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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Tozer, E. C. Articles by Loftus, J. C. Search for Related Content PubMed PubMed Citation Articles by Tozer, E. C. Articles by Loftus, J. C. Related Collections Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, Vol. 93 No. 3 (February 1), 1999: pp. 918-924 A Mutation in the Subunit of the Platelet Integrin IIb3 Identifies a Novel Region Important for Ligand Binding By Eileen Collins Tozer, Elizabeth K. Baker, Mark H. Ginsberg, and Joseph C. Loftus From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, CA.     ABSTRACT Top Abstract Introduction References An unbiased genetic approach was used to identify a specific amino acid residue in the IIb subunit important for the ligand binding function of the integrin IIb. Chemically mutagenized cells were selected by flow cytometry based on their inability to bind the ligand mimetic antibody PAC1 and a cell line containing a single amino acid substitution in IIb at position 224 (DV) was identified. Although well expressed on the surface of transfected cells, IIbD224V3 as well as IIbD224A3 did not bind IIb3-specific ligands or a RGD peptide, a ligand shared in common with v3. Insertion of exon 5 of IIb, residues G193-W235, into the backbone of the v subunit did not enable the chimeric receptor to bind IIb3-specific ligands. However, the chimeric receptor was still capable of binding to a RGD affinity matrix. IIbD224 is not well conserved among other integrin subunits and is located in a region of significant variability. In addition, amino acid D224 lies within a predicted loop of the recently proposed -propeller model for integrin subunits and is adjacent to a loop containing amino acid residues previously implicated in receptor function. These data support a role for this region in ligand binding function of the IIb3 receptor. © 1999 by The American Society of Hematology.     INTRODUCTION Top Abstract Introduction References PLATELET ADHESIVE interactions mediated by the integrin IIb3 are fundamental to the maintenance of normal hemostasis, as illustrated by the inherited bleeding disorder Glanzmann's thrombasthenia. Platelets from patients with this disease either lack the IIb3 receptor, express reduced levels of the receptor, or express receptor variants that lack ligand binding function.1 In areas of vessel or tissue trauma, the IIb3 receptor initiates the formation of a platelet-based plug by binding to soluble fibrinogen, which subsequently forms a bridge to other platelets and facilitates platelet aggregation. Understanding the precise molecular mechanisms underlying the structural basis of ligand-receptor interaction is an important step towards the modulation of platelet function and can provide new insights into the development of novel antithrombotic therapies. Furthermore, understanding the molecular basis of this interaction by IIb3, the prototypic integrin could provide insights into other integrin-ligand interactions in a diverse range of physiological processes. Multiple ligand contact sites have been identified on IIb3. Studies of natural receptor variants, cross-linking of ligand mimetic peptides, and site-directed mutagenesis studies have provided convincing evidence for the importance of two discontinuous regions in the 3 subunit, D119-S123 and D217-E220, in the ligand binding function of IIb3.2-8 The importance of this region of 3 appears to be due to its structural similarity to the integrin  subunit I-domain,9 which has been demonstrated to be critically involved in the ligand binding function of those integrins that contain this structure.10 This region of 3 may also function as a ligand/cation-binding MIDAS-like domain.8 The homologous region of other integrin  subunits is similarly critical for ligand binding function, substantiating that the amino terminal region of integrin  subunits is either directly involved in or structurally important for ligand binding receptor function.11-13 Potential ligand contact sites on integrin  subunits have been identified by mapping epitopes of inhibitory monoclonal antibodies (MoAbs) and subsequent site-directed mutagenesis studies.14-16 Residues of IIb implicated in ligand binding function have also been identified by peptide studies17-19 and by the characterization of mutations present in patients with Glanzmann's thrombasthenia. However, in contrast to many of the mutations identified in 3, nearly all of the naturally occurring mutations identified in IIb reduce IIb3 receptor expression to very low or undetectable levels.1 This suggests that the processing or structural stability of IIb is very sensitive to substitutions. The limited occurrence