Conformational Changes in the Integrin beta A Domain Provide a Mechanism for Signal Transduction via Hybrid Domain Movement*

A. Paul MouldDagger, Stephanie J. Barton, Janet A. Askari, Paul A. McEwan§, Patrick A. Buckley, Susan E. Craig, and Martin J. Humphries

From the Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom

Received for publication, December 23, 2002, and in revised form, February 27, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The ligand-binding head region of integrin beta  subunits contains a von Willebrand factor type A domain (beta A). Ligand binding activity is regulated through conformational changes in beta A, and ligand recognition also causes conformational changes that are transduced from this domain. The molecular basis of signal transduction to and from beta A is uncertain. The epitopes of mAbs 15/7 and HUTS-4 lie in the beta 1 subunit hybrid domain, which is connected to the lower face of beta A. Changes in the expression of these epitopes are induced by conformational changes in beta A caused by divalent cations, function perturbing mAbs, or ligand recognition. Recombinant truncated alpha 5beta 1 with a mutation L358A in the alpha 7 helix of beta A has constitutively high expression of the 15/7 and HUTS-4 epitopes, mimics the conformation of the ligand-occupied receptor, and has high constitutive ligand binding activity. The epitopes of 15/7 and HUTS-4 map to a region of the hybrid domain that lies close to an interface with the alpha  subunit. Taken together, these data suggest that the transduction of conformational changes through beta A involves shape shifting in the alpha 7 helix region, which is linked to a swing of the hybrid domain away from the alpha  subunit.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Integrins mediate a wide variety of essential cell-matrix and cell-cell interactions and also participate in many common disease processes (1, 2). Integrins are heterodimers containing non-covalently associated alpha  and beta  subunits; each subunit has a large extracellular domain linked to a transmembrane segment and a short cytoplasmic tail. Integrins participate in bi-directional signaling; ligand recognition is dynamically regulated by "inside-out" signaling, and ligand occupancy leads to "outside-in" signals that affect cell migration, growth, differentiation, and survival (3-5). Modulation of integrin activity is essential in such processes as leukocyte migration to sites of tissue injury and the aggregation of platelets to form a hemostatic plug. Integrin activation can be mimicked in vitro by divalent cations such as Mn2+ or Mg2+ (6). Three major conformational states of integrins can be distinguished using monoclonal antibodies (mAbs)1: an inactive (resting or low affinity) state, an active (or high affinity) state, and a ligand-occupied state (7). The conformations of the inactive and active states are discriminated by low and high expression, respectively, of activation epitopes (such as those recognized by 12G10, 15/7, and 9EG7 for the beta 1 subunit, see Refs. 8-10). The ligand-occupied conformer expresses high levels of ligand-induced binding site (LIBS) epitopes (which are generally also activation epitopes) and shows decreased expression of ligand-attenuated binding site (LABS) epitopes (such as mAb 13 for the beta 1 subunit, see Ref. 11). The conformational states are in equilibrium; therefore, antibodies that recognize activation epitopes or LIBS tend to cause activation and stabilize the ligand-occupied state. Conversely, antibodies that recognize LABS appear to block ligand binding by preventing conformational changes involved in ligand recognition (7, 11, 12).

The molecular basis of integrin function has been powerfully elucidated by the recent x-ray crystal structures of the extracellular domains of alpha Vbeta 3 in both an unliganded state (13) and in complex with a small peptide ligand (14). Overall, the integrin structure resembles that of a "head" on two "legs." The ligand-binding head region of the integrin contains a seven-bladed beta -propeller in the alpha  subunit, the top face of which is in close juxtaposition with a von Willebrand factor type A domain in the beta  subunit (beta A). beta A consists of seven alpha  helices encircling a central beta -sheet and is connected at its N and C termini to an immunoglobulin-like "hybrid" domain and forms an extensive interface with it. The key regions involved in ligand recognition are loops on the upper surface of the beta -propeller and the upper face of the beta A, which contains a metal ion-dependent adhesion site (MIDAS) and an adjacent MIDAS cation-binding site (13-15). A small number of subtle conformational changes between the unliganded and liganded states were observed. The most important of these appeared to be a shift of the alpha 1 helix in beta A, and a slight closing up of the interface between the upper surface of the beta -propeller and the upper face of the beta A. A surprising feature of the crystal structures was that the two legs are severely bent at the "knees," such that the head is in close contact with lower legs. Because the peptide ligand was soaked into the crystals of unliganded alpha Vbeta 3, it is unclear whether the small conformational changes observed between the unliganded and liganded structures (13, 14) are representative of those that take place upon ligand occupancy of the native integrin. Importantly, no pathway for the transduction of conformational changes from the head to the legs (or from legs to head) was evident. Hence, the molecular basis of both outside-in and inside-out signaling remains to be clarified.

Recently, evidence (16, 17) has been presented that the bent form of the integrin is in the inactive state and that this may undergo a switchblade-like straightening to attain the active conformation. Nevertheless, precisely how this straightening is linked to activation of ligand binding in the head domain is uncertain. A key regulator of integrin activity is known to be the conformation of the beta A domain (15, 18), and we have shown that a movement of the alpha 1 helix activates this domain (8). We hypothesized that the alpha 1 helix could occupy two different positions: position 1 characterized by high binding of the mAb 12G10 (the active conformation), and position 2 characterized by low binding of 12G10 (the inactive conformation). The inward movement of alpha 1 helix observed in the liganded alpha Vbeta 3 structure (14) supports this proposal. Hence, position 1 appears to correspond to the "in" state and position 2 to the "out" state of the alpha 1 helix. About half of the integrin alpha  subunits contain a similar domain (alpha A or I), and in these domains an inward movement of the alpha 1 helix is linked to rearrangement of cation-coordinating residues at the MIDAS and a dramatic downward shift of the C-terminal alpha 7 helix and its preceding loop (19). However, no change in the position of the alpha 7 helix of beta A was observed between the two x-ray structures, and it was suggested that activation of beta A does not involve alpha 7 movement (13-14, 20).

