©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
The Amino-terminal One-third of Defines the Ligand Recognition Specificity of Integrin (*)

(Received for publication, July 7, 1995; and in revised form, October 11, 1995)

Joseph C. Loftus (§) Carol E. Halloran Mark H. Ginsberg Larry P. Feigen (1) Jeffery A. Zablocki (1) Jeffrey W. Smith (2)

From the  (1)Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, Searle Research and Development, Skokie, Illinois 60077, and the (2)La Jolla Cancer Research Foundation, La Jolla, California 92037

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 chimeras to identify regions of alpha that when substituted for the homologous regions of alpha(v) switched the ligand binding phenotype of alpha(v)beta(3) to that of alphabeta(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 reconstituted reactivity with both alphabeta(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.


INTRODUCTION

Integrins are heterodimeric adhesion receptors composed of noncovalently associated alpha and beta subunits. The integrin superfamily consists of at least 20 members that are composed of different combinations of nine beta and more than 15 alpha subunits. The different combinations of alpha and beta 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 beta subunits. Chemical cross-linking, site-directed mutagenesis, and immunological approaches have implicated a highly conserved sequence in the beta subunit in the ligand binding function(6, 7, 8, 9, 10, 11, 12, 13, 14) . A second site in the same region has also been reported to be involved in ligand binding (15, 16) . Six of the integrin alpha subunits contain an additional 200-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 beta 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 beta 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 alpha 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 alpha and alpha(v)(23, 24) . Synthetic peptides (25) as well as a recombinant fragment (26) from this region of alpha have been reported to bind ligand. A homology scanning approach mapped the epitopes of antibodies that block ligand binding to alpha(4) to the NH(2) terminus, but not to the cation binding motifs (27) . Finally, the minimal ligand binding fragments of alphabeta(3) lack the COOH-terminal portions of the receptor, but contain more than half of the entire alpha subunit(28, 29) . Thus, the structures critical for ligand recognition by integrin alpha subunits that lack an I domain remain to be elucidated.

A major difficulty in determining the role of integrin alpha 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 activation-independent(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 alpha subunits that regulate ligand recognition specificity, we exploited the unique tools available for the integrins alphabeta(3) and alpha(v)beta(3). These two integrins share the common beta(3) subunit, and the two alpha 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, 37, 38) . Moreover, true ligand mimetic monoclonal antibodies, PAC1 (39) and OPG2(40) , have been prepared against alphabeta(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 alphabeta(3) is blocked by adhesive protein and small competitive peptide ligands(39, 40) . Neither antibody binds to ligand binding defective mutants of alphabeta(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 alpha 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 alpha subunit was required to transfer ligand recognition specificity.


MATERIALS AND METHODS

Monoclonal Antibodies and Reagents

Murine monoclonal anti-alphabeta(3)-specific antibodies (mAb) 4F10 and 2G12 were kindly provided by Dr. Virgil Woods (University of California, San Diego, CA). The alphabeta(3) complex-specific mAbs AP2 (42) and OPG2 (40) were provided by Dr. Thomas Kunicki (The Scripps Research Institute, La Jolla, CA). The alphabeta(3) complex-specific mAb 10E5 was provided by Dr. Barry Coller (State University of New York, Stonybrook, NY). The alphabeta(3)-specific mAb D57, the anti-beta(3) mAb 15, the mAb anti-LIBS1, and the anti-beta(3) mAb anti-LIBS2 (IgG) have been previously described(30, 41, 43) . mAb PAC1 (IgM) binds specifically to activated alphabeta(3)(39) and was provided by Dr. Sanford Shattil (The Scripps Research Institute). The alpha(v)-specific mAb LM142 (44) was obtained from Chemicon (Temecula, CA). The peptidomimetic Ro 43-5054 (38) was generously provided by Beat Steiner (Hoffman-LaRoche, Basel, Switzerland). The peptidomimetic SC52012 (37) was provided by Searle (Searle Research and Development, Skokie, IL). The synthetic peptides GRGDSP and KYGGHHLGGADQADGV (K16) were prepared by solid phase synthesis on an Applied Biosystems peptide synthesizer (Applied Biosystems, Foster City, CA) using phenylacetamidomethyl resins and t-butoxycarbonyl amino acids purchased from Applied Biosystems. Peptide purity was assessed by reverse phase high performance liquid chromatography and amino acid composition verified by fast atom bombardment mass spectrometry.

