The amino-terminal one-third of alpha IIb defines the ligand recognition specificity of integrin alpha IIb beta 3.

The integrin alpha subunits play a major role in the regulation of ligand binding specificity. To gain further insight into the regions of the alpha subunits that regulate ligand specificity, we have utilized alpha v / alpha IIb chimeras to identify regions of alpha IIb that when substituted for the homologous regions of alpha v switched the ligand binding phenotype of alpha v beta 3 to that of alpha IIb beta 3. We report that the ligand recognition specificity of beta 3 integrins is regulated by the amino-terminal one-third of the alpha subunit. Substitution of the amino-terminal portion of alpha v with the corresponding 334 residues of alpha IIb reconstituted reactivity with both alpha IIb beta 3-specific activation-dependent (PAC1) and -independent (OPG2) ligand mimetic antibodies in addition to small highly specific activation-independent ligands. In contrast, substitution of the amino-terminal portion alone or the divalent cation repeats alone were not sufficient to change ligand binding specificity. These data in combination with previous studies demonstrate that integrin ligand recognition requires cooperation between elements in both the alpha and beta subunits and indicate that the ligand binding pocket is a structure assembled from elements of both the alpha and beta subunits.

The integrin ␣ subunits play a major role in the regulation of ligand binding specificity. To gain further insight into the regions of the ␣ subunits that regulate ligand specificity, we have utilized ␣ v /␣ IIb chimeras to identify regions of ␣ IIb that when substituted for the homologous regions of ␣ v switched the ligand binding phenotype of ␣ v ␤ 3 to that of ␣ IIb ␤ 3 . We report that the ligand recognition specificity of ␤ 3 integrins is regulated by the amino-terminal one-third of the ␣ subunit. Substitution of the amino-terminal portion of ␣ v with the corresponding 334 residues of ␣ IIb reconstituted reactivity with both ␣ IIb ␤ 3 -specific activation-dependent (PAC1) and -independent (OPG2) ligand mimetic antibodies in addition to small highly specific activationindependent ligands. In contrast, substitution of the amino-terminal portion alone or the divalent cation repeats alone were not sufficient to change ligand binding specificity. These data in combination with previous studies demonstrate that integrin ligand recognition requires cooperation between elements in both the ␣ and ␤ subunits and indicate that the ligand binding pocket is a structure assembled from elements of both the ␣ and ␤ subunits.
Integrins are heterodimeric adhesion receptors composed of noncovalently associated ␣ and ␤ subunits. The integrin superfamily consists of at least 20 members that are composed of different combinations of nine ␤ and more than 15 ␣ subunits. The different combinations of ␣ and ␤ subunits produce receptors that often possess a distinct ligand recognition specificity. With regard to integrin ligands, a number of discrete sites recognized by integrins have been identified and high resolution structures have been obtained for a number of integrin ligands (1)(2)(3). An emerging general theme from these structural studies is that integrins recognize protein ligands through interaction with short peptide sequences often presented on extended loops (1)(2)(3)(4)(5).
There is much less precise information concerning the sites within integrins that recognize ligands. A number of potential ligand interactive sites have been identified in the integrin ␤ subunits. Chemical cross-linking, site-directed mutagenesis, and immunological approaches have implicated a highly conserved sequence in the ␤ subunit in the ligand binding function (6 -14). A second site in the same region has also been reported to be involved in ligand binding (15,16). Six of the integrin ␣ subunits contain an additional ϳ200-residue inserted (I) 1 domain, and compelling evidence supports a role for the I domain in ligand binding (17)(18)(19)(20). Mutational evidence and sequence alignment indicates that the I domain and integrin ␤ subunits might utilize a similar mechanism for ligand recognition (10,18,21). These data have led to the hypothesis that the I domain and the conserved ␤ subunit ligand recognition site are structurally related and may define a novel motif essential for integrin receptor function (10,21). A high resolution structure of a recombinant I domain (22) supports this hypothesis.
