell metastasis, and angiogenesis (4-7). The distinctive alpha  subunits, alpha IIb and alpha V, are relatively unique within the integrin family and exhibit only 36% sequence identity (8).

Although both beta 3 integrins recognize the RGD motif, each exhibits a preference for certain RGD-containing ligands. An example is the snake venom disintegrin barbourin (9), containing the KGD sequence, which binds with greater affinity to the platelet integrin alpha IIbbeta 3. Smith et al. (10) have exploited this fact to alter the specificity of an engineered RGD-containing Fab molecule and increase its selectivity for alpha IIbbeta 3 over alpha Vbeta 3. In addition, this fundamental observation has led to the synthesis of a cyclic homoarginine-Gly-Asp (cHarGD)1 peptide that has one of the highest differential affinities for alpha IIbbeta 3 versus alpha Vbeta 3 (11). Nonetheless, each of these ligands, including this peptide, retain some affinity for alpha Vbeta 3, so that they still inhibit cell adhesion mediated by alpha Vbeta 3 (11). The relative specificity of ligands for alpha IIbbeta 3 versus alpha Vbeta 3 is also markedly influenced by divalent cations. For example, fibrinogen binds to alpha Vbeta 3 in the presence of Mn2+ but not in the presence of Ca2+ (12).

To address these factors, Suehiro et al. (13) compared carefully the binding of various RGD ligands and peptides to the beta 3 integrins as a function of divalent cation composition, and grouped ligands into four classes. Class I, represented by RGD peptides and vitronectin, bind equivalently to alpha IIbbeta 3 and alpha Vbeta 3. Class II, represented by cHarGD, fibrinogen, or fibrinogen gamma -chain peptides, bind to both integrins in the presence of Mn2+, but only to alpha IIbbeta 3 in the presence of Ca2+. Class III, such as barbourin, bind exclusively to alpha IIbbeta 3 under any condition. Class IV, represented by osteopontin, bind primarily to alpha Vbeta 3.

To gain further insight into the molecular basis for differences in ligand specificity, we exploited our well characterized, recombinant RGD-containing Fab molecule AP7, and its RYD-containing progenitor OPG2 (14). By the criteria of Suehiro et al. (13), OPG2 is a Class III ligand, binding only to alpha IIbbeta 3 under any condition, while AP7 is a Class II ligand, binding to alpha Vbeta 3 in Mn2+ but not Ca2+ and to alpha IIbbeta 3 in either cation. The single amino acid replacement Asn right-arrow Ala within the RGD-containing loop of AP7 further changes the specificity of the mutagenized Fab molecule. The new Fab molecule, which we designate AP7.4, binds exclusively to alpha Vbeta 3 and belongs to the Class IV RGD ligand group. These findings confirm that selectivity for the beta 3 integrins is determined by precise juxtapositions of the Arg and Asp side chains that are significantly affected by flanking amino acid sequences.


MATERIALS AND METHODS

Synthesis of Recombinant OPG2 Fd and kappa  Chain cDNA

The term Fd denotes the segment of the Ig heavy chain that includes the VH domain, the Cgamma 1 domain, and a portion of the hinge region up to and including the cysteine residue, which participates in a disulfide bond with the carboxyl-terminal cysteine residue of the light chain (14). Fab molecules represent disulfide-linked heterodimers composed of Fd plus kappa  chains. Hexahistidine-tagged Fd cDNAs and kappa  chain cDNA were prepared as described previously (14-16). In each Fd construct, the oligonucleotide sequence (CATCAC-)3 was inserted upstream from a TGA stop codon so as to encode a carboxyl-terminal (His)6 sequence used to purify the Fab or Fd molecules by nickel affinity matrix chromatography, as described (15, 16).