Generation of alpha(v)/alpha Chimeric Subunits

Chimeric alpha subunits, which consisted of the backbone of alpha(v) from which various portions were removed and replaced with the homologous regions of alpha, were constructed utilizing standard techniques. cDNA clones encoding wild type alpha and alpha(v) have been previously described(9, 45) . Oligonucleotide-directed mutagenesis (46) was used to introduce three unique, silent restriction enzyme sites into the cDNA coding for the alpha(v) subunit, resulting in the construct designated alpha(v)MNS. Nucleotide sequence numbering for alpha(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 alpha(v)/alpha chimera is named based on the following convention ``alpha(v)2b(X)'' where 2b(X) designates the portion of alpha that was substituted into the alpha(v) backbone. To generate the chimera alpha(v)2b(1-4C), the MluI/StuI fragment of alpha(v)MNS was replaced with a MluI/StuI fragment of alpha that contained cation binding repeats 1 through 4 of alpha. This fragment was generated by PCR amplification utilizing the wild type alpha cDNA as template and oligo primers that contained the corresponding restriction sites at their 5` ends. The chimera alpha(v)2b(1+2C), which contained cation binding repeats 1 and 2 of alpha, was constructed by digesting alpha(v)MNS with MluI and NheI and ligating the corresponding MluI/NheI alpha fragment generated by PCR. The chimera alpha(v)2b(2+3C), which contained cation binding repeats 2 and 3 of alpha, was constructed by digesting alpha(v)MNS with AflIII (bp 911) and SphI (bp 1328) and ligating the corresponding AflIII/SphI alpha PCR fragment. The chimera alpha(v)2b(3+4C), which contained cation binding repeats 3 and 4 of alpha, was constructed by digesting alpha(v)MNS with NheI and StuI and ligating the corresponding NheI/StuI alpha PCR fragment. alpha(v)2b(L1-Q459) was constructed by digesting alpha(v)2b(1-4C) with HindIII and MluI and ligating the corresponding HindIII/MluI alpha PCR fragment resulting in the intermediate clone designated alpha(v)2bNH(2)`. The MluI site in alpha(v)2bNH(2)` was removed by replacing a 1.4-kilobase pair ClaI fragment contained within the alpha sequence with the same 1.4-kilobase pair ClaI fragment isolated from the wild type alpha cDNA clone BS2b(9) , giving alpha(v)2b(L1-Q459). alpha(v)2b(L1-F223) was constructed by replacing the MluI/StuI fragment of alpha(v)2bNH(2)` with the MluI/StuI fragment of alpha(v)MNS. alpha(v)2b(L1-P334) was constructed by ligating the ClaI/MluI fragment of alpha(v)2bNH(2)` into ClaI/MluI-digested alpha(v)2b(1+2C). alpha(v)2b(R140-F223) was constructed by replacing the amino-terminal KpnI/NotI fragment of alpha(v)2b(L1-P334) with a KpnI/NotI fragment of alpha(v) containing the homologous region of alpha(v). This fragment was generated by PCR using the wild type alpha(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 alpha(v)/alpha alpha subunit construct together with the wild type beta(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 times 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 alphabeta(3) or alpha(v)beta(3) have been described previously(9) .

Binding Assay

[^3H]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/phosphate-buffered 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, and 1 mg/ml bovine serum albumin). Cells were washed three times in binding buffer and suspended at a final concentration of 1 times 10^7 cells/ml and incubated with the indicated concentration of [^3H]SC52012. Binding was performed for 40 min at room temperature. Bound [^3H]SC52012 was separated from free compound by centrifuging the cells through a 200-µl cushion of 20% sucrose. The cell pellet was recovered, resuspended in 200 µl of 2 N NaOH, and then added to 3 ml of scintillation fluid. The amount of [^3H]SC52012 associated with the cell pellet was determined by scintillation spectrometry. Background binding of [^3H]SC52012 to the cells was measured in the presence of 5 mM EDTA. In binding studies between [^3H]SC52012 (500 nM) and cells expressing wild type alphabeta(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-alpha(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.


RESULTS

The Divalent Cation Repeats and the NH(2)-terminal Region of alpha Are Required for an alphabeta(3) Ligand Binding Specificity

To begin to investigate the regions of the alphabeta(3) subunit that regulate ligand recognition specificity of alphabeta(3), alpha(v)/alpha chimeric alpha subunits were generated (Fig. 1). The chimeras designated alpha(v)2b(L1-Q459), alpha(v)2b(L1-P334), and alpha(v)2b(R140-P334) contain portions of both the amino terminus and divalent cation binding motifs of alpha, while the chimera alpha(v)2b(L1-F223) contains only the amino-terminal region of alpha. All of these chimeras were expressed on the cell surface as assayed by flow cytometry with the anti-alpha(v) mAb LM142 (Fig. 2). Moreover, the chimeras alpha(v)2b(L1-Q459), alpha(v)2b(L1-P334), and alpha(v)2b(L1-F223) expressed the alphabeta(3) complex-specific epitopes recognized by the mAbs AP2 and D57 (Table 1). In contrast, the alpha(v)2b(R140-P334) chimera reacted only very weakly with both AP2 and D57 mAbs.