Here we provide evidence that changes in the expression of activation epitopes in the hybrid domain are linked to shape-shifting in the alpha 7 helix region of beta A. This movement appears to participate in the conformational changes involved in both activation and ligand binding. Our data suggest that an outward swing of the hybrid domain is coupled to alpha 7 helix motion, and hence lend support to a recent model of integrin activation (21). There are both strong similarities and some differences between beta A and alpha A domain activation.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Monoclonal Antibodies-- Rat mAbs 16 and 13 recognizing the human alpha 5 and beta 1 subunits, respectively, were gifts from Dr. K. Yamada (NIDCR, National Institutes of Health, Bethesda). Mouse anti-human alpha 5 mAb P1D6 was a gift from Dr. E. Wayner (Fred Hutchinson Cancer Research Center, Seattle, WA). Mouse anti-human alpha 5 mAb SNAKA52 and mouse anti-human beta 1 mAbs 12G10 and 8E3 were produced as described (22, 23). Mouse anti-human mAb TS2/16 was a gift from F. Sánchez-Madrid (Hospital de la Princesa, Madrid, Spain). Mouse anti-human mAbs JB1A and N29 were gifts from J. Wilkins (University of Manitoba, Winnipeg, Canada). Mouse anti-human mAb 15/7 was a gift from T. Yednock (Elan Pharmaceuticals, South San Francisco, CA). Mouse anti-human mAbs 4B4 and HUTS-4 were purchased from Beckman Coulter (High Wycombe, UK) and Chemicon (Harrow, UK), respectively. All mAbs were used as purified IgG.

Expression Vector Construction and Mutagenesis-- C-terminally truncated human alpha 5 and beta 1 constructs encoding alpha 5 residues 1-613 and beta 1 residues 1-455 fused to the hinge regions and CH2 and CH3 domains of human IgGgamma 1 (alpha 5-(1-613)-Fc and beta 1-(1-455)-Fc) were generated as described previously (24). To aid the formation of heterodimers, the CH3 domain of the alpha 5 construct contained a "hole" mutation, whereas the CH3 domain of the beta 1 constructs carried a "knob" mutation as described (24, 25). The L358A and S359A mutations in the beta 1 subunit were carried out using oligonucleotide-directed PCR mutagenesis, as described (24). Oligonucleotides were purchased from MWG Biotech (Southampton, UK). The presence of the mutations (and the lack of any other changes to the wild-type sequence) was verified by DNA sequencing.

Transfection-- Chinese hamster ovary cells L761h variant (24) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, and 1% non-essential amino acids (growth medium). Cells were detached using 0.05% (w/v) trypsin, 0.02% (w/v) EDTA in phosphate-buffered saline, and plated overnight into 6-well culture plates (Costar). Approximately 1 µg of wild-type or mutant beta 1-(1-455)-Fc or beta 1-(121-455)-Fc and 1 µg of wild-type alpha 5-(1-613)-Fc DNA/well was used to transfect the cells using LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's instructions. After 4 days, supernatants were harvested by centrifugation at 1000 × g for 5 min.

For comparison of purified wild-type heterodimers with heterodimers containing the L358A or S359A mutations in beta 1, 75-cm2 flasks of sub-confluent CHOL761h cells were transfected with 5 µg of wild-type or mutant beta 1-(1-455)-Fc and 5 µg of alpha 5-(1-613)-Fc DNA as described above. After 4 days, culture supernatants were harvested by centrifugation at 1000 × g for 5 min. Wild-type or mutant heterodimers were purified using protein A-Sepharose essentially as described before (24).

Proteins-- A recombinant fragment of fibronectin containing type III repeats 6-10 (III6-10) was produced and purified as described previously (12). A mutant fragment in which the RGD integrin-binding sequence is replaced by the inactive sequence KGE (III6-10KGE, see Ref. 26) was produced and purified in the same manner. III6-10 was biotinylated as before (8) using sulfo-LC-NHS biotin (Perbio, Chester, UK). Fab fragments of N29, TS2/16, and 12G10 were prepared by ficin cleavage of purified IgG, followed by removal of Fc-containing fragments using protein A-Sepharose, according to the manufacturer's instructions (Perbio). None of the Fab fragments showed any reactivity with goat anti-mouse IgG (Fc-specific) peroxidase conjugate (Sigma).

Effect of Divalent Cations on 15/7 and HUTS-4 Binding-- 96-Well plates (Costar 1/2-area EIA/RIA, Corning Science Products, High Wycombe, UK) were coated with goat anti-human gamma 1 Fc (Jackson Immunochemicals, Stratech Scientific, Luton, UK) at a concentration of 2.6 µg/ml in Dulbecco's phosphate-buffered saline (50 µl/well) for 16 h. Wells were then blocked for 1-3 h with 200 µl of 5% (w/v) BSA, 150 mM NaCl, 0.05% (w/v) NaN3, 25 mM Tris-Cl, pH 7.4 (blocking buffer). Blocking buffer was removed, and supernatant from cells transfected with wild-type alpha 5-(1-613) and beta 1-(1-455)-Fc diluted 1:1 with 150 mM NaCl, 25 mM Tris-Cl, pH 7.4 (25 µl/well), was added for 1-2 h at room temperature. Wells were then washed three times with 200 µl of 150 mM NaCl, 25 mM Tris-Cl, pH 7.4, containing 1 mg/ml BSA (buffer A). Buffer A was treated with Chelex beads (Bio-Rad) to remove any small contaminating amounts of endogenous Ca2+ and Mg2+ ions. mAbs (1 µg/ml) in buffer A with varying concentrations of Mn2+, Mg2+, or Ca2+ were added to the plate (50 µl/well). The plate was then incubated at 30 °C for 2 h. Unbound antibody was aspirated, and the wells were washed three times with buffer A. Bound antibody was quantitated by addition of 1:1000 dilution of goat anti-mouse IgG (Fc-specific) peroxidase conjugate (Jackson Immunochemicals) in buffer A for 30 min at room temperature (50 µl/well). Wells were then washed four times with buffer A, and color was developed using 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) substrate (50 µl/well). Absorption at 405 nm was measured using a plate reader (Dynex Technologies). Background binding to mAbs to wells incubated with supernatant from mock-transfected cells was subtracted from all measurements. Measurements obtained were the mean ± S.D. of four replicate wells.