A combination of approaches have been utilized to investigate potential ligand binding sites in ␣ subunits that do not contain an I domain; however, the results have been inconsistent. Cross-linking studies have demonstrated that bound ligand was proximal to the four divalent cation binding sites in ␣ IIb and ␣ v (23,24). Synthetic peptides (25) as well as a recombinant fragment (26) from this region of ␣ IIb have been reported to bind ligand. A homology scanning approach mapped the epitopes of antibodies that block ligand binding to ␣ 4 to the NH 2 terminus, but not to the cation binding motifs (27). Finally, the minimal ligand binding fragments of ␣ IIb ␤ 3 lack the COOH-terminal portions of the receptor, but contain more than half of the entire ␣ IIb subunit (28,29). Thus, the structures critical for ligand recognition by integrin ␣ subunits that lack an I domain remain to be elucidated.
A major difficulty in determining the role of integrin ␣ subunits in the regulation of ligand binding specificity is that the binding of most macromolecular ligands is activation-dependent, i.e. the binding of these ligands is highly regulated by the conformational state of the receptor (30,31). In contrast, the binding of small peptide ligand mimetics is often activationindependent (32,33). A limitation of previous studies aimed at identification of ligand binding sites was that a spectrum of both activation-dependent and -independent ligands were not analyzed. To gain further insight into the structures in the ␣ subunits that regulate ligand recognition specificity, we exploited the unique tools available for the integrins ␣ IIb ␤ 3 and ␣ v ␤ 3 . These two integrins share the common ␤ 3 subunit, and the two ␣ subunits are 36% identical in primary sequence (34). They recognize a number of common ligands as well as small peptides containing the Arg-Gly-Asp (RGD) sequence (35). In addition, there exist highly specific small activation-independent ligands (36 -38). Moreover, true ligand mimetic monoclonal antibodies, PAC1 (39) and OPG2 (40), have been prepared against ␣ IIb ␤ 3 . The ligand mimetic property of both mAbs is linked to the tripeptide sequence RYD within the third complementarity-determining region that appears to mimic the RGD recognition sequence (4,5). The binding of both antibodies to ␣ IIb ␤ 3 is blocked by adhesive protein and small competitive peptide ligands (39,40). Neither antibody binds to ligand binding defective mutants of ␣ IIb ␤ 3 (10). However, these two antibodies differ in that the binding of PAC1 is activation-dependent while the binding of OPG2 does not require prior receptor activation. Finally, ligand binding to these receptors can be assessed indirectly by the conformational changes reported by the exposure of LIBS epitopes (41). Utilizing this integrin pair, we have defined the region of the ␣ subunit that regulates recognition specificity for both activation-dependent and -independent ligands. We report here that neither the cation binding repeats or the NH 2 terminus alone is sufficient to control the ligand recognition specificity of this integrin pair. Ligand specificity requires both regions. A minimal sequence encompassing the amino-terminal one third of the ␣ subunit was required to transfer ligand recognition specificity.
Generation of ␣ v /␣ IIb Chimeric Subunits-Chimeric ␣ subunits, which consisted of the backbone of ␣ v from which various portions were removed and replaced with the homologous regions of ␣ IIb , were constructed utilizing standard techniques. cDNA clones encoding wild type ␣ IIb and ␣ v have been previously described (9,45). Oligonucleotidedirected mutagenesis (46) was used to introduce three unique, silent restriction enzyme sites into the cDNA coding for the ␣ v subunit, resulting in the construct designated ␣ v MNS. Nucleotide sequence numbering for ␣ v was according to the published sequence (47). The changes were as follows: bp 759 -764, ACTCGG was changed to ACgCGt to introduce an MluI site; bp 1098 -1103, GCTTCA was changed to GCTagc to introduce a NheI site; and bp 1469 -1474, TGGTCT was changed to aGGcCT to introduce a StuI site. Each ␣ v /␣ IIb chimera is named based on the following convention "␣ v 2b(X)" where 2b(X) designates the portion of ␣ IIb that was substituted into the ␣ v backbone. To generate the chimera ␣ v 2b(1-4C), the MluI/StuI fragment of ␣ v MNS was replaced with a MluI/StuI fragment of ␣ IIb that contained cation binding repeats 1 through 4 of ␣ IIb . This fragment was generated by PCR amplification utilizing the wild type ␣ IIb cDNA as template and oligo primers that contained the corresponding restriction sites at