High Affinity Ligand Binding by Integrins Does Not Involve Head Separation*

Bing-Hao LuoDagger §, Timothy A. SpringerDagger §, and Junichi TakagiDagger ||

From the Dagger  Center for Blood Research, § Department of Pathology and  Pediatrics, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, February 12, 2003, and in revised form, February 20, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Conformational change in the integrin extracellular domain is required for high affinity ligand binding and is also involved in post-ligand binding cellular signaling. Although there is evidence to the contrary, electron microscopic studies showing that ligand binding triggers alpha - and beta -subunit dissociation in the integrin headpiece have gained popularity and support the hypothesis that head separation activates integrins. To test directly the head separation hypothesis, we enforced head association by introducing disulfide bonds across the interface between the alpha -subunit beta -propeller domain and the beta -subunit I-like domain. Basal and activation-dependent ligand binding by alpha IIbbeta 3 and alpha Vbeta 3 was unaffected. The covalent linkage prevented dissociation of alpha IIbbeta 3 into its subunits on EDTA-treated cells. Whereas EDTA dissociated wild type alpha IIbbeta 3 on the cell surface, a ligand-mimetic Arg-Gly-Asp peptide did not, as judged by binding of complex-specific antibodies. Finally, a high affinity ligand-mimetic compound stabilized noncovalent association between alpha IIb and beta 3 headpiece fragments in the presence of SDS, indicating that ligand binding actually stabilized subunit association at the head, as opposed to the suggested subunit separation. The mechanisms of conformational regulation of integrin function should therefore be considered in the context of the associated alpha beta headpiece.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Integrins are major metazoan adhesion receptors that play a fundamental role in cellular organization. They mediate cell-extracellular matrix as well as cell-cell adhesion, connect extracellular cues to the cytoskeleton, and activate many intracellular signaling pathways (1, 2). One unique aspect of integrins is that the affinity of their extracellular domain for biological ligands can be rapidly up-regulated by signals from within the cell. Rapid and precise control of integrin activation is particularly important for leukocytes and platelets, which circulate in the vascular system, where leukocyte emigration and thrombus formation mediated by integrins must be triggered only at the appropriate location. Integrins compose two noncovalently associated type I transmembrane glycoprotein alpha - and beta -subunits (3). A crystal structure of the extracellular domain of the integrin alpha Vbeta 3 revealed a bent conformation, in which there is an acute angle between the headpiece and tailpiece (4), and an extensive headpiece-tailpiece interface (5). Recently, we have shown that the bent conformation represents the low affinity receptor and that activation is associated with a switchblade-like motion of the headpiece resulting in a highly extended conformation (5).

The integrin headpiece contains the ligand-binding site. The headpiece contains the alpha -subunit beta -propeller and thigh domains and the beta -subunit I-like and hybrid domains (Fig. 1), corresponding approximately to the N-terminal two-thirds of the extracellular domain of each subunit. A crystal structure with a bound ligand-mimetic peptide revealed that ligand binds to an interface formed by the beta -subunit I-like domain and beta -sheets 2-4 of the alpha -subunit beta -propeller domain (6) (Fig. 1).

Many experiments support the idea that the inter-subunit association at the cytoplasmic region maintains integrins in low affinity state (7-10). Originally, this notion led to a "hinge hypothesis," where association/dissociation of the cytoplasmic tails caused hinging between the two subunits, and ultimately changed the conformation of the ligand-binding extracellular segments (11). However, the nature of the conformational change that regulates ligand binding by integrins has been controversial (2, 3, 5, 12-15). Hantgan et al. (16, 17), using rotary shadowing EM, reported that binding of RGD peptides induced separation of the headpiece of detergent-solubilized alpha IIbbeta 3. These images suggested a wide separation in the headpiece, with no interaction remaining between the N-terminal halves of the alpha - and beta -subunits. These observations have been highly influential, in part because they seemed to fit with earlier schematics of integrin activation models where a hinge-like motion at the transmembrane domains is transmitted through rigid alpha - and beta -subunit tailpiece segments to the headpiece, resulting in movement apart of the alpha - and beta -subunits in the headpiece (11, 12, 18). However, recent high resolution negative stain EM studies have shown that the alpha -subunit leg can exist in two distinct conformations with respect to the headpiece and that the beta -subunit leg is highly flexible (5). Existence of multiple modular domains (4) also disfavors rigid movement of the entire stalk region. Because the legs are flexible, it is hard to imagine that information can be transmitted to the headpiece as proposed in the hinge model. Furthermore, high resolution EM studies (5), as well as many other EM studies (19-22), have shown that ligand binding to integrins is not accompanied by head separation.