For comparison of the effects of divalent cations on 15/7 and HUTS-4 binding to wild-type heterodimer and the L358A and S359A mutants, plates were coated with anti-human Fc, blocked as described above, and then incubated with supernatant from cells transfected with wild-type or mutant heterodimers. mAb binding was measured as described above in 2 mM EDTA, 2 mM Mn2+, 2 mM Mg2+, or 2 mM Ca2+. Measurements obtained were the mean ± S.D. of four replicate wells.

Effect of mAbs and Ligand on 15/7 and HUTS-4 Binding-- Plates were coated with anti-human Fc and blocked as described above. Wells were then incubated with supernatant from cells transfected with wild-type or mutant heterodimers for 1-2 h at room temperature as above. Wells were washed three times with 200 µl of 150 mM NaCl, 1 mM MnCl2, 25 mM Tris-Cl, pH 7.4, containing 1 mg/ml BSA (buffer B). 15/7 or HUTS-4 (l µg/ml in buffer B) was added to the plates (50 µl/well) either alone or in the presence of Fab fragments of N29, TS2/16, or 12G10 (5 µg/ml), mAb 13 IgG (10 µg/ml), or III6-10 (20 µg/ml). The plates were then incubated at 30 °C for 2 h. Unbound antibody was aspirated, and the wells were washed three times with buffer B. Bound 15/7 or HUTS-4 was quantitated by addition of 1:2000 dilution of goat anti-mouse IgG (Fc-specific, precleared with rat serum proteins) peroxidase conjugate (Sigma) in buffer B for 30 min at room temperature (50 µl/well). Wells were then washed four times with buffer A, and color was developed as above. Background binding of mAbs to wells incubated with supernatant from mock-transfected cells was subtracted from all measurements. Measurements obtained were the mean ± S.D. of four replicate wells.

Comparison of Epitope Expression by Wild-type and Mutant Heterodimers-- Plates were coated with anti-human Fc and blocked as described above. The blocking solution was removed, and cell culture supernatants were added (25 µl/well) for 1-2 h. All supernatants were assayed in triplicate, and supernatant from mock-transfected cells was used as a negative control. The plate was washed 3 times with buffer B (200 µl/well), and anti-alpha 5 or anti-beta 1 mAb (5 µg/ml) was added (50 µl/well). The plate was incubated for 2 h and then washed 3 times in buffer B. Peroxidase-conjugated anti-rat or anti-mouse secondary antibodies (1:1000 dilution in buffer B; Jackson Immunochemicals) were added (50 µl/well) for 30 min, and the plate was washed four times in buffer B, and color was developed as above. All steps were performed at room temperature. Results shown are the mean ± S.D. of three separate experiments.

Effect of L358A and S359A Mutations on III6-10 Binding-- Plates were coated with anti-human Fc and blocked as described above. Wells were then incubated with protein A-purified heterodimers diluted to ~1 µg/ml with 150 mM NaCl, 25 mM Tris-Cl, pH 7.4 (25 µl/well), for 1-2 h at room temperature. Wells were washed three times with 200 µl of buffer B. Biotinylated III6-10 (0.1 µg/ml) in buffer B was added to the plate (50 µl/well) alone or in the presence of N29, TS2/16, or 12G10 (5 µg/ml). The plate was then incubated at 30 °C for 2 h. Unbound ligand was aspirated, and the wells were washed three times with buffer B. Bound ligand was quantitated by addition of 1:500 dilution of ExtrAvidin® peroxidase conjugate (Sigma) in buffer B for 20 min at room temperature (50 µl/well). Wells were then washed four times with buffer B, and color was developed using 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) substrate (50 µl/well). Background binding to BSA-coated wells was subtracted from all measurements. Measurements obtained were the mean ± S.D. of four replicate wells.

Mapping of the 15/7 and HUTS-4 Epitopes-- Substitution of human residues with the corresponding residues in murine beta 1 within the hybrid domain sequence 361-425 was performed using a PCR-based mutagenesis kit (Gene Tailor, Invitrogen) according to the manufacturer's instructions. CHOL761h cells were transfected with wild-type or mutant constructs and supernatants harvested as described above. Binding of 15/7, HUTS-4, and TS2/16 to mutant heterodimers was performed as described above, relative to the wild-type control. Background binding to mAbs to wells incubated with supernatant from mock-transfected cells was subtracted from all measurements. Measurements obtained were the mean ± S.D. of three replicate wells. Results shown are mean ± S.D. of three separate experiments.