of well-expressed natural receptor variants containing IIb mutations has hindered the identification of specific residues in IIb that are critical to the ligand binding function of the receptor. Despite these difficulties, secondary structure predictions have been used to identify several residues in IIb whose substitutions blocked ligand binding but did not significantly affect expression of IIb3.20 To overcome the inefficiencies of mutagenesis approaches in the absence of structural data and the rarity of Glanzmann's thrombasthenia, we recently described a novel method for the identification of induced mutations that affect IIb3 ligand binding function.21 The advantage of this method is its capacity to identify mutations that abolish ligand binding function but do not affect receptor expression. The present study identified a region in IIb that is important for ligand binding and is consistent with the predicted binding sites in the recently proposed structural model22 for the integrin  subunits.     MATERIALS AND METHODS Antibodies and reagents.   The IgM murine antibody PAC1 was kindly provided by Dr Sanford Shattil (The Scripps Research Institute, La Jolla, CA). PAC1 is an activation-specific, fibrinogen-mimetic MoAb that has been extensively characterized elsewhere.23 The IIb3 complex-specific MoAbs AP224 and OPG225 were provided by Dr Thomas Kunicki (The Scripps Research Institute). The IIb3 complex-specific MoAbs 4F10 and 2G12 were kindly provided by Dr Virgil Woods (University of California, San Diego, CA). The IIb3 complex-specific MoAb D57, the anti-3 MoAb 15, and the activating anti-3 MoAbs, anti-LIBS1, anti-LIBS2, and anti-LIBS6, have been described elsewhere.26-28 The anti-v specific MoAb 14229 was purchased from Chemicon (Temecula, CA). The anti-IIb MoAb 98DF6 was obtained from Dr Jari Ylänne (University of Helsinki, Helsinki, Finland). The MoAb D57 was biotinylated using biotin-N-hydroxysuccinimide (Pierce, Rockford, IL) according to the manufacturer's instructions. Fluorescein isothiocyanate-conjugated goat antimouse IgM and IgG were obtained from Biosource (Camarillo, CA). The IIb3 peptidomimetic Ro43-505430 was provided by Beat Steiner (Hoffmann-La Roche, Basel, Switzerland). The GRGDSPK-sepharose column was prepared as described.31 DNA constructs.   The expression constructs pc3a and CD2b encoding wild-type 3 and wild-type IIb, respectively, have been previously described.8,32 A 3.3-kb fragment of IIb containing the entire coding sequence and the 3' untranslated region was excised from CD2b by digestion with Xba I and ligated to the expression vector pcDNA3 (Invitrogen, San Diego, CA). The resulting construct was designated pc2b. The pCDM8 expression vector encoding wild-type v has been previously described.4 An insert containing the entire coding sequence of v was removed from pCDM8 by Xba I digestion and ligated to Xba I-digested pcDNA3. This construct was designated pcv. Construction of the expression plasmids encoding the chimeric subunits IIb6A and 31 has been described elsewhere.28 These chimeric plasmids contain the extracellular and transmembrane domains of human IIb or 3 fused to the cytoplasmic domains of 6A and 1, respectively. CD3hyg is a derivative of CD3a32 containing the hygromycin resistance gene. Amino acid residues of IIb are numbered, with the leucine residue at the amino terminus of the mature protein being residue number 1. The chimeric  subunit, designated pcvIIb, consists of the backbone of v from which amino acids 181-223 were removed and replaced with the corresponding amino acids of exon 5 of IIb (amino acids G193-W235).33 It was constructed using the megaprimer method34 and three consecutive rounds of amplification. Chimeric oligonucleotide primers were constructed in which the 5' ends contained v sequences (30 bp for each primer) and the 3' ends contained the sequences either of the 5' or the 3' end of exon 5 of IIb (27 and 29 bp, 5' and 3' primer, respectively). The first round of polymerase chain reaction (PCR) used pc2b as the template and generated the 129-bp exon 5 of IIb containing 30 bp of v sequence on both the 5' and 3' ends. This fragment was gel-purified and used as a 5' megaprimer together with a 3' v primer corresponding to the sequence 5'-GAATAGCCAAAGCTTGGTGGCATGC-3' in a second round of PCR using pcv as the template to generate a 712-bp fragment containing exon 5 of IIb fused to 3' v sequences. The 712-bp fragment was used as a 3' megaprimer along with a 5' v primer corresponding to the sequence 5'-CCGaGtaagCTTCGGCGATGGCTTTTCCGC-3' in a final round of PCR with pcv as a template. The resulting 1,369-bp fragment contained exon 5 of IIb flanked by 5' and 3' v sequences. This PCR fragment was digested with HindIII and ligated to HindIII-digested pcv. The authenticity of the final construct was confirmed by DNA sequencing of the entire subcloned fragment. Mutagenesis.   