Figure 1: Schematic representation of the alpha(v)/alpha chimeric alpha subunits. Each chimera consists of the backbone of alpha(v) (solid line) from which the indicated portions have been removed and replaced with the homologous region of alpha (shaded boxes). The location of the three silent restriction sites introduced into the wild type alpha(v) sequence to facilitate exchanges and the endogenous SphI site are indicated. Solid black rectangles indicate the position of the four divalent cation binding repeats present in each alpha subunit. The position of alpha residues that delineate chimeras are indicated.




Figure 2: Flow cytometric analysis of stably transfected cell lines expressing alpha(v)/alpha chimeric alpha subunits. CHO cells co-transfected with wild type beta(3) and the indicated alpha subunit and were examined for receptor expression by flow cytometry. Cells transfected with wild type alpha(v) or the indicated alpha(v)/alpha chimeric alpha subunit were stained by indirect immunofluorescence with the anti-alpha(v) mAb LM142. Cells transfected with alpha were stained with the alphabeta(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.





Since several of the substitutions resulted in reactivity with alphabeta(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 alphabeta(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 alphabeta(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 alpha(v)2b(L1-Q459) or alpha(v)2b(L1-P334) specifically bound mAb PAC1 in the presence of activating mAb anti-LIBS2. In contrast, cells expressing wild type alpha(v)beta(3), alpha(v)2b(L1-F223)bulletbeta(3), or alpha(v)2b(1-4C)bulletbeta(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 alpha(v)2b(L1-Q459) and alpha(v)2b(L1-P334) have a ligand binding pocket very similar to alphabeta(3).


Figure 3: Binding of mAb PAC1 to cells stably transfected with alphabeta(3), alpha(v)beta(3), or chimeric alpha(v)/alphabeta(3) receptors. The binding of the alphabeta(3) activation-specific mAb PAC1 to CHO cells stably transfected with beta(3) and the indicated alpha subunit was examined by flow cytometry. Results are depicted as histograms of cell number versus fluorescence intensity. Transfected cells were incubated (activated) in the presence of 8 µM purified IgG anti-LIBS2 for 30 min followed by the addition of mAb PAC1 (IgM). Cells were washed, stained with fluorescein-conjugated goat anti-mouse IgM for 30 min, and analyzed. The binding of mAb PAC1 was analyzed in the presence (open histogram) or absence (solid histogram) of 1 mM GRGDSP peptide.



Interaction of these alpha(v)/alpha chimeras with another ligand mimetic mAb, OPG2, was also examined by flow cytometry (Fig. 4). OPG2 inhibits the binding of adhesive proteins to alphabeta(3) and its binding is blocked by RGD peptides(40) . However, unlike mAb PAC1, the binding of mAb OPG2 to alphabeta(3) is activation-independent. Cells expressing wild type alphabeta(3) stained brightly with mAb OPG2. Consistent with the results obtained with mAb PAC1, mAb OPG2 bound to cells expressing alpha(v)2b(L1-Q459)bulletbeta(3) or alpha(v)2b(L1-P334)bulletbeta(3). No specific mAb OPG2 staining was observed with cells expressing wild type alpha(v)beta(3), alpha(v)2b(L1-F223)bulletbeta(3), or alpha(v)2b(1-4C)bulletbeta(3). The fact that neither mAb bound to the chimera alpha(v)2b(L1-F223) indicates that the NH(2)-terminal region alone does not control ligand binding specificity.


Figure 4: Expression of the OPG2 epitope on recombinant wild type alphabeta(3), alpha(v)beta(3), or chimeric alpha(v)/alphabeta(3) receptors. The binding of the alphabeta(3) complex-specific mAb OPG2 to CHO cells stably transfected with beta(3) and the indicated alpha subunit was examined by flow cytometry. Results are depicted as fluorescence-activated cell sorting histograms. In each panel, the binding of mAb OPG2 (solid histogram) is superimposed on the binding of the anti-alpha(v) mAb 142 (open histogram).