In each assay involving a comparison between different heterodimers, the binding of mAb 8E3 (5 µg/ml) was used to normalize any differences between the amounts of the different heterodimers bound to the wells. For example, normalized A405 for 15/7 binding = (AM15/7 - Am15/7) × ((AWT8E3 - Am8E3)/(AM8E3 - Am8E3)), where AM15/7 = mean absorbance of wells coated with mutant integrin; Am15/7 = mean absorbance of wells coated with mock supernatant; AWT8E3 = mean absorbance of 8E3 binding to wells coated with wild-type integrin; Am8E3 = mean absorbance of 8E3 binding to wells coated with mock supernatant, and AM8E3 = mean absorbance of 8E3 binding to wells coated with mutant integrin. 8E3 recognizes a non-functional epitope in the N-terminal region of the beta 1 subunit (24). Essentially identical results were obtained from normalization using mAb N29 against the PSI domain (Ref. 27, data not shown). In experiments using heterodimers captured from cell culture supernatants, similar results were obtained using protein A-purified heterodimers (data not shown). Each experiment shown is representative of at least three separate experiments.

Homology Modeling of the Head Region of alpha 5beta 1-- A model of the alpha 5-propeller and thigh domains and beta 1A and hybrid domains was built based on an alignment against the alpha Vbeta 3 crystal structure (13), using the same procedures as described previously (8). The PSI domain (residues 1-60 of beta 1) was not included in the model. Representation of the structure was produced using PyMol.2

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of the 15/7 and HUTS-4 Epitopes Is Regulated by Conformational Changes in the beta A Domain-- To investigate the mechanisms of integrin activation, we employed a recently described system for expression of recombinant soluble alpha 5beta 1 (24). For these particular studies, we have used a truncated version of alpha 5beta 1, alpha 5-(1-613) beta 1-(1-455), fused to the Fc region of human IgGgamma 1 (24) (hereafter referred to as tralpha 5beta 1-Fc). This heterodimer contains the alpha  subunit beta -propeller and thigh domain, and the beta  subunit A, hybrid, and PSI domains (13), and has been shown to retain the properties of the full-length receptor (24). This system is particularly useful (a) because it permits the rapid analysis of the effects of mutations, and (b) because conformational changes in the head region can be studied in isolation, i.e. in the absence of any complicating effects due to the presence of the lower leg domains (e.g. unbending, see Refs. 16 and 17) or the cytoplasmic tails (5).

Activation of the integrin head is known to involve conformational changes in beta A, and since beta A is connected at its N and C termini to the hybrid domain, these changes must be transduced from and to the hybrid. HUTS-4 and 15/7 are two previously characterized mAbs whose epitopes lie within this region of the beta 1 subunit (9, 29-31). Expression of the 15/7 and HUTS-4 epitopes by tralpha 5beta 1-Fc was found to be cation-modulated (Fig. 1, A and B). Binding of each mAb was promoted by Mn2+ and to a smaller extent by Mg2+, whereas Ca2+ did not stimulate binding. Importantly, these effects parallel the effects of each divalent ion on the ligand-binding competence of the integrin (32), and they also mirror a conformational change in the beta A domain reported by mAb 12G10 (8). These changes have been shown to be due to cation binding to the MIDAS (8), and in agreement with this, the MIDAS mutation D130A prevented the cation modulation of 15/7 and HUTS-4 binding (data not shown).


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Fig. 1.   Effect of divalent cations on the binding of 15/7 (A) and HUTS-4 (B) to tralpha 5beta 1-Fc. Binding of mAbs was measured in the presence of varying concentrations of Mn2+ (), Mg2+ (black-square), or Ca2+ (black-triangle).

Conformational changes in beta A can also be induced by function-perturbing mAbs with epitopes in this domain. The epitopes of all function-altering anti-human beta 1 mAbs that map to the A domain include one or more residues in the sequence Asn207-Lys218 (33), which is predicted to form the alpha 2 helix region (13). The epitope of mAb 12G10 also includes two arginyl residues that lie near the base of the alpha 1 helix (8). TS2/16 and 12G10 are examples of activating mAbs, whereas 13 is an example of a function-blocking mAb (11). The mAb N29, whose epitope lies in the PSI domain, was used as a control.3 15/7 and HUTS-4 binding to tralpha 5beta 1-Fc was increased by TS2/16 and 12G10 but markedly decreased by mAb 13 (Fig. 2). Hence, conformational changes in the alpha 1/alpha 2 helix region of beta A appear to modulate 15/7 and HUTS-4 binding. As ligand recognition is also known to cause shape-shifting in beta A and to stabilize the active conformation of this domain (8), we tested the effect of ligand binding on the expression of the 15/7 and HUTS-4 epitopes. A recombinant fragment of fibronectin containing the alpha 5beta 1 recognition sites (26) stimulated 15/7 and HUTS-4 binding (Fig. 2) to a similar extent as TS2/16 and 12G10.


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Fig. 2.   Effect of function-perturbing mAbs and III6-10 fibronectin fragment on binding on 15/7 and HUTS-4 to tralpha 5beta 1-Fc. Binding of 15/7 or HUTS-4 was measured in the absence of other mAbs or ligand (Con) or in the presence of Fab fragments of the non-function-perturbing N29, or the activating TS2/16 or 12G10, or in the presence of IgG of the function-blocking mAb 13, or in the presence of III6-10 fragment of fibronectin (FN). The experiment was performed in the presence of 1 mM Mn2+. In control experiments the binding of 15/7 or HUTS-4 was not affected by control rat IgG or by a III6-10 fragment of fibronectin lacking the RGD integrin binding sequence (data not shown).

Taking these data together, the active conformation of beta A (stabilized by Mn2+/Mg2+, activating mAbs, or ligand) leads to increased expression of the 15/7 and HUTS-4 epitopes. Conversely, the inactive conformation of beta A (stabilized by Ca2+ or function-blocking mAbs) leads to decreased expression of the 15/7 and HUTS-4 epitopes. These effects may be linked to a motion of the alpha 1 helix (8). Our data on the effects of cations, function-perturbing mAbs, and ligand are in broad agreement with previous characterization of 15/7 and HUTS-4 as mAbs recognizing activation/LIBS epitopes (9, 29, 31).