Site-directed mutagenesis of selected IIb residues was performed using splice overlap extension as previously described.35 PCR-generated fragments containing IIb point mutations were digested with EcoRI and Cla I, gel-purified, and ligated to EcoRI-Cla I-digested pc2b. All amplified fragments were sequenced in their entirety to verify the introduction of the mutation and the absence of any other substitutions. The Py stable cell line was generated by transfecting CHO cells with three plasmids: IIb6A, 31, and a plasmid encoding the neomycin resistance gene.21 Chemical mutagenesis of the Py stable cell line was performed by treating 2 × 106 cells with 300 µg/mL ethyl methane sulfonate (EMS; Sigma, St Louis, MO) for 15 to 19 hours. After 1 week, approximately 1 × 108 cells were harvested and ligand binding mutants were selected by flow cytometry. Flow cytometry.   Chemically mutagenized cells were individually sorted on a FAC-STAR Plus (Becton Dickinson, Mountain View, CA) using two-color flow cytometry with MoAbs D57 and PAC1 as described in detail.36 Cells that exhibited positive staining for receptor expression (D57 positive) but weak or absent PAC1 staining were individually sorted into 96-well plates and cultured for further analysis. Surface expression of transfected integrins was analyzed by flow cytometry as described.27 PAC1 binding to transfected cells was analyzed by two-color flow cytometry as described.36 PAC1 binding was analyzed only on the subset of cells positive for IIb3 receptor expression that were gated using biotinylated D57. Some samples also contained 4 µmol/L of one of the anti-3 MoAbs anti-LIBS1, anti-LIBS2, or anti-LIBS6. These antibodies bind to distinct epitopes on 3 and directly induce PAC1 binding to IIb3.37 Other samples contained 2 µmol/L Ro43-5054. Reverse transcriptase-PCR (RT-PCR).   Total cellular RNA was isolated from the selected cell lines using TRIzol (GIBCO/BRL, Grand Island, NY). cDNA was synthesized using oligo(dT) primers and the cDNA Cycle kit (Invitrogen). Resulting cDNA was amplified for 30 cycles with IIb-specific primers. The PCR products were directly sequenced using fluorescent automated sequencing (Applied Biosystems, Foster City, CA). Cell culture and transfections.   Cell lines were routinely passaged in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 1% nonessential amino acids, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin. cDNAs were transfected into CHO cells with Lipofectamine (Life Technologies, Gaithersburg, MD) as previously described.36 cDNAs encoding vIIb, IIbD224V, or selected IIb mutants were transfected into CHO cells that had been previously transfected with the expression plasmid CD3hyg. Stably transfected cells were established by selection with 700 µg/mL G418. Clonal cell lines expressing vIIb3 were selected by flow cytometry using the antihuman v-specific MoAb 142. Clonal lines expressing IIbD224V3 were sorted with the anti-IIb3 complex-specific antibody, D57. Affinity chromatography.   Lysates of 3 × 107 cells expressing either wild-type IIb3, IIbD224V3, wild-type v3, or vIIb3 were prepared and applied to a GRGDSPK-Sepharose 4B column (1 mL bed volume) as described.31 The column was washed with 10 mL of lysis buffer and then eluted with 3 mL of buffer containing 1 mg/mL GRGDSPK. One-milliliter fractions were collected and resolved on 8% sodium dodecyl sulfate (SDS) polyacrylamide gels under nonreducing conditions. Proteins were transferred to nitrocellulose and blotted with the IIb-specific MoAb, 98DF6 (3.5 µg/mL), for the IIb3 and IIbD224V3 receptors; with the v-specific MoAb, 142 (1:200 dilution of ascites), for the v3 and vIIb3 receptors; or with the 3-specific MoAb, MoAb15 (3.5 µg/mL).     RESULTS Selection and identification of ligand binding defective mutants.   We have previously demonstrated that fusing the cytoplasmic domains of 6A and 1 to the extracellular and transmembrane domains of IIb and 3, respectively, caused IIb3 to assume the high-affinity state as defined by constitutive binding of the ligand mimetic antibody PAC1.28 Therefore, to isolate cell lines with disrupted integrin ligand binding function, CHO cells stably transfected with the active receptor variant IIb6A31 were exposed to the chemical mutagen, EMS. Mutagenized cells were then sorted by two-color flow cytometry to isolate those cells that failed to bind PAC1 but continued to express IIb3 based on the binding of the MoAb D57 as described.21 Selected cells were individually sorted and subsequent analysis of the clonal cell lines