The Divalent Cation Binding Repeats Alone Do Not Determine Ligand Binding Phenotype

Integrin alpha subunits contain seven homologous repeats, of which the last three or four appear to be divalent cation binding sites. Since a peptide derived from the carboxyl terminus of the Fgn chain binds preferentially to alphabeta(3) rather than alpha(v)beta(3)(30, 50) and cross-links to an alpha fragment that spans the second divalent cation binding site in alpha(23) , we investigated the contribution of the divalent cation binding repeats to ligand recognition specificity. In these chimeras, alpha domains consisting of all four cation binding repeats together (2b1-4C) or consisting of two adjacent cation binding repeats (2b1+2C, 2b2+3C, and 2b3+4C) were substituted for the corresponding domains in alpha(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 alpha(v)2b(1-4C), alpha(v)2b(1+2C), alpha(v)2b(2+3C), and alpha(v)2b(3+4C) all exhibited strong staining with the anti-alpha(v) mAb LM142, none of these chimeras reacted with the alphabeta(3) complex-specific mAbs 10E5, 4F10, 2G12, or D57 (Table 1). An exception was the complex-specific mAb AP2, which exhibited very weak but reproducible reactivity with the chimeras alpha(v)2b(1-4C), alpha(v)2b(1+2C), and alpha(v)2b(2+3C). 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 1).

To determine the capacity of the chimeras containing substitutions of the divalent cation repeats to bind small activation-independent ligands specific for alphabeta(3), we examined the capacity of the alphabeta(3)-selective peptidomimetic Ro 43-5054 (38, 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 alphabeta(3) but not to cells expressing alpha(v)beta(3) (Fig. 5). Similarly, Ro 43-5054 failed to stimulate the binding of mAb anti-LIBS1 to cells expressing the chimeras alpha(v)2b(2+3C) or alpha(v)2b(3+4C), indicating lack of binding to the receptor. Unexpectedly, mAb anti-LIBS1 bound maximally to cells expressing the chimeras alpha(v)2b(1+2C) or alpha(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.


Figure 5: Flow cytometric analysis of the capacity of transfected cells to bind an alphabeta(3)-specific peptidomimetic. The binding of the alphabeta(3)-selective peptidomimetic Ro 43-5054 (38) to cells stably transfected with beta(3) and the indicated alpha subunits was examined with the mAb anti-LIBS1. For LIBS1 binding analysis, cells were incubated with or without Ro 43-5054 (5 µM) and LIBS1 mAb (0.1 µM) for 30 min on ice. Cells were washed and incubated with fluorescein-conjugated goat anti-mouse Ig. Results are expressed as histograms of cell number versus fluorescence intensity. In each panel, the binding of LIBS1 in the presence of Ro 43-5054 (solid histogram) is overlaid on the binding of LIBS1 in the absence of Ro 43-5054 (open histogram).



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-alpha(v) mAb. Eluted fractions of the control alphabeta(3)-expressing cells were immunoprecipitated with anti-alpha antiserum(30) . Precipitated proteins were then resolved by SDS-polyacrylamide gel electrophoresis. Consistent with previous reports(48) , wild type alpha(v)beta(3) was poorly eluted by the K16 peptide (data not shown). While 64% of the bound wild type alphabeta(3) was eluted from the affinity matrix by the chain peptide K16, the chimeras alpha(v)2b(1-4C) (8.4%), alpha(v)2b(1+2C) (3.4%), alpha(v)2b(2+3C) (7.5%), or alpha(v)2b(3+4C) (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 alphabeta(3) and alpha(v)beta(3) receptors and all the chimeras were readily eluted from the affinity matrix by RGD peptide (data not shown). The fact that the chimeras alpha(v)2b(1-4C) and alpha(v)2b(1+2C) 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 alpha(v) divalent cation binding regions with the corresponding regions from alpha was not sufficient to change the ligand binding specificity of alpha(v)beta(3) to that of alphabeta(3).


Figure 6: Fibrinogen chain peptide K16 does not displace alpha(v)beta(3) or the alpha(v)/alpha divalent cation binding repeat chimeras from a RGD affinity matrix. CHO cells stably expressing the wild type alphabeta(3) or chimeric alpha(v)/alpha 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-alpha(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.



Direct Binding of an alphabeta(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 alpha(v)2b(L1-Q459)bulletbeta(3) and alpha(v)2b(L1-P334)bulletbeta(3) had acquired the capacity to bind alphabeta(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 of 42 nM(37) . SC52012 is also highly selective for alphabeta(3)versus alpha(v)beta(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 [^3H]SC52012 demonstrated specific binding to cells expressing alphabeta(3) and the chimeras alpha(v)2b(L1-Q459)bulletbeta(3) and alpha(v)2b(L1-P334)bulletbeta(3). The number of molecules SC52012 bound to cells expressing alphabeta(3) was within the number of receptors (138,000-440,000 sites/cell) previously determined for this cell line(30) . No specific binding of [^3H]SC52012 was observed to cells expressing alpha(v)beta(3) or to any of the other chimeras. This result confirms that the chimeras alpha(v)2b(L1-Q459)bulletbeta(3) and alpha(v)2b(L1-P334)bulletbeta(3) exhibit a ligand binding specificity identical to alphabeta(3).