A Mutation in the beta A Domain alpha 7 Helix (L358A) Results in Constitutively High Expression of the 15/7 and HUTS-4 Epitopes-- The transduction of conformational changes from beta A to the hybrid domain must take place at the interface between these two modules. At its C terminus beta A is joined to the hybrid domain by the alpha 7 helix, and mutations in this region of alpha  subunit A domains cause activation by favoring a downward shift of alpha 7 (34, 35). We therefore tested whether similar mutations in the alpha 7 helix of beta A could affect 15/7 and HUTS-4 binding. A mutation Leu358 to Ala was found to cause increased expression of the 15/7 and HUTS-4 epitopes, whereas the control mutation S359A had little effect (Figs. 3 and 4). Expression of the 15/7 and HUTS-4 epitopes by the L358A mutant was constitutively high compared with the wild-type receptor, and Mn2+/Mg2+ only caused a small enhancement of the level of binding seen in the absence of divalent ions (Fig. 3, A and B). The high level of 15/7 binding to the L358A mutant was only slightly increased by activating mAbs TS2/16 or 12G10 and was relatively resistant to inhibition by mAb 13 (Fig. 4); similar results were obtained for HUTS-4 (data not shown). In contrast to the wild-type receptor, ligand binding did not increase 15/7 epitope expression by the L358A mutant (Fig. 4). The mutation S359A had little effect on the ability of mAbs or ligand to modulate 15/7 binding (results similar those for wild-type tralpha 5beta 1-Fc, see Fig. 2). These findings suggest that the L358A mutation constrains the beta A domain in an active conformation and that the alpha 7 helix region is involved in conveying conformational changes from beta A to the hybrid domain.


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Fig. 3.   Effect of L358A and S359A mutations on modulation of 15/7 (A) and HUTS-4 (B) binding to tralpha 5beta 1-Fc by divalent cations. 15/7 or HUTS-4 binding was measured in the presence of 2 mM EDTA (open bars), 2 mM Mn2+ (filled bars), 2 mM Mg2+ (gray bars), or 2 mM Ca2+ (diagonally shaded bars).


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Fig. 4.   Effect of L358A and S359A mutations on modulation of 15/7 binding to tralpha 5beta 1-Fc by function-perturbing mAbs and III6-10 fibronectin fragment. Binding of 15/7 or HUTS-4 to the L358A or S359A mutants was measured in the absence of other mAbs or ligand (Con), or in the presence of Fab fragments of N29, TS2/16, or 12G10, or IgG of the function-blocking mAb 13, or the III6-10 fragment of fibronectin (FN). The experiment was performed under the same conditions as that shown in Fig. 2.

The L358A Mutant Mimics the Ligand-occupied Conformation-- We next tested whether the L358A mutant caused any other conformational changes associated with activation or ligand binding. For this purpose we used a panel of mAbs recognizing epitopes on both the alpha 5 and beta 1 subunits (Fig. 5A). The results showed that the L358A mutation increased the expression of the 12G10 epitope and decreased the expression of the 13 and 4B4 epitopes in beta A. The mutation also attenuated the expression of the epitopes of function-blocking mAbs SNAKA52, 16, and P1D6, which lie at or near the top of the alpha 5 beta -propeller domain (23), close to the beta A/propeller interface (13). Although, as shown above, the L358A mutation increased the expression of the 15/7 and HUTS-4 epitopes, it did not alter the expression of the JB1A epitope, which maps to a different region of the hybrid domain (36). Furthermore, the expression of epitopes that are not affected by the activation state, such as TS2/16 and JB1A, was not altered by the L358A mutation. The control mutation S359A had no significant effect on the expression of any of the epitopes tested (Fig. 5B). Taken together, these results show that the L358A mutation specifically increases the expression of all activation/LIBS epitopes (12G10, 15/7, and HUTS-4, see Refs. 8, 9, and 31) and decreases the expression of all LABS epitopes (SNAKA52,4 16, P1D6, 13, and 4B4, see Refs. 11, 12, and 37). Hence, the data suggest that L358A mutant adopts a conformation that is similar to the ligand-occupied state. Furthermore, a conformational change in the alpha 7 helix region of beta A caused by the mutation appears to be linked to movements in the alpha 1/alpha 2 helix region (the location of the 12G10, 13, and 4B4 epitopes) and the proximity of the beta A/propeller interface (the location of the SNAKA52, 16, and P1D6 epitopes).


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Fig. 5.   Expression of alpha  and beta  subunit epitopes by L358A mutant (A) and S359A mutant (B). Binding of anti-alpha 5 and anti-beta 1 mAbs to tralpha 5beta 1-Fc with the mutations L358A or S359A in beta 1 is expressed as a percentage of the binding to wild-type tralpha 5beta 1-Fc. Experiments were performed in the presence of 1 mM Mn2+. Results are mean ± S.D. of at least three separate experiments. *, p < 0.05 by Student's t test.

The L358A Mutation Causes Activation of Ligand Binding-- If the conformation of the L358A mutant is akin to the ligand-occupied state, it would be predicted that this mutant should be constitutively active for ligand binding (38). Tralpha 5beta 1-Fc has low constitutive ligand binding activity when captured onto enzyme-linked immunosorbent assay plates using goat anti-human Fc, but the same protein has similar activity to recombinant integrin containing the complete extracellular domains of alpha 5 and beta 1 when stimulated with mAbs such as 12G10 (24). We compared the ligand binding activity of wild-type tralpha 5beta 1-Fc with the L358A and S359A mutants (Fig. 6). The results showed that compared with the wild-type receptor, the L358A mutant had high constitutive ligand binding activity, which was only slightly enhanced by activating mAbs TS2/16 or 12G10. In contrast, the S359A mutant had constitutively low activity, similar to wild-type levels.