grouped the mutants into three phenotypic classes: (1) ligand binding mutants, (2) integrin activation mutants, and (3) cellular activation mutants.21 Individually sorted clonal lines that failed to bind PAC1 in the presence of the activating MoAb anti-LIBS6 were defined as ligand binding defective mutants. To identify which subunit contained a mutation resulting in the loss of ligand binding function, isolated cell lines with a ligand binding defect were transfected individually with the original IIb6A or 31 subunit and then reanalyzed for restoration of PAC1 binding. For most of the ligand binding defective mutants, PAC1 binding was restored after retransfection with the parental  subunit 31, indicating the causative mutation was present in the  subunit. In contrast, PAC1 binding was reconstituted in one cell line only after retransfection with IIb6A, indicating that the defect was likely contained in the IIb subunit. This cell line was subjected to further analysis. Total RNA was isolated from this cell line and subjected to RT-PCR. The entire IIb6A subunit was amplified in three separate fragments and the resulting PCR products were sequenced directly. Analysis of the resulting sequence identified a single AT mutation resulting in substitution of D224 by valine. Characterization of ligand binding defective mutant.   To confirm that this single amino acid substitution was responsible for the observed loss of ligand binding function, this mutation was introduced into wild-type IIb cDNA and transfected into cells stably expressing the wild-type 3 subunit. Whether transiently or stably transfected, the IIbD224V3 receptor was expressed on the cell surface and bound a panel of complex-specific anti-IIb3 MoAbs, including D57 (Fig 1A), 2G12, 4F10, and AP2, suggesting the mutation exerted minimal structural effects. However, cells expressing IIbD224V3 did not bind MoAb PAC1 in the presence or absence of several activating antibodies, including anti-LIBS6 (Fig 1D), anti-LIBS1, anti-LIBS2, and AP5. The inability of these MoAbs to activate PAC1 binding was not due to a loss of antibody epitopes, because all of these MoAbs bound to IIbD224V3, as assayed by flow cytometry (data not shown). Furthermore, cells expressing IIbD224V3 exhibited minimal binding of the activation-independent ligand mimetic MoAb OPG2 compared with cells expressing wild-type IIb3 (Fig 1B). These results confirm that substitution of IIbD224 alone was sufficient for the functional defect. View larger version (36K): [in this window] [in a new window]   Fig 1. Substitution of IIb amino acid residue D224 results in a loss of ligand binding function. FACS histograms depicting the binding of the IIb3 complex-specific MoAb D57, the activation-independent ligand mimetic MoAb OPG2, or the ligand mimetic MoAb PAC1 to CHO cells transfected with wild-type IIb3 or IIbD224V3. (A) MoAb D57 staining of cells transfected with IIb3 (shaded histogram) or IIbD224V3 (open histogram). Untransfected CHO cells are depicted by the dotted histogram. (B) MoAb OPG2 staining of the IIb3 (shaded histogram) or IIbD224V3 (open histogram) transfected cells. Untransfected CHO cells are depicted by the dotted histogram. Cells expressing wild-type IIb3 (C) or IIbD224V3 (D) were activated by incubation with 4 µmol/L anti-LIBS6 for 30 minutes followed by the addition of MoAb PAC1 (IgM). Cells were washed, stained with fluorescein-conjugated goat antimouse IgM for 30 minutes, and analyzed by FACS. The binding of PAC1was analyzed in the presence (open histogram) and absence (shaded histogram) of the competitve inhibitor Ro43-5054. The failure of IIbD224V3 to bind the IIb3-specific ligands PAC1 and OPG2 could represent the loss of IIb3-specific ligand recognition or the loss of ligand binding function in general. To discriminate between these two possibilities, the RGD recognition function of IIbD224V3 was examined by affinity chromatography. Lysates of IIbD224V3-expressing or wild-type IIb3-expressing cells were applied to a GRGDSPK Sepharose column, washed, and then eluted with GRGDSPK peptide. Eluted fractions were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then immunoblotted with anti-IIb or anti-3 MoAbs (Fig 2). Wild-type IIb3 bound to the matrix and was specifically eluted by GRGDSPK peptide. In contrast, the mutant IIbD224V3 showed only minimal binding to the affinity matrix. The failure of IIbD224V3 to bind to the RGD peptide indicates that this substitution results not only in the loss of binding of IIb3-specific ligands such as PAC1 and OPG2, but also the loss of binding to ligands shared in common with other integrins. View larger version (75K): [in this window] [in a new window]   Fig 2. Binding of IIb3 and IIbD224V3 to a GRGDSPK-Sepharose 4B column. Lysates of CHO cells stably transfected with the indicated receptors were loaded onto a 1 mL GRGDSPK-Sepharose column. The column was washed then eluted with 1 mmol/L GRGDS peptide (3 mL). One-milliliter column fractions were collected, resolved on a 8% nonreducing polyacrylamide gel, transfered onto nitrocelluose, and immunoblotted with the IIb-specific MoAb 98DF6 (3.5 µg/mL). L, lysate; FT, flow through; CE, RGD eluted fraction. Site-directed mutagenesis and analysis.   