their 5Ј ends. The chimera ␣ v 2b(1ϩ2C), which contained cation binding repeats 1 and 2 of ␣ IIb , was constructed by digesting ␣ v MNS with MluI and NheI and ligating the corresponding MluI/NheI ␣ IIb fragment generated by PCR. The chimera ␣ v 2b(2ϩ3C), which contained cation binding repeats 2 and 3 of ␣ IIb , was constructed by digesting ␣ v MNS with AflIII (bp 911) and SphI (bp 1328) and ligating the corresponding AflIII/SphI ␣ IIb PCR fragment. The chimera ␣ v 2b(3ϩ4C), which contained cation binding repeats 3 and 4 of ␣ IIb , was constructed by digesting ␣ v MNS with NheI and StuI and ligating the corresponding NheI/ StuI ␣ IIb PCR fragment. ␣ v 2b(L1-Q459) was constructed by digesting ␣ v 2b(1-4C) with HindIII and MluI and ligating the corresponding HindIII/MluI ␣ IIb PCR fragment resulting in the intermediate clone designated ␣ v 2bNH 2 Ј. The MluI site in ␣ v 2bNH 2 Ј was removed by replacing a 1.4-kilobase pair ClaI fragment contained within the ␣ IIb sequence with the same 1.4-kilobase pair ClaI fragment isolated from the wild type ␣ IIb cDNA clone BS2b (9), giving ␣ v 2b(L1-Q459). ␣ v 2b(L1-F223) was constructed by replacing the MluI/StuI fragment of KpnI/NotI fragment of ␣ v containing the homologous region of ␣ v . This fragment was generated by PCR using the wild type ␣ v cDNA as template. The authenticity of each construct was confirmed by DNA sequencing of all junctions to verify that the correct reading frame was intact. All PCR-generated fragments were sequenced in their entirety to verify the absence of any other substitutions. A fragment containing the complete coding sequence of each chimera was subcloned into the expression vector CDNeo (15).
Cell Transfection and Flow Cytometry-Stably transfected CHO cell lines were established by electroporation with a chimeric ␣ v /␣ IIb ␣ subunit construct together with the wild type ␤ 3 construct CD3a (45) as described previously (10). Surface expression of recombinant integrins was analyzed by flow cytometry with specific antibodies as described previously (41). Briefly, 5 ϫ 10 5 cells were incubated on ice for 30 min with primary antibody, washed, and then incubated on ice for 30 min with fluorescein-conjugated goat anti-mouse second antibody (Tago, Burlingame, CA). Cells were pelleted, resuspended and analyzed on a FACScan (Becton Dickinson, Mountain View, CA). Stably transfected cell lines expressing wild type ␣ IIb ␤ 3 or ␣ v ␤ 3 have been described previously (9).
Binding Assay-[ 3 H]SC52012 (63.4 Ci/mmol) was used to assess the ligand binding phenotype of the stably transfected cell lines. Cells were harvested from tissue culture flasks with 1.2 mM EDTA/phosphatebuffered saline at room temperature. Cells were resuspended in binding buffer (Hank's balanced salt solution containing 50 mM Hepes, pH 7.4, 1 mM Ca 2ϩ , and 1 mg/ml bovine serum albumin). Cells were washed three times in binding buffer and suspended at a final concentration of 1 ϫ 10 7 cells/ml and incubated with the indicated concentration of [ 3 H]SC52012. Binding was performed for 40 min at room temperature. Bound [ 3 H]SC52012 was separated from free compound by centrifuging the cells through a 200-l cushion of 20% sucrose. The cell pellet was recovered, resuspended in 200 l of 2 N NaOH, and then added to 3 ml of scintillation fluid. The amount of [ 3 H]SC52012 associated with the cell pellet was determined by scintillation spectrometry. Background binding of [ 3 H]SC52012 to the cells was measured in the presence of 5 mM EDTA. In binding studies between [ 3 H]SC52012 (500 nM) and cells expressing wild type ␣ IIb ␤ 3 , 10,000 -20,000 cpm were routinely bound with a standard error of less than 12%.
Affinity Chromatography and Immunoprecipitation-Stably transfected cells were surface-labeled by the lactoperoxidase-glucose oxidase method and solubilized, and detergent lysates of labeled cells were applied to a KYGRGDSP-Sepharose column (1 ml bed volume) as described (48). The lysates were incubated with the resin overnight at 4°C then the column was washed with five volumes of lysis buffer. The column was eluted with 3 ml of 1.5 mM fibrinogen fragment K16, washed with 3 ml of lysis buffer, and then eluted with 3 ml of 5 mM EDTA. Aliquots of the eluted fractions were precleared by incubating with protein G-Sepharose (Pharmacia Biotech Inc.) and then immunoprecipitated with the monoclonal anti-␣ v mAb LM142 as described previously (49). Immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis (non-reducing 7% acrylamide gels). Gels were dried and precipitated proteins visualized by autoradiography. Densitometric analysis was performed with the NIH Image software program.