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Fig. 6.   Effect of L358A and S359A mutations on binding of III6-10 fibronectin fragment. Binding of III6-10 was measured in the absence of mAbs (open bars) or in the presence of the non-function-perturbing mAb N29 (horizontally hatched bars), or the activating mAbs TS2/16 (diagonally hatched bars), or 12G10 (cross-hatched bars).

The Epitopes of 15/7 and HUTS-4 Map to a Region of the Hybrid Domain Close to an Interface with the alpha  Subunit-- The above results suggest that the transduction of conformational changes from beta A to the hybrid domain involves a shift of the alpha 7 helix. To understand these changes more fully, we fine-mapped the epitopes of 15/7 and HUTS-4. Both antibodies bind to human beta 1 but not to mouse beta 1, and their epitopes have been shown to reside within amino acid residues 355-425 (30, 31). These residues in human beta 1 show 10 differences with the equivalent sequence in murine beta 1 (Table I). Mutant proteins containing single point mutations at each of these positions were expressed and tested for binding of 15/7 and HUTS-4, using TS2/16 as a control. The mutations E371D and K417N abrogated both 15/7 and HUTS-4 binding, whereas the mutation S370P completely blocked 15/7 binding and strongly inhibited HUTS-4 binding. The other mutations had either no effect or showed a partial inhibition (Table I). None of the mutations affected binding of TS2/16. Ser370 and Glu371 map to the C-D loop, and Lys417 maps to the neighboring E-F loop of the hybrid domain (13). The spatial proximity of this triplet of residues is consistent with them forming an antibody epitope.


                              
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Table I
Analysis of 15/7 and HUTS-4 reactivity with beta 1 hybrid domain substitution mutants
CHO L761h cells were transfected with alpha 5-(1-613)-Fc and wild-type or mutant beta 1-(1-455)-Fc. Cell culture supernatants were analyzed for reactivity with anti-beta 1 mAbs by sandwich enzyme-linked immunosorbent assay. Results are expressed as a percentage of wild-type binding and are mean ± S.D. from three separate experiments (except for the S422T mutant, from two separate experiments). A value of 0% indicates that mAb reactivity was identical to, or slightly lower than, reactivity with supernatant from mock-transfected cells. All the mutants bound well to the hybrid domain mAb JB1A, and none of the mutations affected recognition of the III6-10 fragment of fibronectin (data not shown).

Comparison with the crystal structures of alpha Vbeta 3 (13, 14) shows that these epitopes map to a region of the hybrid domain that faces the alpha  subunit beta -propeller and are very close to residues that form a small interface with it. To estimate the antibody-accessible surface, we rolled a 20-Å sphere over the structure (16). The results (not shown) demonstrated that Lys417 would be accessible in this conformational state, but Ser370 and Glu371 would not. However, Ser370 and Glu371 would be available for antibody binding if the hybrid domain moves away from the beta -propeller.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

By using the conformation-sensitive mAbs 15/7 and HUTS-4 and site-directed mutagenesis, we have studied conformational changes in integrin beta 1 A domain, and we investigated how these relate to signal transduction in the integrin head region. Our results show the following: (i) beta A domain activation involves a conformational change in the region of alpha 7 helix; (ii) this shape-shifting results in increased exposure of the 15/7 and HUTS-4 epitopes in the hybrid domain and is also associated with other conformational changes in beta A and the top of the alpha  subunit beta -propeller; (iii) the 15/7 and HUTS-4 epitopes map to a portion of the hybrid domain that is likely to be partly masked (in the inactive receptor) due to its close proximity to the beta -propeller. Taking these results together with previous data showing that a movement of the alpha 1 helix is important for activation of beta A (8), we propose the model of affinity regulation shown in Fig. 7.


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Fig. 7.   Model of conformational changes involved in activation of integrin alpha  and beta  head domains. A model of the alpha 5-propeller and thigh domains and beta 1 A and hybrid domains was built as described under "Experimental Procedures." For the beta  subunit, beta  strands are shown in blue, and alpha  helices are shown in red, except for alpha 7 (in yellow) and alpha 2 (in orange, partly hidden by alpha 1). The divalent ion at the MIDAS site of beta A is depicted by a magenta sphere. The metal ion at the adjacent to MIDAS site of the beta A domain (13) is omitted for the sake of clarity. For the alpha  subunit, secondary structure elements are shown in gray. In the model, Ser370, Glu371, and Lys417, which form the 15/7 and HUTS-4 epitopes, are depicted by green space fill. In the transition from the inactive to the active form of the beta A domain appears to be both an inward movement of the alpha 1 helix and a downward shift of the alpha 7 helix (solid arrows). A downward motion of the alpha 7 helix would be coupled to an outward swing of the hybrid domain (open arrow 1), but this movement of the hybrid domain could also push on the lower face of beta A (open arrow 2), stabilizing the in position of the alpha 1 helix and tilting beta A toward the top face of the beta -propeller (open arrow 3). The alpha 1/alpha 2 helix region contains the epitopes of activating and inhibitory anti-beta 1 mAbs (8, 33, 47), whereas loops at the top of the beta -propeller (indicated by arrowheads) contain the epitopes of function-blocking anti-alpha 5 mAbs (23). The tilt of beta A toward the beta -propeller could partially attenuate the anti-alpha 5 mAb epitopes because it closes up the beta A/propeller interface.