Figure 7: Direct binding of an alphabeta(3)-selective peptidomimetic. The binding of the alphabeta(3)-specific peptidomimetic SC52012 (37) to stably transfected cell lines expressing alphabeta(3), alpha(v)beta(3), or the indicated chimeric alpha(v)/alphabulletbeta(3) receptor was determined by incubating transfected cells with [^3H]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 ± S.D. of triplicates.




DISCUSSION

The major findings of the present study are as follows. 1) 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 amino acid residues of alpha switched the ligand recognition specificity of alpha(v)beta(3) to that of alphabeta(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 amino-terminal region or the cation binding repeats alone is sufficient to control ligand specificity. Chimeras that omit the amino-terminal 140 residues or first two divalent cation binding repeats of alpha fail to change ligand specificity. Thus, the ligand binding pocket of alphabeta(3) is a structure that contains elements of both the alpha and beta subunits.

Previous studies have suggested that the regions that control ligand binding to alphabeta(3) reside in the amino-terminal portion of alphabeta(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 alpha. The chimera designated alpha(v)2b(L1-Q459) contained the amino-terminal portion and all four divalent cation repeats of alpha and reacted with several alphabeta(3) complex-specific mAbs. In addition, this chimera specifically bound small activation-independent alphabeta(3)-specific peptidomimetics and both activation-dependent (PAC1) and activation-independent (OPG2) ligand mimetic mAbs. The chimera alpha(v)2b(L1-P334) retains the amino-terminal portion of alpha but contains only the first two divalent cation repeats of alpha. This chimera also exhibited a ligand binding phenotype consistent with that of alphabeta(3) in that it bound specific peptidomimetics, the ligand mimetic mAbs PAC1 and OPG2, and several alphabeta(3)-specific mAbs. These results indicate that the ligand specificity of alphabeta(3) can be reconstituted with the first 334 amino acid residues of alpha and does not require the third or fourth divalent cation repeats of alpha.

Chimeras that omit the 140 amino-terminal residues or the first two divalent cation motifs of alpha fail to change the ligand specificity of alpha(v)beta(3) to that of alphabeta(3). The chimera alpha(v)2b(1-4C) contains a substitution of the entire divalent cation repeat region of alpha(v) with the corresponding region of alpha. 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 alphabeta(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 alpha(v)2b(R140-P334) did not bind the ligand mimetic mAbs PAC1 and OPG2 or the alphabeta(3)-specific mimetic peptidomimetic. This chimera contains the first two divalent cation repeats of alpha but is missing the first 140 amino-terminal residues of mature alpha. 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 alpha(4) by mapping the epitopes for function blocking antibodies to the amino-terminal portion, but not the divalent cation repeats of alpha(4)(19) . In the present study, we demonstrated that the mAb AP2, which blocks ligand function(42) , binds strongly to the chimera alpha(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 alphabeta(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 beta subunit in ligand binding to alphabeta(3). Single amino acid substitutions in a highly conserved region of beta(3) completely block the ligand binding function of alphabeta(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 alphabeta(3) is controlled by the first 334 amino acid residues of the alpha subunit. Together, these results indicate that ligand recognition requires cooperation between elements in both the alpha and beta subunits and indicates that the ligand binding pocket is a topographical structure that is assembled from regions of both the alpha and beta subunits.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL42977 (to J. C. L.) and HL48728 (to J. C. L. and M. H. G.), AR27214 (to M. H. G.), and CA56483 and AR42750 (to J. W. S.). This work was done during the tenure of an Established Investigatorship of the American Heart Association (J. C. L. and J. W. S.). This is publication 9374-VB from The Scripps Research Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 619-784-7120; Fax: 619-784-7343.

(^1)
The abbreviations used are: I, inserted; bp, base pair(s); PCR, polymerase chain reaction; mAb, monoclonal antibody; CHO, Chinese hamster ovary.


ACKNOWLEDGEMENTS

We gratefully acknowledge the superb technical assistance of Kahuku Oades. We thank Charles Markos of Searle Radiochemistry for synthesis of [^3H]SC52012.


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