There are other reasons for the popularity of the head separation model. First, it has been suggested that residues that have been implicated in ligand binding are buried in the headpiece and that separation could expose them, resulting in higher affinity binding (2). Second, there is the mystery of the synergy site in fibronectin type III module 9 of fibronectin, which is distant from the RGD site in module 10. It has been proposed that separation of the headpiece (2, 15) would facilitate simultaneous binding of the alpha -subunit to the synergy site and the beta -subunit to the RGD site (23). However, the liganded alpha Vbeta 3 crystal structure shows that the Arg of RGD binds to the alpha -subunit and the Asp of RGD binds to the beta -subunit (6), strongly suggesting that headpiece separation would disrupt binding to RGD. Third, there are structural homologies between integrins and G proteins (4, 24, 25). By taking this analogy further, it has been suggested that upon integrin activation, the beta -propeller and I-like domains might dissociate analogously to the G protein beta - and alpha -subunits (2, 15).

An alternative model for integrin activation has been proposed that is supported by high resolution EM, physicochemical studies, ligand binding assays, introduction of disulfide bonds that lock in the bent conformation, and localization of epitopes that become exposed after integrin activation (5, 26). In this model, activation is regulated by the conformational equilibrium between three states as follows: a bent conformation with low affinity, an extended conformation with a closed headpiece with intermediate affinity, and an extended conformation with an open headpiece with high affinity. Binding of RGD peptide was found not to induce head separation but to induce a dramatic change in angle between the beta -subunit I-like and hybrid domains, leading to the swing-out of the hybrid domain away from the alpha -subunit (5). The prominence of the hybrid domain in the interface between the headpiece and the tailpiece in the bent conformation provides a mechanism for linking the change in angle upon ligand binding to the equilibrium between the bent and extended integrin conformations (5).

Definitive experimental testing of the head separation model is important. Therefore, we have used mutagenesis to introduce disulfide bonds between the alpha -subunit beta -propeller domain and the beta -subunit I-like domain, and we have tested the effect of preventing head separation on activation of alpha IIbbeta 3 and alpha Vbeta 3 integrins on the cell surface. Furthermore, we test the effect of ligand-mimetic compounds on the association between the alpha - and beta -subunits in native integrins on the cell surface and in soluble integrin fragments that contain only the headpiece.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Monoclonal Antibodies-- Mouse monoclonal antibody (mAb)1 PT25-2 recognizing human alpha IIb subunit (27) was a gift from Dr. M. Handa (Keio University, Tokyo, Japan). Mouse mAb 10E5 recognizing the human alpha IIbbeta 3 complex (28) was a gift from Dr. B. S. Coller (Rockefeller University, New York). Mouse anti-beta 3 AP3 was from American Type Culture Collection. All other mAbs were obtained from the Fifth International Leukocyte Workshop (29).

Plasmid Construction, Transient Transfection, and Immunoprecipitation-- Plasmids coding for full-length human alpha IIb, alpha V, and beta 3 were subcloned into pcDNA3.1/Myc-His(+) or pEF/V5-HisA as described previously (5). Mutants were made using site-directed mutagenesis, and DNA sequences were confirmed before being transfected into 293T cells using calcium phosphate precipitation. Transfected cells were metabolically labeled with [35S]cysteine/methionine as described (5). Labeled cell lysates were immunoprecipitated with anti-beta 3 AP3, eluted with 0.5% SDS, and subjected to nonreducing or reducing SDS-7.5% PAGE and fluorography.