The Fd construct AP7.4 was generated by splice overlap extension PCR (14, 16), using AP7 Fd cDNA (14) as a template. The 5' cDNA fragment of AP7.4 Fd was obtained as a BglII/SacII fragment from AP7 Fd cDNA. The primer 5'-CTTCTACCGCGGCGACGGGGGAGCTTACTATGCTATGG-3' (AP74FOR) changes Asn108 within AP7 H3 to Ala, while retaining a SacII site. AP74FOR was used in combination with the oligonucleotide 5'-CTA<UNL>TCTAGA</UNL>TCAATCCCTGGGCAC-3' (HREV) to produce the 3' fragment of AP7.4 Fd cDNA. Both fragments were then digested with SacII and ligated. Ligated cDNA served as template for subsequent PCR reactions using the oligonucleotide 5'-CTC<UNL>AGATCT</UNL>ACAATGGACTTCGGGCTC-3' (HFOR) and HREV to amplify AP7.4 Fd cDNA. A BglII-NcoI digest of the cDNA product was then ligated into pVL1392 containing a portion of the murine constant region, the carboxyl-terminal cysteine, and the (CATCAC-)3 sequence followed by the TGA stop codon. The cloned, recombinant virus containing this new Fd construct is designated pVL7.4Fd.

The Fd construct of AP7.7 was generated by PCR-based overlap extension using FdOPG2 as template. The 5' fragment was produced with the primer HFOR and the oligonucleotide 5'- GGTCCATAGCATAGTAAGCTCCCCCGTCGTACC-3' (AP77REV). The 3' fragment was generated using the oligonucleotide 5'-GGTACGACGGGGGAGCTTACTATGCTATGGACC-3' (AP77FOR) and the primer HREV. The cDNA products were gel-purified and added together in a PCR reaction with no additional oligonucleotide primers. The DNA fragments were allowed to anneal and complimentary sequences were produced by extension from the overlapping sequences for 10 thermocycles. Subsequently, the primers HFOR and HREV were added, and an additional 25 cycles were carried out. The product was then digested with BglII and NcoI, purified, and ligated into the transfer vector employed for AP7.4 cDNA, as described above. The cloned, recombinant virus containing this insert is designated pVL7.7Fd.

Cloning and Analysis of Recombinant Baculoviruses

Recombinant viruses were cloned by infection of Sf9 cells (Invitrogen) (2 × 106 in 2 ml of complete Grace's medium) seeded in T25 culture flasks, as described (14-16). The sequence of each recombinant clone was confirmed prior to its use, using Sequenase version 2.0 (U.S. Biochemicals, Inc.). Recombinant viruses were used to coinfect High Five insect cells (Invitrogen, Inc.), and Fab molecules were harvested from the media, normally after 72-h cultures, as described (14-16). Recombinant Fd and kappa  chains were detected by a quantitative Western blot assay using rabbit polyclonal anti-murine Fd+kappa antibody, developed in our laboratory (14, 16). Fab molecules were purified by adsorption to Ni-NTA resin (QIAGEN, Chatsworth, CA) and elution with imidazole buffer, as described (16). The purity of Fab molecules was assessed by silver staining of eluted proteins separated by electrophoresis on 10% SDS-polyacrylamide gel electrophoresis slab gels (16). Purified Fab molecule concentration was determined by optical density at 280 nm using an extinction coefficient of 1.4.

Purified Integrin ELISA

Integrin alpha IIbbeta 3 was purified as a functional heterodimer from human platelets as described by Fitzgerald et al. (17), except that protease inhibitors were included in the final buffer, namely 0.4 mM phenylmethylsulfonyl fluoride, 100 µg/ml leupeptin, 0.02 µg/ml pepstatin A, and 10 mM benzamidine. The vitronectin receptor alpha Vbeta 3 was purified as a functional heterodimer from human placentas by immunoaffinity chromatography using the murine monoclonal antibody LM609, as described (18). Purified integrin heterodimers were adsorbed onto the wells of Immulon II microtiter plates (Dynatech, Inc., Chantilly, VA), and the ability of murine monoclonal Fab molecules or recombinant proteins to bind to each integrin was assessed by ELISA (19).

Flow Cytometry Analysis of Platelets and M21 Cells

Platelet-rich plasma was obtained by differential centrifugation of ACD(A)-anticoagulated whole blood. Platelet-rich plasma was harvested, and platelets were gently pelleted by centrifugation at 950 × g for 11 min at ambient temperature. The pellet was immediately resuspended in HEPES-modified, Tyrode's buffer, pH 6.5, containing 0.1% bovine serum albumin and 0.1% dextrose. The platelet suspension was applied to a Sepharose 2B column, and fractions containing platelets were collected. The recombinant Fab molecules in Tyrode's buffer were added to 5 × 105 platelets in the presence of either 20 ng/ml prostaglandin E1 or 0.2 µM phorbol myristate. After a 15 min incubation at ambient temperature, fluorescein isothiocyanate-labeled goat anti-mouse IgG F(ab')2 (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) was added. After an additional 15-min incubation in the dark, samples were diluted 10-fold with Tyrode's buffer and analyzed on a Becton Dickinson FACScan apparatus, as described (14, 15).