Since several of the substitutions resulted in reactivity with ␣ IIb ␤ 3 -specific mAbs, the binding of the ligand mimetic mAb PAC1 was examined utilizing flow cytometry. The binding of mAb PAC1 is activation-dependent (39). Therefore, while resting ␣ IIb ␤ 3 exhibited low reactivity with mAb PAC1, activation with the mAb anti-LIBS2 significantly increased the binding of mAb PAC1 (Fig. 3). mAb anti-LIBS2 acts directly upon ␣ IIb ␤ 3 , provoking high affinity ligand binding function (30). The binding of mAb PAC1 was specific since it was completely blocked by GRGDSP peptide. Similarly, cells expressing the chimeras ␣ v 2b(L1-Q459) or ␣ v 2b(L1-P334) specifically bound mAb PAC1 in the presence of activating mAb anti-LIBS2. In contrast, cells expressing wild type ␣ v ␤ 3 , ␣ v 2b(L1-F223)⅐␤ 3 , or ␣ v 2b(1-4C)⅐␤ 3 failed to bind mAb PAC1 after activation with the mAb anti-LIBS2. The lack of mAb PAC1 binding to these chimeras was not due to the failure of anti-LIBS2 to bind to the chimeric receptor as the epitope was present on each of these receptors as assayed by flow cytometry (data not shown). These data suggest that the chimeras ␣ v 2b(L1-Q459) and ␣ v 2b(L1-P334) have a ligand binding pocket very similar to ␣ IIb ␤ 3 .
Interaction of these ␣ v /␣ IIb chimeras with another ligand mimetic mAb, OPG2, was also examined by flow cytometry (Fig. 4). OPG2 inhibits the binding of adhesive proteins to ␣ IIb ␤ 3 and its binding is blocked by RGD peptides (40). However, unlike mAb PAC1, the binding of mAb OPG2 to ␣ IIb ␤ 3 is activation-independent. Cells expressing wild type ␣ IIb ␤ 3 stained brightly with mAb OPG2. Consistent with the results CHO cells co-transfected with wild type ␤ 3 and the indicated ␣ subunit and were examined for receptor expression by flow cytometry. Cells transfected with wild type ␣ v or the indicated ␣ v /␣ IIb chimeric ␣ subunit were stained by indirect immunofluorescence with the anti-␣ v mAb LM142. Cells transfected with ␣ IIb were stained with the ␣ IIb ␤ 3 -specific mAb D57. Results are depicted as histograms with the log of the fluorescence intensity on the abscissa and the cell number on the ordinate.

TABLE I
Summary of monoclonal antibody reactivity with recombinant ␤ 3 integrins CHO cells stably transfected with the indicated wild type receptors or chimeric ␣ v /␣ IIb receptors were analyzed for mAb reactivity by flow cytometry as described under "Materials and Methods." ϩ, positive staining; Ϫ, staining identical to that obtained with the negative control (normal mouse Ig); Ϯ, weak staining consistently above the negative control. ND, not determined.  pear to be divalent cation binding sites. Since a peptide derived from the carboxyl terminus of the Fgn ␥ chain binds preferentially to ␣ IIb ␤ 3 rather than ␣ v ␤ 3 (30,50) and cross-links to an ␣ IIb fragment that spans the second divalent cation binding site in ␣ IIb (23), we investigated the contribution of the divalent cation binding repeats to ligand recognition specificity. In these chimeras, ␣ IIb domains consisting of all four cation binding repeats together (2b1-4C) or consisting of two adjacent cation binding repeats (2b1ϩ2C, 2b2ϩ3C, and 2b3ϩ4C) were substituted for the corresponding domains in ␣ v (Fig. 1). In addition to the cation binding motifs themselves, these substitutions included flanking sequences. All of these chimeras were expressed on the cell surface as assessed by flow cytometry (Fig.  2). While the chimeras ␣ v 2b(1-4C), ␣ v 2b(1ϩ2C), ␣ v 2b(2ϩ3C), and ␣ v 2b(3ϩ4C) all exhibited strong staining with the anti-␣ v mAb LM142, none of these chimeras reacted with the ␣ IIb ␤ 3 complex-specific mAbs 10E5, 4F10, 2G12, or D57 (Table I). An exception was the complex-specific mAb AP2, which exhibited very weak but reproducible reactivity with the chimeras ␣ v 2b(1-4C), ␣ v 2b(1ϩ2C), and ␣ v 2b(2ϩ3C). None of these chimeras bound the activation-dependent ligand mimetic mAb PAC1. This was not due to a defect in activation as none of these chimeras bound the activation-independent ligand mimetic mAb OPG2 (Table I).