Two-color Ligand Binding and Flow Cytometry-- Binding of fluorescein-labeled human fibrinogen and fibronectin were performed as previously described (5). To test the effect of EDTA treatment on mAb epitope expression, transiently transfected 293T cells were preincubated in 20 mM Tris-buffered saline, pH 8.4, containing either 1 mM Ca2+, 1 mM Mg2+, or 5 mM EDTA at 37 °C for 30 min, followed by washing and resuspension in 20 mM Hepes, 150 mM NaCl, 5.5 mM glucose, 1% bovine serum albumin, and 1 mM Ca2+, 1 mM Mg2+ (HBS). Cells were then incubated with mAbs on ice for 30 min, followed by staining with FITC-conjugated anti-mouse IgG and flow cytometry. To test the effect of RGD peptide on mAb epitope expression, cells in HBS were incubated with 100 µM GRGDSP peptide at room temperature for 30 min before adding mAbs and staining as above.

Stability of the alpha IIbbeta 3 Headpiece in SDS in the Presence of an RGD-mimetic Compound-- An integrin headpiece fragment comprising alpha IIb residues 1-621 and beta 3 residues 1-472 was produced in Chinese hamster ovary Lec 3.2.8.1 cells stably transfected with plasmids coding for each fragment. Acid-base alpha -helical coiled-coil peptides were fused to the C termini of the alpha - and beta -subunits to increase the stability of the heterodimer. Methods were as described previously (8). A hexahistidine tag was also attached to the C terminus of the beta -subunit to facilitate purification by nickel chelate chromatography (8). The purified headpiece fragment was treated with 10 µg/ml chymotrypsin for 16 h at 25 °C to remove the C-terminal clasp, and incubated with the high affinity RGD-mimetic compound L738,167 (gift from Dr. G. D. Hartman, Merck) (30) at 10 µM for 30 min at 37 °C. The mixture was cooled to room temperature; an equal volume of sample buffer containing 0.1% SDS was added, and samples were immediately subjected to nonreducing SDS-PAGE on a 4-20% gradient gel.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Introduction of Disulfide Bonds between the beta -Propeller and I-like Domains-- To make mutant integrins that were unable to undergo head separation, we mutationally introduced cysteine residues at the interface between the alpha IIb beta -propeller and beta 3 I-like domains (Fig. 1). Inspection of the structure of alpha Vbeta 3 and a model of alpha IIbbeta 3 made from the alpha Vbeta 3 template identified candidate positions for disulfide bonds where Calpha distances between beta -propeller and I-like domain residues were within 7 Å. Disulfide-forming efficiency was assessed by transient transfection of 293T cells followed by immunoprecipitation and nonreducing SDS-PAGE. Preliminary experiments revealed that the efficiency varied from 20 to 100%, depending on the combination of residues chosen (data not shown). In alpha Vbeta 3, the combination of the alpha V-M400C and beta 3-Q267C mutations gave ~100% formation of an intersubunit disulfide bridge as indicated by the formation of a high molecular weight band in nonreducing SDS-PAGE (Fig. 2, compare wild type in lane 1 and double mutant in lane 2), whereas the alpha V- and beta 3-subunits migrated separately in reducing SDS-PAGE (Fig. 2, lanes 3 and 4). The combination of alpha V-E311C and beta 3-G293C mutations resulted in slightly less efficient disulfide formation (~70%, data not shown). Since alpha IIb lacks the long loop containing M400 in alpha V, the mutation corresponding to alpha V-E311C (alpha IIb-E324C) was tested and shown to form an efficient disulfide when coupled with the beta 3-G293C mutation (Fig. 2, compare lane 6 with wild type in lane 5). Under reducing conditions, the disulfide-linked complex was reduced into individual alpha - and beta -subunits indistinguishable from the wild type subunits (Fig. 2, lanes 7 and 8). The mutations link beta -propeller blade 5 or the connection between blades 6 and 7 to the I-like domain, whereas the ligand-binding interface locates on the opposite side of the interface where blades 2-4 of the beta -propeller domain contact the I-like domain (Fig. 1). The mutations should therefore not directly affect the ligand-binding site or disrupt conformational changes in loops near the ligand-binding site. A panel of mAbs directed against the head regions of alpha Vbeta 3 and alpha IIbbeta 3 bound identically to mutant and wild type receptors (data not shown), suggesting that the mutant receptors adopt a native fold.