Mechanism of beta A Activation-- Recent debate concerning the mechanism of activation of beta A has centered on whether or not the alpha 7 helix moves. Three distinct models have been proposed. (i) Based on the crystal structures of alpha Vbeta 3 (13, 14) in which there is no movement of alpha 7, and the MIDAS site is unoccupied in the absence of ligand, it was suggested that beta A is regulated by unmasking of the MIDAS site (20, 39). (ii) Based on a separation of the head domains observed in RGD-occupied alpha IIbbeta 3 after rotary shadowing/electron microscopy (40), and on a similarity between the quaternary structures of integrins and heterotrimeric G-proteins (13), it was hypothesized that activation involves a rotation of beta A away from its contact with the beta -propeller (41). In this scenario, the position of the alpha 7 helix is fixed but the rotation of beta A resulted in the same net movement of alpha 7 seen in alpha A domains. (iii) Based on studies of inactive (Ca2+-occupied) and active (Mn2+-occupied) alpha Vbeta 3 by negative staining/electron microscopy (17), it was suggested that activation of the head region is regulated by an outward swing of the hybrid domain, which is predicted to be coupled to downward shift of the alpha 7 helix equivalent to that in alpha A domains (19, 42). Model i appears unlikely because our results (this work and see Ref. 8) and the results of others (17, 18) show that conformational changes take place through cation binding to the MIDAS in the absence of ligand occupancy. Model ii is improbable because beta A and the beta -propeller appear to move closer together, rather than farther apart, upon ligand recognition because ligand binding requires close apposition of residues on both subunits (14, 39). Instead, our data supply strong support for model iii because they suggest that a movement in the alpha 7 helix region is important for activation and that this movement is linked to a change in the position of the hybrid domain such that it moves away from the alpha /beta subunit interface. The existence of the swing-out motion of the hybrid is further supported by the finding that a section of the beta 3 hybrid domain encompassing residues 393-423 (equivalent to residues 402-432 in beta 1) is exposed in the active but not the resting form of alpha IIbbeta 3 (43). This portion of the hybrid domain encompasses part of the 15/7 and HUTS-4 epitopes (Lys417).

Why was no movement of the alpha 7 helix seen in the crystal structure of the liganded form of alpha Vbeta 3 (13)? The likely explanation is that motion of alpha 7 would be prevented because the hybrid domain is paralyzed by lattice contacts and by its contacts with the leg domains (16, 17). Some conformational changes are observed in the liganded beta A domain; these include rearrangement of the loops that coordinate the MIDAS cation, leading to an inward movement of the alpha 1 helix. These changes are very similar, both in direction and form, to those seen in alpha A domains; however, as pointed out above, the subsequent downward motion of the alpha 7 helix that takes place in alpha A domains is probably prohibited. Thus, the structural changes observed in the liganded beta A also favor the hypothesis that the unliganded structure represents the inactive form of beta A (44).

We found that a mutation in the alpha 7 helix, L358A, caused activation of the beta A domain. In alpha A domains mutation of a highly conserved isoleucine residue at the same position favors the active state. This is apparently because this residue fits into a hydrophobic pocket (known as "socket for isoleucine," SILEN), and this interaction favors the inactive form (34). However, unlike the alpha A domains, mutation of residues that form the hydrophobic pocket surrounding the alpha 7 helix in the beta 1 A domain (Leu125, Leu149, Leu253, and Ile314) did not affect the activation state of tralpha 5beta 1-Fc.5 Hence, the mechanism that regulates alpha 7 movement may differ slightly from that of alpha A domains. Nevertheless, mutation of Leu358 may favor the active state of beta A because this residue is likely to be more exposed in the "down" position of the alpha 7 helix than in the "up" position. Hence, mutation of the leucine residue to the less hydrophobic alanine would be predicted to lower the energy of the active state. We cannot rule out the possibility that the L358A mutation activates the receptor by altering the beta A/hybrid interface. However, mutation of Ser359 (also at the interface) did not cause activation, and furthermore, this paradigm would not explain how activating anti-beta A mAbs cause activation and hybrid domain movement in a similar manner to the L358A mutation. In contrast, a linked movement of the alpha 1 and alpha 7 helices as seen in beta A domains can explain the mechanism of action of the anti-beta A domain mAbs (see below).

Similarities and Differences between Activation of beta A and alpha A Domains-- In the activation of alpha A domains a change in cation coordination at the MIDAS is linked to an inward movement of the alpha 1 helix. This movement pinches the hydrophobic core, squeezing out residues in the loop that precedes the alpha 7 helix, and results in an ~10-Å downward motion of alpha 7 (19). A similar link between alpha 1 and alpha 7 helix movement in beta A is suggested by our findings. For example, occupancy of the MIDAS by Mn2+ or the binding of activating mAbs, which stabilize the in position of the alpha 1 helix (8), also promotes the downward motion of the alpha 7 helix (reported by increased exposure of the 15/7 and HUTS-4 epitopes).

Although the overall mechanisms of beta A and alpha A activation now appear to be closely related, there are some subtle differences. For example, for alpha A domains Mn2+ and Mg2+ are equally effective for promoting ligand binding to the MIDAS (45, 46). In contrast, Mn2+ is much more effective than Mg2+ for promoting ligand binding to the beta A MIDAS (8). This property of Mn2+ may be due to the fact that it binds with much higher affinity than Mg2+ to the beta A MIDAS, whereas the affinities of alpha A domains for these two ions are more comparable (44, 45). Similar to alpha A domains, movement of the alpha 7 helix appears to form an essential part of the activation mechanism of beta A because alpha 7 movement closely parallels the activation state. However, as noted above, the regulation of alpha 7 motion may be slightly different to that in alpha A domains.

Allosteric Mechanism of Function-perturbing mAbs-- We have shown previously (11, 12, 37) that most function-blocking anti-alpha 5 and anti-beta 1 mAbs have an allosteric mode of action. They recognize epitopes attenuated by ligand recognition and appear to perturb ligand binding by preventing a conformational change involved in the formation of the integrin-ligand complex (7). The epitopes of function-blocking anti-beta 1 A domain mAbs map to the alpha 2 or alpha 1 helix regions (13, 33, 47), which lie adjacent to each other. Similarly, the epitopes of function-blocking anti-beta 2 A domain mAbs have been shown to include residues in the alpha 1/alpha 2 or alpha 7 helix regions (48, 49). It is likely that these mAbs function allosterically by stabilizing alpha 1 in the inactive (out) location and/or alpha 7 in the inactive (up) position. The epitopes of function-blocking anti-alpha subunit mAbs map to loops on the top face of the beta -propeller domain (indicated by arrowheads in Fig. 7), and the binding of these mAbs may prevent conformational rearrangements required for ligand binding (14). Hence both anti-alpha and anti-beta subunit function-blocking mAbs appear to impede conformational changes involved in ligand recognition, as proposed previously (7).