To determine the capacity of the chimeras containing substitutions of the divalent cation repeats to bind small activation-independent ligands specific for ␣ IIb ␤ 3 , we examined the capacity of the ␣ IIb ␤ 3 -selective peptidomimetic 51) to increase the binding of mAb anti-LIBS1 by flow cytometry. Since the mAb anti-LIBS1 binds preferentially to the occupied conformation of the receptor (41), increased binding of mAb LIBS1 is evidence of receptor-ligand interaction. In the presence of Ro 43-5054, there was an increase in the binding of anti-LIBS1 to cells expressing ␣ IIb ␤ 3 but not to cells expressing ␣ v ␤ 3 (Fig. 5). Similarly, Ro 43-5054 failed to stimulate the binding of mAb anti-LIBS1 to cells expressing the chimeras ␣ v 2b(2ϩ3C) or ␣ v 2b(3ϩ4C), indicating lack of binding to the receptor. Unexpectedly, mAb anti-LIBS1 bound maximally to cells expressing the chimeras ␣ v 2b(1ϩ2C) or ␣ v 2b(1-4C) even in the absence of ligand. This result suggested that these two chimeras possessed a structure that is slightly altered from that of the wild type receptors. Although the anti-LIBS1 epitope was exposed on these two chimeras, additional data (see below) indicate that their ability to bind ligand was not impaired.
To test whether the chimeric receptors containing substitutions of the cation binding repeats possessed an intact RGD ligand recognition function and to test their capacity to distinguish between the RGD and fibrinogen ␥ chain sequence, the ligand binding function of the recombinant receptors was analyzed by affinity chromatography (Fig. 6). Detergent lysates of radiolabeled, transfected cells were applied to an RGD affinity column and eluted with the fibrinogen ␥ chain peptide K16, followed by elution with EDTA. The eluted fractions were then immunoprecipitated with an anti-␣ v mAb. Eluted fractions of the control ␣ IIb ␤ 3 -expressing cells were immunoprecipitated with anti-␣ IIb antiserum (30). Precipitated proteins were then resolved by SDS-polyacrylamide gel electrophoresis. Consistent with previous reports (48), wild type ␣ v ␤ 3 was poorly eluted by the K16 peptide (data not shown). While 64% of the bound wild type ␣ IIb ␤ 3 was eluted from the affinity matrix by the ␥ chain peptide K16, the chimeras ␣ v 2b(1-4C) (8.4%), FIG. 6. Fibrinogen ␥ chain peptide K16 does not displace ␣ v ␤ 3 or the ␣ v /␣ IIb divalent cation binding repeat chimeras from a RGD affinity matrix. CHO cells stably expressing the wild type ␣ IIb ␤ 3 or chimeric ␣ v /␣ IIb receptors were radioiodinated and lysed, and the extract was applied an GRGDSPK-Sepharose 4B column. After incubation and washing, the bound proteins were sequentially eluted with 1.5 mM K16, followed by 5 mM EDTA. The eluted fractions were immunoprecipitated with the anti-␣ v mAb LM142. The immunoprecipitated proteins were resolved by SDS-polyacrylamide gel electrophoresis on 7% nonreducing acrylamide gels and detected by autoradiography. Lanes 1, immunoprecipitate of K16-eluted material; lanes 2, immunoprecipitate of subsequent EDTA-eluted material.