To determine whether the observed functional defect was due to the substitution with valine, site-directed mutagenesis was used to replace D224 by alanine. When transiently transfected into CD3a cells, IIbD224A formed a complex with 3 and was expressed on the cell surface as assayed by flow cytometry. However, similar to cells expressing the IIbD224V3 mutant, cells expressing IIbD224A3 did not bind PAC1 in the presence or absence of the activating antibody, anti-LIBS6 (Fig 3A). View larger version (34K): [in this window] [in a new window]   Fig 3. PAC1 binding to the IIb3-mutant receptors. The binding of the activation-dependent ligand mimetic antibody PAC1 to CHO cells transfected with the indicated IIb mutant and the 3 subunit was examined by flow cytometry. To determine PAC1 binding, transfected cells were incubated (activated) with 4 µmol/L anti-LIBS6, followed by the addition of PAC1. The binding of PAC1 was analyzed in the presence (open histogram) and absence (shaded histogram) of the competitve inhibitor Ro43-5054. Untransfected CHO cells stained with PAC1 in the presence of anti-LIBS6 are depicted by the dotted histogram. The region surrounding D224 consists of a number of amino acids containing oxygenated side chains, particularly serine. This grouping of serine residues resembles the alignment of Asp and Ser residues that constitute a portion of the MIDAS motif found in the I domains of certain integrin  subunits and in the related structure present in all integrin  subunits. A number of these serine residues (S220, S222, and S226) were replaced by alanine and the effect on ligand binding was examined after transient and stable transfection of CD3a cells. The receptors IIbS220A3, IIbS222A3, and IIbS226A3 were all expressed on the cell surface and retained their ability to bind MoAb PAC1 (Fig 3B through D). Interestingly, Ro43-5054 did not inhibit the binding of PAC-1 to IIbS222A3. The mechanism for this loss of inhibition is unknown. Ligand binding specificity of IIb3.   Alignment of the integrin  subunits shows that the region corresponding to IIbD224 is not well conserved (Fig 4). Because other known ligand binding regions of the integrin receptors often exhibit a high degree of similarity, this lack of conservation suggested that this region might be important in determining the ligand recognition specificity of IIb3. To test this hypothesis, a chimeric  subunit was constructed by inserting exon 5 of IIb, which contains D224, into the corresponding location in the related v subunit. The vIIb chimeric subunit was stably expressed in CD3a cells and assayed for the capacity to bind the IIb3-specific ligand PAC1. The chimeric receptor was well expressed on the cell surface (Fig 5A); however, these cells did not exhibit binding to PAC1 in the presence or absence of the activating antibody anti-LIBS6 (Fig 5B). Similarly, binding to the activation-independent ligand mimetic antibody OPG2 was not observed (data not shown). Introduction of the IIb sequence into v did not disrupt ligand binding in general, because the chimeric receptor retained the capacity to bind to a RGD affinity matrix (Fig 5C). View larger version (22K): [in this window] [in a new window]   Fig 4. Alignment of integrin subunits between the type III and IV homologous repeats. The alignment was created using the UWGCG program "pretty." The amino acid sequences of the integrin subunits are shown using the single letter code. Alanine substitutions are indicated (*). The boundaries of exon 5 of IIb are indicated by vertical lines. Bold letters indicate residues with oxygen containing side chains. CN, consensus sequence. View larger version (36K): [in this window] [in a new window]   Fig 5. Analysis of the ligand binding function of the vIIb3 chimeric receptor. CHO cells stably transfected with vIIb3 were stained with the anti-v MoAb 142 (A) or the IIb3-specific ligand mimetic antibody PAC1 (B). To analyze PAC1 binding, cells were incubated with the activating antibody anti-LIBS6 and washed and then PAC1 binding was analyzed in the presence (open histogram) and absence (shaded histogram) of the inhibitor Ro43-5054. Stained, untransfected CHO cells are depicted by the dotted histogram. (C) Lysates of CHO cells stably transfected with vIIb3 were loaded onto a 1-mL GRGDSPK-Sepharose column. The column was washed and then eluted with 1 mmol/L GRGDS peptide (3 mL). Fractions were resolved on a 8% nonreducing polyacrylamide gel, transferred to nitrocellulose, and immunoblotted with MoAb 142. L, lysate; FT, flow through; LW, last wash fraction; CE, eluted fraction.     