The epitopes of activating anti-beta A domain mAbs map to the same regions as inhibitory mAbs (8, 18, 33) and are likely to function by stabilizing alpha 1 in the active (in) location and/or alpha 7 in the active (down) position. It has been suggested that 12G10 activates by affecting the beta A/hybrid domain interface (44), rather than by an effect on the alpha 1 helix (8). However, we consider this proposal to be incorrect because (i) the mechanism of 12G10 action is likely to be closely overlapping with that of other activating anti-beta A mAbs (whose epitopes are not close to the interface), and (ii) 12G10 has the same properties in a recombinant integrin that lacks a large part of the hybrid domain, suggesting that it directly affects the conformation of beta A (24). The activating effect of the 15/7 and HUTS mAbs (9, 31) is likely to be due to their preferential binding to the active form of the integrin in which the hybrid domain is shifted away from the beta -propeller.

Implications for Inside-out and Outside-in Signaling-- A recent NMR study of a complex between the intracellular segments of alpha IIb and beta 3 suggests that integrin activation is prevented by a "handshake" between alpha  and beta  cytoplasmic tails, and that unclasping of this handshake by proteins such as talin represents the first stage of activation (5). Separation of the cytoplasmic domains may then be linked to unbending of the integrin legs, which in turn is coupled to activation of head domains (16, 17). In this scenario, hybrid domain movement is severely restricted by its interface with the leg domains in the inactive, bent form of the integrin. Unbending causes activation because it is coupled to the release of the hybrid domain from these constraints, allowing swinging of the hybrid to take place, which, in turn, would cause pulling on the alpha 7 helix of beta A. Our findings generally support this model of activation. However, since removal of the lower leg domains (in the truncated integrin) did not favor the active state (24), hybrid domain movement (rather than unbending per se) appears to be the essential requirement for activation. Hybrid domain motion provides the conduit for the transduction of signals to and from the head region. The attenuation of the epitopes on the beta -propeller in the L358A mutant also suggests that the outward swing of the hybrid domain is coupled to a conformational rearrangement of the beta A/propeller interface that is involved in ligand recognition (14). Similarly, ligand binding reinforces and stabilizes the conformational changes associated with activation (e.g. the movements of the alpha 1 and alpha 7 helices and hybrid domain). This feature of ligand recognition may explain the well known ability of integrin ligands to cause activation that persists after dissociation of the complex (50, 51). Outside-in signaling could result, in part, from stabilization of the active conformation, allowing the separated cytoplasmic tails to stably interact with cytoskeletal and signaling molecules. In addition, integrin clustering is also a major contributor to this signaling (52, 53).

In summary, we have shown that a conformational shift in the alpha 7 helix region of the beta A domain is involved in the regulation of integrin activity. This movement is coupled to a swing-out of the hybrid domain and thereby provides a pathway for signal transduction. Integrins are important therapeutic targets in many inflammatory and cardiovascular disorders (28), and our findings suggest a novel way in which highly specific regulators of integrin activity could be developed (e.g. by stabilizing the hybrid/propeller interface). A more complete understanding of the signaling mechanisms will require further characterization and crystallization of an integrin in defined conformational states.

    ACKNOWLEDGEMENTS

We thank A. Coe, P. Stephens, and M. Robinson for the alpha 5 and beta 1 constructs. We are grateful to J. Wilkins, T. Yednock, K. Yamada, E. Wayner and F. Sánchez-Madrid for mAbs; K. Yamada and S. Aota for the III6-10 and III6-10 KGE constructs; E. Symonds for assistance with integrin purification and cell culture; A. Arnaout for the coordinates of the alpha Vbeta 3 structures; and J. Bella for advice on molecular modeling.

    FOOTNOTES

* This work was supported in part by grants from the Wellcome Trust (to M. J. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Rd., Manchester M13 9PT, UK. Tel.: 44-161-275-5649; Fax: 44-161-275-5082; E-mail: paul.mould@man.ac.uk.

§ Supported by a studentship from the Biotechnology and Biological Sciences Research Council.

Published, JBC Papers in Press, March 3, 2003, DOI 10.1074/jbc.M213139200

2 W. L. Delano, The Pymol Molecular Graphics System, Delano Scientific, San Carlos, CA (www.pymol.org).

3 Although previously characterized as an activating mAb (27), N29 did not cause any activation of tralpha 5beta 1-Fc and was used as a negative control.

4 K. Clark, R. Pankov, J. A. Askari, A. P. Mould, S. E. Craig, P. Newham, K. M. Yamada, and M. J. Humphries, manuscript in preparation.

5 A. P. Mould, J. A. Askari, S. J. Barton, and M. J. Humphries, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: mAb, monoclonal antibody; beta A, beta subunit von Willebrand factor type A domain; alpha A, alpha subunit von Willebrand factor type A domain; MIDAS, metal-ion dependent adhesion site; BSA, bovine serum albumin; tralpha 5beta 1-Fc, recombinant soluble integrin heterodimer containing C-terminally truncated alpha 5 and beta 1 subunits (alpha 5 residues 1-613 and beta 1 residues 1-455) fused to the Fc region of human IgGgamma 1; LIBS, ligand-induced binding site; LABS, ligand-attenuated binding site.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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