␣ v 2b(1ϩ2C) (3.4%), ␣ v 2b(2ϩ3C) (7.5%), or ␣ v 2b(3ϩ4C) (13%) were poorly eluted by the K16 peptide from the RGD affinity matrix (Fig. 6). Each of these receptors bound to the RGD matrix and was readily eluted from the matrix by EDTA. Both wild type ␣ IIb ␤ 3 and ␣ v ␤ 3 receptors and all the chimeras were readily eluted from the affinity matrix by RGD peptide (data not shown). The fact that the chimeras ␣ v 2b(1-4C) and ␣ v 2b(1ϩ2C) bound to the RGD affinity matrix and were specifically eluted by EDTA or RGD peptide indicates that the alteration in structure reported by anti-LIBS1 did not affect the ligand binding function of these receptors. These data show that all chimeras containing substitutions of the cation binding motifs can recognize the RGD sequence, but that substitution of the ␣ v divalent cation binding regions with the corresponding regions from ␣ IIb was not sufficient to change the ligand binding specificity of ␣ v ␤ 3 to that of ␣ IIb ␤ 3 .
Direct Binding of an ␣ IIb ␤ 3 -selective Peptidomimetic to Chimeric Integrins-To directly test the ability of the chimeras to bind small activation-independent ligands and further verify that the chimeric receptors ␣ v 2b(L1-Q459)⅐␤ 3 and ␣ v 2b(L1-P334)⅐␤ 3 had acquired the capacity to bind ␣ IIb ␤ 3 -specific ligands, we examined the binding of the peptidomimetic SC52012 to cells expressing chimeric receptors. SC52012 is a high affinity RGD mimetic that inhibits ADP-induced platelet aggregation with an IC 50 of 42 nM (37). SC52012 is also highly selective for ␣ IIb ␤ 3 versus ␣ v ␤ 3 (Fig. 7). All of the cell lines were assayed by flow cytometry prior to the binding assay to confirm that all cell lines expressed similar numbers of receptors. Direct binding assays with [ 3 H]SC52012 demonstrated specific binding to cells expressing ␣ IIb ␤ 3 and the chimeras ␣ v 2b(L1-Q459)⅐␤ 3 and ␣ v 2b(L1-P334)⅐␤ 3 . The number of molecules SC52012 bound to cells expressing ␣ IIb ␤ 3 was within the number of receptors (138,000 -440,000 sites/cell) previously determined for this cell line (30). No specific binding of [ 3 H]SC52012 was observed to cells expressing ␣ v ␤ 3 or to any of the other chimeras. This result confirms that the chimeras ␣ v 2b(L1-Q459)⅐␤ 3 and ␣ v 2b(L1-P334)⅐␤ 3 exhibit a ligand binding specificity identical to ␣ IIb ␤ 3 .

DISCUSSION
The major findings of the present study are as follows. 1) Ligand recognition specificity of ␤ 3 integrins is regulated by the amino-terminal one-third of the ␣ subunit. Substitution of the amino-terminal portion of ␣ v with the corresponding 334 amino acid residues of ␣ IIb switched the ligand recognition specificity of ␣ v ␤ 3 to that of ␣ IIb ␤ 3 . This change in ligand specificity was observed with an activation-dependent ligand mimetic antibody, an activation-independent ligand mimetic antibody, and small activation-independent ligands. 2) Neither the aminoterminal region or the cation binding repeats alone is sufficient to control ligand specificity. Chimeras that omit the aminoterminal 140 residues or first two divalent cation binding repeats of ␣ IIb fail to change ligand specificity. Thus, the ligand binding pocket of ␣ IIb ␤ 3 is a structure that contains elements of both the ␣ and ␤ subunits.
Previous studies have suggested that the regions that control ligand binding to ␣ IIb ␤ 3 reside in the amino-terminal portion of ␣ IIb ␤ 3 , but the minimal structures identified in these studies encompassed more than one half of constituent subunits (28,29). In the present study, we have mapped the regions that regulate ligand specificity to a smaller region of ␣ IIb . The chimera designated ␣ v 2b(L1-Q459) contained the amino-terminal portion and all four divalent cation repeats of ␣ IIb and reacted with several ␣ IIb ␤ 3 complex-specific mAbs. In addition, this chimera specifically bound small activation-independent ␣ IIb ␤ 3 -specific peptidomimetics and both activation-dependent (PAC1) and activation-independent (OPG2) ligand mimetic mAbs. The chimera ␣ v 2b(L1-P334) retains the amino-terminal portion of ␣ IIb but contains only the first two divalent cation repeats of ␣ IIb . This chimera also exhibited a ligand binding phenotype consistent with that of ␣ IIb ␤ 3 in that it bound specific peptidomimetics, the ligand mimetic mAbs PAC1 and OPG2, and several ␣ IIb ␤ 3 -specific mAbs. These results indicate that the ligand specificity of ␣ IIb ␤ 3 can be reconstituted with the first 334 amino acid residues of ␣ IIb and does not require the third or fourth divalent cation repeats of ␣ IIb .