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Fig. 1.   The alpha Vbeta 3 integrin headpiece and location of introduced disulfide bonds. The stereo view of the alpha Vbeta 3 headpiece is based on the crystal structure of the alpha Vbeta 3 extracellular domain bound to a ligand-mimetic RGD peptide (6). The alpha V beta -propeller and thigh domains are red; the beta 3 I-like and hybrid domains are blue, and engineered disulfide bonds are in gold. The bound RGD peptide is magenta, and its Arg and Asp side chains are shown. Backbones are shown as a worm-like trace. Note that the disulfide bonds introduced by mutations alpha V-M400C/ beta 3-Q267C and alpha V-E311C/beta 3-G293C (which corresponds to alpha IIb-E324C/beta 3-G293C) are located on the side of the beta -propeller and I-like domain interface opposite from the ligand-binding site. Figure was prepared with Ribbons (44).


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Fig. 2.   Formation of intersubunit disulfide bonds in the headpieces of alpha Vbeta 3 and alpha IIbbeta 3. Lysates were prepared from [35S]methionine- and -cysteine-labeled 293T cells that had been transiently transfected with wild type alpha Vbeta 3 (lanes 1 and 3), alpha V-M400C/beta 3-Q267C (lanes 2 and 4), wild type alpha IIbbeta 3 (lanes 5 and 7), or alpha IIb-E324C/beta 3-G293C (lanes 6 and 8), immunoprecipitated with mouse mAb AP3 to human beta 3, and subjected to SDS-7.5% PAGE under nonreducing (lanes 1, 2, 5, and 6) or reducing (lanes 3, 4, 7, and 8) conditions followed by fluorography. Positions of molecular size markers are shown on the left.

High affinity binding of alpha Vbeta 3 and alpha IIbbeta 3 transfectants to fluorescent, soluble ligands was measured with simultaneous staining of surface-expressed alpha Vbeta 3 or alpha IIbbeta 3 using two-color flow cytometry (5). As described previously, wild type alpha IIbbeta 3 bound soluble fibrinogen when activated by Mn2+ and activating mAb PT25-2 but not in the absence of activation (Fig. 3A). The alpha IIb-E324C/beta 3-G293C mutant could not bind soluble fibrinogen in the resting state but showed full activity when treated with Mn2+ and PT25-2 (Fig. 3A). Thus, the ligand-binding phenotype of alpha IIbbeta 3 containing a disulfide-linked headpiece is identical to wild type.


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Fig. 3.   Ligand binding by disulfide-linked receptors. 293T cells expressing alpha IIbbeta 3 (A) or alpha Vbeta 3 (B and C) integrins were incubated with (filled bars) or without (open bars) 1 mM Mn2+ and the activating mAbs PT25-5 for alpha IIbbeta 3 or AP5 for alpha Vbeta 3. Binding of FITC-fibrinogen (A and B) or FITC-fibronectin (C) was determined and expressed as the percentage of mean fluorescence intensity relative to immunofluorescent staining with Cy3-labeled AP3 mAb. wt, wild type.