Cell lines that differentially express the integrins of interest to this study were maintained as described previously (20). These include: M21, which expresses alpha Vbeta 3 and alpha 5beta 1 (21, 22); M21-L, which expresses alpha 5beta 1 but not alpha Vbeta 3 (21, 22); and UCLA-P3, which expresses alpha Vbeta 5 and not alpha Vbeta 3 (23). M21 and UCLA-P3 cells were used with permission of Dr. D. L. Morton (UCLA, Los Angeles, CA), and M21-L cells were used with permission of Dr. D. A. Cheresh (The Scripps Research Institute, La Jolla, CA). Cells were washed twice in 0.5 mM EDTA and resuspended in 0.02 M Tris, 0.15 M NaCl, pH 7.4, containing the desired combination of 1 mM CaCl2, 1 mM MgCl2, or 0.1 mM MnCl2. The cell suspensions were then incubated for 30 min at ambient temperature with selected murine monoclonal antibodies: 50 µg/ml recombinant OPG2, AP7, AP7.2, or AP7.4 Fab molecules; 10 µg/ml anti-alpha 5beta 1 (P1D6) (24), anti-alpha Vbeta 3 (LM609) (25), or anti-alpha V (AV-8) (26). To these mixtures, 50 µl of secondary antibody were added (fluorescein isothiocyanate-labeled goat anti-mouse Fab; 1:50 dilution in Tris-buffered saline; Jackson Immunoresearch Laboratories, Inc., West Grove, PA). Following another 30 min of incubation, bound Fab molecules were detected by flow cytometry, as described (16, 20).


RESULTS

The RGD-containing Fab ligand AP7 binds the integrin alpha IIbbeta 3 equivalently on both resting or activated platelets (14, 16). On the premise that flanking sequences can influence integrin specificity, our goal was to determine the smallest possible mutation within the AP7 H3 loop that would change its specificity from alpha IIbbeta 3 to alpha Vbeta 3.

Since each Fd construct employed in this study contains the (His)6 coding sequence, Fab molecules were purified to >= 99% homogeneity by Ni-NTA resin chromatography. Purified protein concentrations were determined by adsorption at 280 nm. The average yield of Fab molecules per 72 h of culture was essentially equal for each construct: OPG2, 20.1 ± 2.1 µg/ml (mean ± S.D., n = 5) AP7, 19.4 ± 1.1 µg/ml (n = 5); AP7.4, 16.4 ± 3.4 µg/ml (n = 2); and AP7.7, 20.9 ± 0.9 µg/ml (n = 2).

Binding of Recombinant Fab Molecules to Platelets

The binding of AP7, AP7.4, and AP7.7 Fab molecules to gel-filtered platelets was compared by flow cytometry as a function of extracellular divalent cation composition and platelet activation with phorbol myristate. Equivalent results were obtained in the presence of either 10 µM Mn2+ (Fig. 1) or 1 mM Ca2+ plus 1 mM Mg2+ (data not shown). As we have shown in the past (14, 16), the binding of AP7 Fab molecules to platelets was saturable and could be completely inhibited by RGDW (>= 10 µM) or EDTA (5 mM) (data not shown). AP7.4 and AP7.7, on the other hand, do not bind to platelets in the presence of any combination of divalent cations and regardless of the extent of platelet activation (Fig. 1).