Chimeras that omit the 140 amino-terminal residues or the first two divalent cation motifs of ␣ IIb fail to change the ligand specificity of ␣ v ␤ 3 to that of ␣ IIb ␤ 3 . The chimera ␣ v 2b(1-4C) contains a substitution of the entire divalent cation repeat region of ␣ v with the corresponding region of ␣ IIb . This chimera was expressed on the cell surface and could bind ligand as demonstrated by its ability to bind to an RGD affinity matrix. However, this chimera was poorly displaced from the matrix by a fibrinogen ␥ chain peptide and did not bind the ligand mimetic mAbs PAC1 and OPG2 or an ␣ IIb ␤ 3 -specific peptidomimetic. These data indicate that substitution of the divalent cation repeats alone is not sufficient to change the ligand binding specificity. Similarly, the chimera ␣ v 2b(R140-P334) did FIG. 7. Direct binding of an ␣ IIb ␤ 3selective peptidomimetic. The binding of the ␣ IIb ␤ 3 -specific peptidomimetic SC52012 (37) to stably transfected cell lines expressing ␣ IIb ␤ 3 , ␣ v ␤ 3 , or the indicated chimeric ␣ v /␣ IIb ⅐␤ 3 receptor was determined by incubating transfected cells with [ 3 H]SC52012 (500 nM) at room temperature. After 40 min, bound ligand was separated from free ligand by centrifugation through 20% sucrose. The pellet associated counts were determined by liquid scintillation spectrometry. Background binding was measured in the presence of 5 mM EDTA. Shown are representative results of three separate assays. Results shown are mean Ϯ S.D. of triplicates. not bind the ligand mimetic mAbs PAC1 and OPG2 or the ␣ IIb ␤ 3 -specific mimetic peptidomimetic. This chimera contains the first two divalent cation repeats of ␣ IIb but is missing the first 140 amino-terminal residues of mature ␣ IIb . This result suggests a requirement for residues near the amino terminus and indicates that an extended portion of the receptor is required for ligand specificity.
The approach of homolog-scanning mutagenesis (52,53) is of general use for the identification of functional domains. A recent report used this technique to localize the putative ligand binding domains of ␣ 4 by mapping the epitopes for function blocking antibodies to the amino-terminal portion, but not the divalent cation repeats of ␣ 4 (19). In the present study, we demonstrated that the mAb AP2, which blocks ligand function (42), binds strongly to the chimera ␣ v 2b(L1-F223). However, a ligand binding domain cannot be ascribed to this region since this chimera did not bind the ligand mimetic mAbs PAC1 and OPG2 and did not bind an ␣ IIb ␤ 3 -specific peptidomimetic. Thus, our results based on the interaction of true ligand mimetics demonstrates the inherent limitations of relying solely on the localization of the epitopes of function blocking mAbs to map ligand binding sites.
Previous studies have clearly demonstrated a role for the ␤ subunit in ligand binding to ␣ IIb ␤ 3 . Single amino acid substitutions in a highly conserved region of ␤ 3 completely block the ligand binding function of ␣ IIb ␤ 3 (9,10). This loss of ligand binding is not due to an effect on the activation state of the receptor as the mutations also block the activation-independent binding of mAb OPG2 and small ligand mimetics. However, our present results demonstrate that the specificity for the binding of PAC1, OPG2, and specific peptidomimetics to ␣ IIb ␤ 3 is controlled by the first 334 amino acid residues of the ␣ subunit. Together, these results indicate that ligand recognition requires cooperation between elements in both the ␣ and ␤ subunits and indicates that the ligand binding pocket is a topographical structure that is assembled from regions of both the ␣ and ␤ subunits.