DISCUSSION We report the identification of a novel mutation in the integrin IIb subunit (D224V) that results in loss of IIb3 adhesion receptor function. Unlike most of the mutations identified in the IIb subunit, this mutation did not significantly affect expression of IIb3 on the cell surface. However, IIbD224V3 did not bind the activation-dependent ligand mimetic antibody, PAC1, and bound only minimally to the activation-independent ligand mimetic antibody, OPG2. Substitution of D224 by alanine also resulted in a loss of receptor function. Insertion of exon 5 of IIb containing D224 into the backbone of the v subunit did not enable the resulting chimera to bind IIb3-specific ligands; however, it did not interfere with ligand binding to v3. These data suggest that IIbD224 may play an important role in ligand binding specific to IIb3. An unbiased genetic approach was used to identify chemically induced mutations that result in the loss of IIb3 ligand binding activity. A key feature of this approach is the ability to isolate cells that expressed IIb3 on the cell surface but failed to bind the activation-dependent, ligand mimetic antibody PAC1.21 Whereas the majority of the isolated clones contained substitutions in the 3 subunit, PAC1 binding was restored in one clone only after retransfection with the parental  subunit. This mutant receptor contained a single basepair change resulting in substitution of D224 by valine. This receptor was expressed on the cell surface as assayed with a panel of anti IIb3 MoAbs, indicating that the substitution likely had minimal effects on the processing or structure of this subunit. This finding is in sharp contrast to most of the identified naturally occurring mutations in IIb that significantly reduce or abolish receptor expression.1 The effect of the mutation at D224 on ligand binding to IIb3 was not restricted to IIb3-specific ligands such as PAC1 and OPG2. This mutation also significantly decreased the binding of a RGD peptide. Thus, this mutation affects the binding of ligands specific to IIb3 and the binding of ligands shared in common with other integrins. D224 is located in a group of serine residues distributed in a pattern that resembles a portion of the MIDAS motif known to be important for cation and ligand binding in the I domain of integrin  subunits and in a related motif in the integrin  subunits.8,9,11-13,38,39 However, substitution of several of these serine residues (S220, S222, and S226) with alanine did not result in the loss of PAC1 binding, suggesting that they do not directly play a critical role in the ligand binding process. Surprisingly, the substitution of S222 did appear to inhibit the capacity of the peptidomimetic Ro43-5054 to block PAC1 binding. The mechanism of the effect of substitution of D224 on ligand binding is unclear. It is unlikely to be the result of a gross structural alteration, because the mutant receptor bound a panel of complex-specific anti-IIb3 antibodies. Other possibilities include direct interaction of D224 with ligand or, alternatively, through an indirect method by interacting cooperatively with other regions present in IIb or in the 3 subunit. The precise functional assignment of this residue cannot be defined without high resolution structural data for the IIb3 receptor. The ligand recognition specificity of the integrins is determined in large part by the  subunits. In the case of IIb3, the specificity of ligand recognition maps to the first 334 residues of the IIb subunit.40 The location of D224 is consistent with this data. Alignment between the integrin  subunits demonstrates that the region surrounding D224 is not well conserved, suggesting that this region of IIb may be involved in a specific rather than general role of ligand recognition. Previous work from this laboratory had demonstrated that a larger region of IIb (ie, amino acids R140-P334) did not confer IIb3 ligand binding specificity to an vIIb chimeric  subunit.40 In the present study, a smaller chimera (IIb amino acids 193-235) was constructed consistent with IIb intron/exon boundaries. Although this chimeric integrin consisting of the v subunit and exon 5 of IIb was well expressed, it did not bind the IIb3-specific ligand PAC1. Moreover, disruption of the native v region with the IIb sequences did not abolish the binding of RGD