Fig. 1. Fab molecule binding to gel-filtered platelets determined by flow cytometry. Purified Fab molecules at the concentration indicated on the abscissa were mixed with nonactivated platelets (open symbols) or platelets pretreated with phorbol myristate (closed symbols). In the depicted assay, platelets were maintained in buffers containing 1 mM Ca2+ plus 1 mM Mg2+ throughout exposure to the Fab molecules and subsequent flow cytometry, but identical results were obtained in the presence of 10 µM Mn2+. The number of Fab molecules bound after a 30-min incubation is represented by the mean fluorescence intensity (ordinate). The added Fab molecules were: AP7 (open circle , bullet ), AP7.4 (triangle , black-triangle), and AP7.7 (diamond , black-diamond ). Each entry is the mean of triplicate measurements in a single representative assay.
[View Larger Version of this Image (13K GIF file)]


Binding of Recombinant Fab Molecules to Human Cell Lines

The parent Fab molecule OPG2 fails to bind to alpha Vbeta 3 expressed by the melanoma cell M21 under any conditions (Fig. 2A), a finding that is consistent with our previous reports that this antibody is selective for the integrin alpha IIbbeta 3 (27, 28). However, the comparative binding of AP7 and its derivatives to M21 cells is more complex and obviously influenced by divalent cation composition. While AP7.7 Fab molecules also fail to bind to M21 cells under any conditions (data not shown), differential binding of both AP7 and AP7.4 Fab molecules is observed. In general, we found that 1 mM Ca2+ exerts an inhibitory effect on the binding of either AP7 or AP7.4 Fab molecules, that the binding of each is markedly augmented by the presence of >= 100 µM Mn2+, and that 1 mM Mg2+ has neither a supportive nor inhibitory influence (Fig. 2A). In the case of AP7, a synergistic effect is observed in the presence of both 1 mM Mg2+ and 100 µM Mn2+, so that maximal binding equivalent to that seen with AP7.4 is observed. This is not true of AP7.4 itself, which shows maximal binding in the presence of either 100 µM Mn2+ alone or both 1 mM Mg2+ and 100 µM Mn2+. In any case, binding that would otherwise be supported by Mn2+ is significantly attenuated in the presence of 1 mM Ca2+. Under optimum divalent cation conditions, the binding of either AP7 or AP7.4 Fab molecules to M21 melanoma cells is completely inhibited in a dose-dependent manner by GRGDSPK but not GRGESPK (Fig. 2B).


Fig. 2. Fab molecule binding to M21 melanoma cells determined by flow cytometry. A, M21 cells rinsed in 0.5 mM EDTA and suspended in Tris-buffered saline were incubated for 30 min with a saturating concentration of Fab molecules (50 µg/ml) in the absence of divalent cations (sample to the far left) or the presence of 1 mM Ca2+, 1 mM Mg2+, or 100 µM Mn2+, added individually or in combinations, as indicated on the abscissa. The binding of three Fab molecules is compared: AP7.4 (solid bars), AP7 (diagonally striped bars), and OPG2 (open bars). Each entry represents the mean ± S.D. of triplicate measurements. B, M21 cells were incubated for 30 min in the presence of AP7.4 (solid bars) or AP7 (diagonally striped bars), 1 mM Mg2+ plus 100 µM Mn2+, and the peptides GRGDSPK or GRGESPK at the concentrations indicated on the abscissa. The amount of bound Fab molecules was then determined and is proportional to the MFI indicated on the ordinate. Each entry represents the mean ± S.D. for three measurements.
[View Larger Version of this Image (24K GIF file)]


Despite the observed changes in selectivity for alpha IIbbeta 3 versus alpha Vbeta 3, the AP7 and AP7.4 Fab molecules remained selective for only these beta 3 integrins and did not bind to either M21-L cells, which express alpha 5beta 1 but not alpha Vbeta 3, or UCLA-P3 cells, which express alpha Vbeta 5 but not alpha Vbeta 3 (data not shown).

Binding of Recombinant Fab Molecules to Purified alpha IIbbeta 3 or alpha Vbeta 3

The selectivity of each Fab molecule for alpha IIbbeta 3 or alpha Vbeta 3 and the dependence of binding on the RGD sequence were further investigated using the purified integrins in an ELISA (Fig. 3).