Similarly, the mutant alpha Vbeta 3 receptor was tested for activation-dependent binding to soluble fibrinogen (Fig. 3B) and fibronectin (Fig. 3C). As for alpha IIbbeta 3, the ligand binding activities of wild type and mutant alpha Vbeta 3 receptors were indistinguishable. Both showed little or no basal ligand binding and strong binding of both ligands upon activation by Mn2+ and activating mAb AP5 (Fig. 3, B and C). These data show that disulfide bond formation between the beta -propeller and I-like domains has no effect on high affinity ligand binding by beta 3 integrins, strongly arguing against the notion that head separation is required for conversion to high affinity.

Introduced Disulfide Bond Blocks EDTA-induced Subunit Dissociation-- EDTA treatment at pH 8.4 at 37 °C is known to induce dissociation of the integrin alpha IIbbeta 3 into the alpha IIb- and beta 3-subunits on the platelet surface (31-33). We confirmed that alpha IIbbeta 3 expressed on 293T transfectants is similarly susceptible to the EDTA-induced subunit dissociation. Incubation of cells expressing wild type alpha IIbbeta 3 with 5 mM EDTA at pH 8.4 completely abolished binding of the alpha beta complex-specific mAbs AP2 (34) and 10E5 (34) but had no effect on binding of the beta 3-specific mAb AP3 (Fig. 4A). The complex-specific mAbs require the presence of a complex between alpha IIb- and beta 3-subunits for recognition, and loss of reactivity is thus an excellent indicator of subunit dissociation. In contrast to the wild type receptor, binding of the mAbs AP2 and 10E5 to the mutant alpha IIb-E324C/beta 3-G293C receptor was unaffected by EDTA treatment (Fig. 4B) showing that the disulfide bond conferred resistance to EDTA-induced subunit dissociation. Thus, maintenance of a covalent connection between the beta -propeller and I-like domains is sufficient to protect the epitopes of the AP2 and 10E5 mAbs, suggesting that the deleterious effect of EDTA on these epitopes is a consequence of subunit separation, rather than a direct stabilizing effect of divalent cations on residues within the epitopes.


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Fig. 4.   Effect of EDTA treatment and RGD peptide on the binding of complex-specific alpha IIbbeta 3 mAbs. Transiently transfected 293T cells expressing wild type alpha IIbbeta 3 (A) or mutant alpha IIbE324C/beta 3G293C (B) were stained with the indicated mAbs in the presence of 1 mM Ca2+/Mg2+ (open bars), 5 mM EDTA (filled bars), or 1 mM Ca2+/Mg2+ plus 100 µM GRGDSP peptide (shaded bars, wild type only) as described under "Experimental Procedures." Binding is expressed as the percentage of positive cells after subtraction of background staining by ×63 IgG control.

RGD Peptide Binding Does Not Induce Head Separation-- We used the AP2 and 10E5 mAbs to determine whether RGD-mimetic peptides, like EDTA, could induce head separation of cell surface alpha IIbbeta 3. Incubation of wild type alpha IIbbeta 3 293T cell transfectants with 100 µM GRGDSP peptide resulted in full exposure of cryptic epitopes called ligand-induced binding sites (data not shown), as described previously (35, 36), suggesting that all of the alpha IIbbeta 3 on the cell surface bound GRGDSP peptide. However, preincubation with and the continued presence of 100 µM RGD peptide had no effect on binding of AP2 and 10E5 mAbs to alpha IIbbeta 3 (Fig. 4A). This result not only establishes that RGD peptide and these mAbs bind to independent sites on the alpha IIbbeta 3 headpiece but also strongly suggests that the two subunits stay associated in the headpiece when RGD peptide is bound.