peptides to this chimera. Thus, the region of IIb that contains D224 may fully substitute for the analogous v sequences or the analogous v region is not similarly important for ligand binding to v3. Although the lack of conservation between the  subunits in this region makes it difficult to accurately align residues with D224, these data do indicate that D224 is important for ligand binding function of IIb3. A structural model for the N-terminal approximately 440 amino acids of the integrin  subunits has recently been proposed.22 This model predicts that the seven homologous sequence repeats found in the  subunits adopt the fold of a -propeller domain. Enzymes with known -propeller folds have their active sites at the top of the -propeller, typically where adjacent loops run in opposite directions, such as seen between strands 2 and 3 within a  sheet and strands 4 and 1 between two sheets. The residue identified in this study, IIbD224, lies between repeats III and IV in the IIb subunit and is located in the large 4-1 loop region located at the top of the  propeller between the W3 and W4  sheets. In a previous study, the IIb region, G184-193, was implicated as important for ligand binding and is located in the adjacent 2-3 loop found in the W3 sheet at the top of the -propeller.20 Thus, these two loop regions could act synergistically to form a portion of the ligand binding pocket between the IIb and 3 subunits. The recent identification of an IIb L183P mutation in a Glanzmann's thrombasthenia patient41 that produces quantitative and qualitative abnormalities in the receptor also substantiates the importance of this region in ligand binding. The identification of residues critical for 41-ligand interactions14 and for the binding of fibronectin to the integrin 5116 further supports a role for the loop structures of the  subunits on the upper face of the predicted -propeller model in ligand binding specificity. The region identified in this study is located between the homologous repeats III and IV found in all integrin  subunits. Interestingly, this region is known to be alternatively spliced in 6 and 7 and has been postulated to be alternatively spliced in 3.42,43 In 6 and 7, alternative splicing produces two variants, one of which (the 7X2 variant) contains a Asp residue homologous to D224 in IIb (see Fig 4). The 7 splice variants are both developmentally regulated and tissue specific. Although no functional significance has yet been determined for these different variants, it has been speculated that this region may be involved in ligand specificity and/or different ligand binding affinities. Alternative splicing between the third and fourth repeats has also been reported for the Drosophila integrin PS2 subunit.44,45 Together, the differences among such variants could suggest a general function for this region in ligand recognition specificity consistent with the low conservation of this region among different  subunits. In summary, this study has identified a novel region in IIb that is important for ligand binding. This location of this region is within a predicted ligand binding site in the  propeller model proposed for the  subunits. This region is not highly conserved among the  subunits, and substitutions in the analogous regions of v did not affect ligand binding function of v3. These results indicate that this region of IIb may play a role in ligand binding that is specific to IIb3.     FOOTNOTES Submitted July 27, 1998; accepted October 5, 1998. Supported in part by National Heart, Lung and Blood Institute Grant No. HL42977 (J.C.L.) and a fellowship from the National Institute of Health (1F32HL09321; to E.C.T.). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact. Address reprint requests to Joseph C. Loftus, PhD, Mayo Clinic Scottsdale, 13400 E Shea Blvd, Scottsdale, AZ 85259; e-mail: loftus.joseph{at}mayo.edu.     REFERENCES Top Abstract Introduction References 1. French DL, Coller BS: Hematologically important mutations: Glanzmann thrombasthenia. Blood Cells Mol Dis 23:39, 1997[Medline] [Order article via Infotrieve] 2. D'Souza SE, Ginsberg MH, Burke TA, Lam SC, Plow EF: Localization of an Arg-Gly-Asp recognition site within an integrin adhesion receptor. Science 242:91, 1988[Abstract/Free Full Text] 3. Charo IF, Nannizzi L, Phillips DR, Hsu MA, Scarborough RM: Inhibition of fibrinogen binding to GP IIb-IIIa by a GP IIIa peptide. J Biol Chem 266:1415, 1991[Abstract/Free Full Text] 4. Loftus JC, O'Toole TE, Plow EF, Glass A, Frelinger A, Ginsberg MH: A 3 integrin mutation abolishes ligand binding and alters divalent cation-dependent conformation. Science 249:915, 1990[Abstract/Free Full Text] 5. 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