Fig. 3. Binding of purified Fab molecules to purified integrins. Depicted are the results of binding to alpha IIbbeta 3 in the presence of 1 mM Ca2+ plus 1 mM Mg2+ (A), alpha Vbeta 3 in the presence of 1 mM Ca2+ plus 1 mM Mg2+ (B), alpha IIbbeta 3 in the presence of 10 µM Mn2+ (C), and alpha Vbeta 3 in the presence of 10 µM Mn2+ (D). The concentration of Fab molecules added (µg/ml) is indicated on the abscissa, and the amount of bound Fab molecules is proportional to the OD at 405 nm depicted on the ordinate. The Fab molecules tested are OPG2 (black-square), AP7 (open circle ), AP7.4 (bullet ), and AP7.7 (diamond ). Each entry represents the mean ± S.D. of triplicates.
[View Larger Version of this Image (20K GIF file)]


In the presence of 1 mM each of Ca2+ and Mg2+, AP7 or OPG2 Fab molecules exhibit a strong affinity for alpha IIbbeta 3 (Fig. 3A) but fail to bind to alpha Vbeta 3 (Fig. 3B). Binding of either Fab molecule is completely inhibited by 1 mM EDTA or >= 10 µM RGDW but not by <= 2 mM RGEW (data not shown). Under the same divalent cation conditions, both AP7.4 and AP7.7 Fab molecules fail to bind to either alpha IIbbeta 3 (Fig. 3A) or alpha Vbeta 3 (Fig. 3B).

In the presence of >= 10 µM Mn2+, the binding of each Fab molecule to purified alpha IIbbeta 3 is unchanged (Fig. 3C). However, AP7 and AP7.4 Fab molecules now bind strongly to alpha Vbeta 3 in the presence of Mn2+, while OPG2 Fab molecules fail to bind (Fig. 3D). The binding of AP7 or AP7.4 Fab molecules to alpha Vbeta 3 in this cell-free system is completely inhibited by 1 mM EDTA or >=  10 µM RGDW, but not at all by up to 2 mM RGEW (data not shown). As a negative control, AP7.7 Fab molecules (50 µg/ml) fail to bind to either integrin under any conditions (Fig. 3, A-D).


DISCUSSION

Using the recombinant murine Fab molecule OPG2 as a versatile framework, our results validate the hypothesis that the amino acid composition immediately flanking an RGD tripeptide can profoundly influence the specificity and divalent cation modulation of ligand binding to beta 3 integrins. The relevant sequences of the recombinant Fab molecules, OPG2, AP7, AP7.4, and AP7.7, and their comparative specificities, as determined by this study, are summarized in Table I.

Table I.

Comparative binding of recombinant Fab molecules to beta 3 integrins


Fab H3 sequence Binds in the presence of
Ca2+ plus Mg2+
Mn2+
 alpha IIbbeta 3  alpha Vbeta 3 Platelets M21  alpha IIbbeta 3  alpha Vbeta 3 Platelets M21

OPG2 HPFYRYDGGN Yes No Yes No Yes No Yes No
AP7 HPFYRGDGGN Yes No Yes No Yes Yes Yes Yes
AP7.4 HPFYRGDGGA No No No No No Yes No Yes
AP7.7 HPFYRYDGGA No No No No No No No No

The model that we have developed using OPG2 and the AP7 series of recombinant Fab molecules provides a unique opportunity to compare both RGD and RYD analogs of the same ligand and to predict the impact of single amino acid substitutions on specificity based upon known side chain interactions defined by x-ray crystallography of the parent Fab molecule (28). In the case of each Fab molecule in our series, a single amino acid substitution within the third complementarity-determining region of the heavy chain results in a profound change in specificity. For example, there is the complete loss of binding to either beta 3 integrin that is characteristic of AP7.7 created by the replacement of Asn108 by an Alanine within the OPG2 H3 sequence. The major reason for our selection of Asn108 as a target for substitution is the fact that, in the crystal structure of the OPG2 Fab molecule (28), the side chain of Asn108 is in a position to form a hydrogen bond with that of Asp105, i.e. the distance between Asp105-OD1 and Asn108-ND1 is 2.8 or 3.2 Å in each of two alternate conformers of the H3 loop. We reasoned that disruption of such a side chain interaction would likely influence the juxtaposition of the remaining side chains, particularly those of Arg103 and Asp105. This hypothesis is borne out by our experimental evidence, and our results provide strong support for the presence of a hydrogen bond between these side chains. It follows that this side chain interaction probably holds the Asp105 carboxyl group in a unique orientation with respect to the amino group of Arg103 such that OPG2 is recognized exclusively by alpha IIbbeta 3.