RGD-mimetic Compound Stabilizes Rather than Destabilizes Integrin Headpiece Association-- High affinity ligand-mimetic compounds can stabilize integrin alpha beta complexes and make them resistant to SDS-induced dissociation during gel electrophoresis (37, 38). A wide variety of integrins can be stabilized, including alpha IIbbeta 3 (17, 39, 40). In these studies, the integrins were either native or were recombinant integrins containing the entire extracellular domain. In the bent integrin conformation, there are substantial interactions between the alpha - and beta -subunits in both the headpiece and tailpiece (4, 5). To clarify whether ligand-mimetic peptide binding stabilizes the alpha beta interface present within the headpiece, we designed a truncated alpha IIbbeta 3 integrin fragment composed only of headpiece segments. It contains the alpha IIb beta -propeller and thigh domains and the beta 3 plexin-semaphorin-integrin (PSI), I-like, hybrid, and the first integrin-epidermal growth factor-1 domains; the only alpha -beta interface present in this fragment is between the beta -propeller and I-like domains (Fig. 1). When the purified alpha IIbbeta 3 headpiece fragment was incubated with SDS sample buffer at room temperature and subjected to SDS-PAGE, two distinct bands corresponding to the truncated alpha IIb- and beta 3-subunits were found (Fig. 5, lane 1). By contrast, when the alpha IIbbeta 3 headpiece fragment was incubated with 10 µM of the RGD-mimetic compound L738,167 prior to SDS-PAGE, a single band with higher molecular mass was found with no free alpha IIb or beta 3 fragments (Fig. 5, lane 2). Thus, the alpha IIbbeta 3 headpiece becomes resistant to dissociation by SDS after incubation with a ligand-mimetic peptide. The ligand-mimetic compound strengthens association between the beta -propeller domain of alpha IIb and the I-like domain of beta 3 probably by providing an additional interaction between them, as seen in the crystal structure in which an RGD-mimetic peptide binds to alpha V through its Arg and beta 3 through its Asp (6). Stabilization by the ligand-mimetic compound of alpha beta headpiece association is in strong contradiction to the notion that ligand-mimetic peptides cause dissociation of the alpha - and beta -subunits in the headpiece.


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Fig. 5.   Ligand-induced stabilization of intersubunit association in the headpiece. The alpha IIbbeta 3 headpiece fragment comprising alpha IIb residues 1-621 and beta 3 residues 1-472 was incubated without (lane 1) or with 10 µM L738,167 (lane 2) and subjected to SDS 4-20% gradient PAGE and Coomassie Blue staining. Positions of molecular size markers are shown on the left.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our study definitively establishes that head separation is not required for activation of ligand binding by integrins, as shown with both alpha IIbbeta 3 and alpha Vbeta 3 by introduction of a covalent linkage between the alpha -subunit beta -propeller domain and the beta -subunit I-like domain. Despite the covalent linkage, binding of alpha IIbbeta 3 to fibrinogen, and alpha Vbeta 3 to fibrinogen and fibronectin, was fully activable. We used the AP2 and 10E5 mAbs, which bind to epitopes contained wholly within the alpha IIbbeta 3 headpiece and are specific for the alpha IIbbeta 3 complex, to monitor separation between alpha IIb and beta 3 in the headpiece within intact alpha IIbbeta 3 on the cell surface. EDTA dissociated the alpha IIbbeta 3 headpiece as shown by loss of both epitopes. However, the ligand-mimetic GRGDSP peptide did not dissociate the headpiece, despite saturation binding to alpha IIbbeta 3 as shown by full exposure of ligand-induced binding site epitopes. Finally, a high affinity RGD-mimetic compound was found to stabilize association between alpha IIb and beta 3 in a fragment containing only the headpiece. These results completely contradict the idea that RGD-mimetics induce headpiece separation. A model for I domain-containing integrins, in which separation between the alpha -subunit beta -propeller and the beta -subunit I-like domain allows the alpha -subunit I domain to bind to the beta -propeller domain in place of the I-like domain (15), also seems highly unlikely in view of our results, because integrins that contain and lack I domains are activated by similar mechanisms (3).

By contrast, the results are completely consistent with an alternative model of integrin activation, in which the integrin headpiece stays associated and ligand binding affinity is linked to the equilibrium between bent and extended integrin conformations, and between two headpiece conformations that differ in the angle between the I-like and hybrid domains (5). In contrast to the headpiece separation model, the bent-extended model has received experimental support. Disulfide bonds that stabilize the bent conformation inhibit integrin activation (5), and an introduced N-glycosylation site designed to wedge open the angle between the I-like and hybrid domains activates ligand binding (36).