The most dramatic and novel finding of our study is the change in specificity created by the engineering of the AP7.4 Fab molecule. The sole difference between the Fab molecule AP7, which binds preferentially to alpha IIbbeta 3 in the presence of calcium ions, and AP7.4, which binds solely to alpha Vbeta 3 in the presence of manganese ions, is a single amino acid substitution adjacent to the RGD motif within the H3 loop (HPFYRGDGGN in AP7 versus HPFYRGDGGA in AP7.4). To our knowledge, this is the first published report of a complete change in specificity of an RGD ligand from alpha IIbbeta 3 to alpha Vbeta 3 as a result of a single amino acid substitution. While others have shown that single amino acid differences in RGD peptides or an Fab molecule can increase their relative affinities for alpha IIbbeta 3 (10, 11, 29, 30), the engineered AP7.4 molecule represents the first instance in which a dramatic decrease in affinity for alpha IIbbeta 3 and reciprocal increase in affinity for alpha Vbeta 3 has been produced. This change in relative affinities is so extreme that the binding of the Fab molecule to alpha IIbbeta 3 has fallen below the level of detection. Apparently, because Tyr104 of OPG2 has been replaced by Gly in AP7, the subsequent replacement of Asn108 with Ala no longer results in the dramatic loss of binding to either integrin that was observed with the creation of AP7.7 (see above). In the case of AP7.4, the elimination of the putative hydrogen bond and the accommodation of this change by the mutated H3 loop must increase the flexibility of the Arg103 and Asp105 side chains and facilitate the increased selectivity of AP7.4 for the integrin alpha Vbeta 3. Our results would argue that both alpha IIbbeta 3 and alpha Vbeta 3 are highly restrictive with respect to the Arg and Asp side chain orientations that each will recognize.

There are at least two mechanisms that may be involved in the divalent cation regulation of ligand binding to integrins. On one hand, integrin conformation is likely influenced by cations, particularly Mn2+. As an example, the monoclonal antibody 9EG7 binds to a Mn2+-induced epitope and stimulates beta 1 integrin functions (31). On the other hand, there is substantial evidence that divalent cations support an initial ternary complex with integrin and ligand. As the ligand-integrin binding becomes stabilized, the divalent cation is displaced (32). Replacement of Asn108 with Ala may eliminate the ability of AP7.4 to disrupt cation coordination upon contact with an integrin. This would not explain, however, why AP7.4 then binds selectively to alpha Vbeta 3, an integrin whose recognition of RGD ligands is equally regulated by divalent cations.

Osteopontin was the first RGD ligand identified that has a substantial preference for alpha Vbeta 3 relative to alpha IIbbeta 3 (33). It is particularly relevant that Ca2+ is a strong inhibitor of osteopontin binding to alpha Vbeta 3, while Mn2+ enhances this interaction (33). In the microenvironment of bone tissue, osteoclasts liberate Ca2+ from demineralized bone during resorption such that levels of free Ca2+ increase locally. This would then favor detachment of osteoclasts from bone by inhibition of the osteopontin binding to alpha Vbeta 3. Conversely, increases in the relative level of Mg2+ compared to Ca2+ in areas of bone growth would favor osteopontin-mediated cell attachment. Micromolar levels of Mn2+ found in many tissues, including bone and liver, would support cell attachment via alpha Vbeta 3 (12). Clearly, the engineered, recombinant Fab molecule AP7.4 behaves precisely as does osteopontin with respect to its selectivity for the integrin alpha Vbeta 3 and the influence of divalent cations on its binding properties. Thus, AP7.4 is a powerful new tool to investigate the role of the integrin alpha Vbeta 3 in various biological processes, including bone resorption.


FOOTNOTES

*   This study was supported by R01 Grant HL-46979 (to T. J. K.) from the NHLBI, National Institutes of Health and R27 Grant CA-67988 (to B. F.-H.) from the NCI, National Institutes of Health. This is manuscript 10132-MEM from The Scripps Research Institute. 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    To whom all correspondence should be addressed: Dept. of Molecular and Experimental Medicine, The Scripps Research Institute, 10666 N. Torrey Pines Rd., Maildrop SBR13, La Jolla, CA 92037. Tel.: 619-784-2668; Fax: 619-784-7124.
1    The abbreviations used are: cHar, cyclic homoarginine; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay.

Acknowledgments

We thank Dr. E. Wayner (University of Minnesota, St. Paul, MN) for monoclonal antibody P1D6 and Dr. D. Cheresh (The Scripps Research Institute) for monoclonal antibody LM609.


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