The crystal structure of alpha Vbeta 3 bound to an RGD ligand-mimetic peptide was obtained by soaking the peptide into a crystal containing the bent conformer of alpha Vbeta 3 (6). Some movement was seen at the beta -propeller interface with the I-like domain upon ligand binding, but its magnitude was small and compatible with the disulfide bonds we have introduced. Thus, the distances between Calpha atoms before and after ligand binding, respectively, are 6.7 and 6.8 Å for alpha V-M400/beta 3-Q267, and 5.8 and 5.8 Å for alpha V-E311/beta 3-G293. In the absence of restraining crystal lattice contacts, ligand-mimetic peptide binding induces adoption of the high affinity, extended conformation of alpha Vbeta 3 and a change in angle between the I-like and hybrid domains (5). Our disulfide bond-linked mutants are fully competent for high affinity ligand binding. Therefore, any further change in orientation of the beta -propeller domain with respect to the I-like domain between the ligand-bound bent and extended conformations must be relatively small.

Apart from complete head separation, the possibility has been raised of integrin activation by a marked tilt of the I-like domain so that it separates from the beta -propeller domain at the ligand binding interface but not on the opposite side of the interface, near where we have introduced disulfide bridges (2, 15). We note that the alpha IIb-E324C/beta 3-G293C mutation involves residues that are buried in the beta -propeller I-like domain interface and that the surrounding region is highly conserved in alpha V and alpha IIb. Therefore, pivoting around the introduced disulfide bond to open the ligand-binding site seems unlikely, because on the side of the pivot opposite from the ligand-binding site residues are already closely packed. Thus, beta 3 residue Glu-297 would clash with alpha V residue Phe-337, and beta 3 residues Leu-324 and Pro-326 would clash with alpha V residues Lys-308 and Leu-309; all of these alpha V residues are highly conserved in alpha IIb. Our results therefore rule out a significant tilting motion between the beta -propeller and I-like domains that would open up the ligand binding interface, as well as complete separation of these domains. This conclusion is in accord with high resolution EM projection averages that show no gross change in orientation between the beta -propeller and I-like domains upon ligand binding (5). Indeed, the only inter-domain movement observed within the headpiece upon ligand binding is between the I-like and hybrid domains (5). Partial head separation is also inconsistent with the crystal structure of RGD peptide bound to alpha Vbeta 3, because the Arg and Asp side chains of RGD are already extended in opposite directions (Fig. 1) (6), and separation at this interface would abolish one or the other of the highly specific interactions that these side chains make with the alpha V- and beta 3-subunits, respectively.

We have suggested that swinging of the hybrid domain pulls down the C-terminal helix of the I-like domain and as a consequence activates the metal ion-dependent adhesion site analogously to I domain activation (3, 36). Since physiological ligands use many residues other than the potential metal ion-dependent adhesion site-coordinating residues to interact with integrins, it is natural to expect that conversion to the high affinity conformation involves rearrangement of residues of the alpha -beta interface outside of the metal ion-dependent adhesion site. In fact, there are activation-reporting mAbs that map to this region (41-43). These rearrangements may involve loop remodeling and some reorientation between the beta -propeller and I-like domains, but in a more subtle degree than proposed by head separation/tilting models. Precise determination of the subtle conformational changes responsible for affinity regulation of integrins awaits further study using atomic resolution analysis.

    ACKNOWLEDGEMENTS

We thank Drs. Barry S. Coller, Makoto Handa, and George D. Hartman for the generous gifts.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL48675.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Center for Blood Research, Harvard Medical School, 200 Longwood Ave., Rm. 238, Boston, MA 02115. Tel.: 617-278-3200; Fax: 617-278-3232; E-mail: takagi@cbr.med.harvard.edu.

Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M301516200

    ABBREVIATIONS

The abbreviations used are: mAb, monoclonal antibody; FITC, fluorescein isothiocyanate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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